VisiScope Confocal-FRAP Cell Explorer - unifr.ch€¦ · VisiScope Confocal-FRAP Cell Explorer Live...

Visitron Systems GmbH Tel. 089 / 890245 - 0 Gutenbergstr. 9 Fax 089 / 890245-18 D-82178 Puchheim, FRG Info @ Visitron .de

VI ITRON S GmbHYSTEMS Imaging � Microscopy

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VisiScope Confocal-FRAP

Cell Explorer

Live Cell Imaging System User Manual

Rev. 2.5 03-2014



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Notices Visitron Systems GmbH, 2014 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Visitron Systems GmbH as governed by international copyright laws. Edition Edition: 03-2014 Release: Vers. 2.5.0. Printed in Germany Visitron Systems GmbH Gutenbergstraße 9 82179 Puchheim Germany Acknowledgements Windows is a U.S registered trademark of Microsoft Corporation. Warranty The material contained in this document is provided as is, and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Visitron Systems GmbH disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Visitron Systems GmbH shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.



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Contents System VisiScope / VisiFRAP Fluorescence Imaging System 1. Safety Instructions and Installation Precautions 2. Introduction to the VisiScope System 2.1 Overview 2.2 Parts of the VisiScope System 3. Installing the of the VisiFRAP-DCSystem 3.1 Installing 3.2 Connecting the Cables 3.2.1 Overview 3.2.2 Overview of the cables required 3.3 Optical Alignment / Adapting to Microscope 3.4 Control unit scanner 3.4.1 Control by VS-Trigger-Control Unit 3.5 UV Laser 355nm passively Q-switched 3.6 UV Laser 405nm 3.7 VIS Laser and Coupling 4. Camera System 4.1 Coolsnap-EZ / ES Camera 4.2 Evolve Camera 4.3 PCO Edge 5.5 sCMOS Camera 4.4 PCO Edge 4.2 sCMOS Camera 5. Confocal and Laser Systems 5.1 CSU-W1 Spinning Disk Confocal 5.2 VS-Laser Power Control 5.3 VS Laser-Merge-System 5.3.1 VS Laser-Merge-System LMS6 with Dual Output 5.3.2 Additional Laser Output with Galvo 5.4 VS AOTF Control 5.5 VS AOM Control 5.5.1 VS-110 DAC-Trigger Box 5.6 Fiber Coupling Adapter - OZ Coupler 5.6.1 Fiber Coupling Adapter KineFlex Coupler 6.0 VS-Laser Safety 6.1 for Eye Piece, TIRF sample plate cover, microscope illumination arm



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7. Imaging Software for Operation of the VisiScope 7.1 VisiView® Software Installation 7.2 How to Update VisiView® Software 7.2 VisiView® Software 7.4 VisiView® Hot-Key Function 8. Microscope Peripherals 8.1 Filter Wheel System 8.2 XYZ- Motorized Stage with Piezo insert 8.2.1 Z-Piezo Insert for XY-Stage 8.3 Filterset 8.4 LED Illumination PE-300 White 8.5 LED Illumination PE-100 9. Incubation of the VisiScope System 9.1 okolab Incubation Chamber – overview only 10. EC Declaration of Conformity



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1. Safety Instructions and Installation Precautions Safety Terms A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met. CAUTION A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

Waste of Electrical and Electronic Equipment (WEEE)

This symbol on the product or on its packaging indicates that this product must not be disposed of with your other household waste. Instead, it is your responsibility to dispose of your waste equipment by handing it over to a designated collection point for the recycling of waste electrical and electronic equipment. The separate collection and recycling of your waste equipment at the time of disposal will help to conserve natural resources and ensure that it is recycled in a manner that protects human health and the environment. For more information about where you can drop off your waste equipment for recycling, please contact your local city office, your household waste disposal service or the vendor where you purchased the product. CAUTION No part of the user’s manuals may be transferred or reproduced without prior written consent from Visitron Systems GmbH. Visitron Systems continues to improve the product’s performance and features, and for this reason, the specifications and information herein are subject to change at any time and without notice. The authors and publishers of this manual have used their best efforts in preparing this document, but make no representation or warranties with respect to the accuracy, applicability, fitness, or completeness of the contents of this manual. If you have noticed any problems or have any suggestions for the manual, please contact us.



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2. Introduction of the VisiScope System

Multidimensional Acquisition and Analysis of Living Cells

Modern cell biology on living cells and tissues has reached revolutionary success in biological, medical and pharmacological research in recent years. With the VisiScope system you can reliably document living organisms and intracellular processes, even over a period of several days. While someone has to work with none-automated microscopes resulting in restricted application possibilities in the past, nowadays fully automated fluorescence microscopes with multidimensional control have been established. The VisiScope Cell Explorer, based on Visitron imaging software, solves all these new challenges easily and user friendly with highest precision. 2.1 Overview and Parts of the VisiScope System Overview of System Components

� High-performance microscope with superior optics and automation � High resolution digital scientific camera with highest sensitivity and best

cooling � Fully automated xy-table with best position accuracy and reproducibility � Precise z-axis control and acquisition (optionally: piezo z-focus drive) � Powerful Visitron imaging software including multidimensional control of

all hardware / motorized components � Incubation chambers controlling cell culture environment

Basic Setup:

� scientific grade CCD camera � upright / inverted microscope � high quality ET Filter-Sets � HBO / Metal Halide lamp system � motorized XY-stage or XYZ-stage � incubation chamber � CO² control

PC Computer

� 24” Monitor � VisiView Imaging Software



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Peripherals Option:

� Excitation / emission filter wheel system � Piezo Focus Control

Options:

� vibration isolation platform for microscope



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3. Installing the of the VisiFRAP-DC System with integrated Laser 3.1 Installing

If the system has been kept at low temperature allow warm up to room temperature before installation to avoid condensation water.

Place the VisiFRAP-DC System onto a robust and stable table. Best would be a vibration free microscope table.

1. Unpack carefully all delivered packages and put the equipment on a save table.

2. Take a look that the delivered equipment is conform to the ordered configuration and the Visitron delivery note

3. Mounting the optical components carefully in regards of the instruction from the Visitron support engineers.



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3.2 Connecting the Cables 3.2.1 Overview standard scanner with illumination The latest model extension of the Visitron System 2D-FRAP scanner family convinces by its compactness and flexibility. The new design of the VisiFRAP-DC allows the direct coupling of up to two lasers in the Galvo-scan-head without laser fiber. An additional VIS laser fiber input offers the combination of the system with other external VIS-laser lines if required. This unique new scan head opens new dimension in biological studies of living cells.



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Laser and Epi illumination ports at VisiFRAP-DC Direct microscope coupling by Visitron adapter plate with internal Laser combination and additional VIS-Laser input. Figure 01: internal laser 355nm, VIS laser Input option and Epi-port Figure 02: Dichroic slider In / Out with positioning / adjustment hex-driver



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3.2.2 Overview of the cables required There are two cables X and Y on the scan-head which bust be connected on the back side FRAP controller (X-Y labeled connector). On additional small connector A1 is for the analog voltage control and must by connected to the control box of the PC multi I/O board.

„X-connector“ „Y-connector“

„D1 analog input“



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3.3 Optical Alignment / Adapting to Microscope Overview internal optics and alignment options

2D Scanner

355nm UV laser

VIS Laser Input

EPI- Illumination



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XY-Alignment of 2D scan-head The scan head can be aligned in X and Y to get the laser beam centered and the correct symmetry. The 4 screws must be carefully a little bit opened at each side, then carefully moving the head by observing the laser beam on the computer screen. The screws has to be locked again after alignment procedure.

„X-position“

„Y-position“



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3.4 Control unit scanner The LED`s on the front panel of the FRAP scanner control unit indicates that the power is on. The power switch is on the back side of the controller. The control unit should be positioned on a place where enough air flow is available for cooling.

Power on Option: Laser

Option: Laser Key

You may switch on the required laser lines e.g. 405nm by turning the appropriate key switch in the on (horizontal) position. The corresponding laser warning light will go on immediately. However, the laser will actually go on only some seconds later for safety reasons.



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On the back panel a couple of connector available for synchronization of the scanner. Also the connector for the power cable for the XY-scanner and the analog interface for computer communication.

Option: Laser control

Connection to Galvo Scanner

Connection to PC analog out

Power switch



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3.4.1 Control by VS-Trigger-Control Unit The 2D Scanner are controlled by analog voltage in regards of the required positioning. Therefore an multi I/O DAC PC board including VS-101 “Trigger-Control Unit” is required which get delivered with the system.

The FRAP Channel output cable get connected on the rear panel of the FRAP controller Analog-In. For single laser systems without AOTF the Qswitch gets connected with the “Trigger-Input” of the laser. Additional Trigger Options:

Beside of the analog voltage control there are additional TTL Input trigger available for controlling shutters or TTL illumination systems The triggers are fully supported by the VisiView Imaging Software.



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3.5 UV Laser 355nm passively Q-switched For generating high peak power ultraviolet pulses of a few hundred picoseconds, microchip lasers are economical, compact, and reliable. Microjoule UV pulses are generated from the harmonic conversion of the emissions from a passively Q-switched Nd: YAG microchip engine. The SNV series are designed for high average power, either from pulse energies as high as 2�J or from free running repetition rates up to 40kHz. The SFV and STV triggered series allow users to trigger the laser externally from 10Hz to 4kHz with 2 different optimization strategies : STV :continuous control over 10Hz-2kHz range SFV : up to 3 defined frequencies over the full range 10Hz-4kHz



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UV Laser 355nm Control Unit



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4.0 Camera System 4.1 Coolsnap-EZ / ES Camera Introduction The CoolSNAPcf , CoolSNAPcf 2, CoolSNAPEZ, and CoolSNAP ES2, from, are ideal cameras for digital microscopy and many other biological applications. The simple, compact CoolSNAPcf and CoolSNAP cf2 incorporate a highquality CCD (charge-coupled device), a 12-bit scientific digitizer, and low-noise electronics to produce high-quality 12-bit monochrome or 36-bit digital color images at greater than 1k x 1k resolution. The CoolSNAPEZ and CoolSNAP ES2 offer higher sensitivity (especially in the red region) and lower read noise to produce highquality 12-bit monochrome images. All of these hardware components should be included with your installed VisiScope Fluorescence Imaging System and PC computer system. Your Coolsnap-cf/EZ/ES camera system has the following hardware components:

� Camera head � FireWire interface card � Data cable � Power supply

CCD Specifications of the Coolsnap

� Resolution 1392 x 1040 � Pixel Size 6,45 um x 6,45 um � Readout Amplifiers; 20MHz � Digitalization (Readout): 12 Bit Dynamic



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Precautions

The CCD and other system electronics are extremely sensitive to electrostatic discharge (ESD). To avoid permanently damaging the system, please observe the following precautions: • If you are using high-voltage equipment (such as an arc lamp) with your camera system, be sure to turn the camera power on last and power the camera off first. • Never connect or disconnect any cable while the camera system is powered on. • Although you should switch off the camera’s power supply before disconnecting any camera system cable, you do not need to power off your computer to detach the cables. • Use caution when triggering high-current switching devices (such as an arc lamp) near your system. The CCD can be permanently damaged by transient voltage spikes. If electrically noisy devices are present, an isolated, conditioned power line or dedicated isolation transformer is highly recommended. • Always leave one inch of space around the camera’s external cooling fins for air flow. • Never open the camera. There are no user-serviceable parts inside the CoolSNAP camera. Opening the camera voids the warranty. • Use only the PCI card, cables, and power supply designated for this camera system. Using non-CoolSNAPHQ2 cables, PCI cards, or power supplies may result in permanent damage to your system. • Do not use a C-mount lens that has optics that extend behind the flange of the lens. • Severe power line disruptions may cause the camera to lock-up. Cycling power will correct this. Environmental Requirements The CoolSNAP camera system should be operated in a clean, dry nvironment. The camera system’s ambient operating temperature is 0°C to 30°C with less than 80% relative humidity, noncondensing. Storage Requirements Store the CoolSNAP camera system in its original containers. To protect the system from excessive heat, cold, and moisture, store at an ambient temperature between -20°C and 60°C with a relative humidity of 0%-90%, noncondensing. Microscopes, Lenses, and Tripods The camera has a standard threaded video mount and can be mounted to any microscope that accepts a standard C-mount adapter. The camera also allows you to install any lens that is compatible with a standard threaded video mount as long as its optics do not extend behind the flange of the lens. The CoolSNAP camera can be mounted to a tripod using the tripod mounting attachment located on the bottom of the camera. Cleaning



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Clean exterior surfaces of the camera with a dry, lint-free cloth. To clean the camera’s imaging window, use only a filtered compressed-air source. Hand-held cans are not recommended, as they may spray propellant onto the window. Do not touch the window. Connecting the CoolSNAP Camera

The CoolSNAP cable connects your CoolSNAP camera to the interface card. This cable is designed to serve as a conduit both for data and power.

located on the back of the camera. To connect your CoolSNAP IEEE-1394 camera: 1. Connect either end of the CoolSNAP IEEE-1394 cable to the CoolSNAP IEEE-1394 interface card that you have installed in the host computer. 2. Connect the other end of the CoolSNAP IEEE-1394 interface cable to either DATA connector located on the back of the camera (shown below).

The following connectors and display lights are located on the back of the CooSNAP camera.

� �DATA connectors: 6-pin IEEE-1394 connectors for data transfer and power.

� �EXPOSE OUT connector: BNC connector; the signal at the BNC � connector will go to a TTL high level when the exposure begins; useful for

synchronizing an external shutter in the illumination pathway; will not power the shutter.



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� �XFER display light: amber LED illuminates during data transfer.

� �PWR display light: green LED illuminates hen camera is powered on. � �EXT PWR connector: 5.5 x 2.1 mm DC jack for auxiliary CoolSNAP

power supply. Note: CoolSNAP IEEE-1394 cameras draw power from the IEEE-1394 bus or from the external power supply. If no power exists on the IEEE-1394 bus, the external power supply must be used. In either case, the camera does not supply power to other devices connected to the IEEE- 1394 bus. CCD Specifications and Orientations:

CoolSNAPcf CoolSNAPEZ CoolSNAPES2

Image Type Mono Mono Mono Sony (mono) ICX205AL ICX285AL ICX285AL Resolution 1392 x 1040 1392 x 1040 1392 x 1040 Pixel Size 4.65 x 4.65 �m 6.45 x 6.45 �m 6.45 x 6.45 �m Digitization Rate 20 MHz 20 MHz 20 MHz Readout Noise 10 e- rms 8 e- rms 7 e- rms Cooling 5°C below ambient 5°C below ambient 0°C Auxiliary Power Supply:

Usage Requirements The auxiliary CoolSNAP power supply (shown below) provides power to your CoolSNAP camera, but is required only under certain conditions. If your CoolSNAP camera is connected to an IEEE-1394 port that does not supply power (such as a 4-pin connector on a laptop computer) you need to use the auxiliary power supply. The data ports on the CoolSNAP camera do not pass power. If the CoolSNAP camera is connected in a daisy chain configuration, and it is not the first device in the daisy chain, you need to use the auxiliary power supply. If the CoolSNAP camera is connected to a PC that supplies power through the 6-pin IEEE-1394 port, the auxiliary power supply is not needed; however, connecting the power supply will not damage the camera. CoolSNAP ES2 only: The power supply is required for camera cooling. If the power supply is not connected, the fan will not turn and the camera will not cool. Warning: Use the auxiliary power supply that shipped with your system ONLY. Do not use third-party power supplies.



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CoolSNAP-EZ Power Supply CoolSNAPE-S2 Power Supply Installation The auxiliary power supply ships with four unique wall plugs. You need to install one of the plugs on the power supply before using it. Select the correct plug for your wall socket and press it into the power supply. The supplied plugs include: Specifications:

CoolSNAPES2 All other CoolSNAP IEEE-1394 cameras Input Voltage 100-240 VAC / 47-63 Hz Output Voltage 9 V 12 VDC Output Current (Max) 3.3 A 1250 mA



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4.2 Evolve Camera Introduction The Evolve 512 camera system components are linked by the data cable and controlled by your host computer system. All of these hardware components should be included with your installed VisiFRAP Fluorescence Imaging System and PC computer system. Your Evolve 512 camera system has the following hardware components:

� Camera head � FireWire interface card � Data cable � Power supply

CCD Specifications Evolve 512

� Window UV grade fused-silica, broadband MgF2 anti-reflective coating on both surfaces

� CCD Array CCD e2v CCD97-00 CCD Process Back Illuminated

� Resolution 512 x 512 � Pixel Size 16 um x 16 um � Readout Amplifiers; 2 ports � Digitalization (Readout): 16 Bit Dynamic � Rate: 10 MHz, 5 MHz, 1.25 MHz



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Introduction On-Chip Multiplication Gain

As explained previously, the Evolve 512 uses a unique CCD capable of multiplying the charge (electrons) generated in the pixels. When the multiplication is sufficiently high, it is possible to see extremely low-light events. The amount of multiplication is controlled by the voltage applied to multiplication register clocks. These are certified by a quantitative multiplication gain slider which has 0-100 steps. The calibration feature of the camera maps the gain slider to a voltage which will provide the electron multiplication level selected. Offset (bias)

CCD cameras are typically designed to produce a certain level of offset (also known as bias) when no light is present and the exposure time is set to zero (0). Typically, the user subtracts an offset (bias) from the sample image for quantitative measurement. Since the offset can change based on several factors such as multiplication gain, speed, etc., it is recommended that a fresh offset (bias) image be taken with the same settings as the sample image and then be subtracted from the sample image. The Evolve 512 has an automatically adjusting offset switch. When a speed/gain setting is altered, the bias valve is kept as close to a pre-selected offset as possible. This enhances the quantitative stability of the camera. Exposure- Readout Modes

The Evolve 512 camera uses a frame transfer CCD and supports Non-Overlap and Overlap exposure and readout modes. These modes are further described in the sections that follow. Note: A frame transfer CCD has both a light-sensitive area (sensor area) and a storage area (frame transfer area). Since the image can be quickly transferred from the sensor area to the frame transfer area, there is no need for a mechanical shutter.



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Non-Overlap Mode Non-Overlap mode allows you to expose the array for the exposure time specified in the software and is similar in performance to a normal, full-frame device. The operational sequence for this mode is: 1. Clearing the CCD 2. Exposing for the specified exposure time 3. Shifting the image from the sensor area to the frame-transfer area 4. Reading out the CCD Steps 1-4 are repeated for each frame in a sequence. Steps 1 and 3, clearing the CCD and shifting the image, are usually very short and typically have minimum impact on the frame rate.

Calibration The Evolve 512 has incorporated a calibration routine which allows the camera to adjust voltages of the ADC offsets such that the electron multiplication gain input given to the camera reflects the actual gain provided by the device. Settings 1 to 1000 on the electron multiplication gain slider are mapped linearly to provide the actual multiplication gain requested by the user. Due to the impact-ionization method used with the electronmultiplication gain register the actual gain realized by the detector with time will slowly be reduced. By using the calibration routine the camera is able to re-establish the electron multiplication gain slider such that it will provide the gain which is input on the gain slider. This ensures the quantitative nature of the camera over time. Even though the camera is capable of delivering large multiplication gain factors, multiplication gain should only be used as needed to preserve as much dynamic range as necessary and to prolong the device’s.



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4.3 PCO Edge 5.5 sCMOS Camera Introduction The new pco.edge is a breakthrough in scientific Imaging cameras, due to its distinctive ability to simultaneously deliver extremely low noise, fast frame rates, wide dynamic range, high quantum efficiency, high resolution and a large field of view - all in one image. The camera's main features are:

• Iow noise: 1.4 electrons • high resolution: 5.5 megapixel • high dynamic range: 22000:1 • high speed: 100 fps • flexibility: user selectable choice of rolling or global shutter • free of drift: stabilized Peltier coolling In order to avoid any drift phenomena in image sequences



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4.4 PCO Edge 4.2 sCMOS Camera Introduction

The pco.edge sCMOS cameras feature temperature stabilized Peltier cooling, allowing for continuous operation free of drift phenomena in image sequences capture. This is achieved by the proper selection and sophisticated combination of electronics and FPGA algorithms. As the measurement result shows while running at full speed of 100 frames/s over 4 hours measuring time the camera doesn’t show any significant drift (figure on the right side). This degree of stability enables long-term measuring series, which should be quantitatively evaluated and processed. For example, in PCR (Polymerase Chain Reaction) applications, when so-called melting curves must be measured, the fluorescence in multi-well plates with different samples is recorded over a longer time at different sample temperatures. Here all the images are used for processing, which is only possible if the offset is stable and the camera is free of drift. A 4.2 Mpixel resolution in combination with a moderate chip size (18.8 mm diagonal, 6.5 �m pixel pitch) benefits microscopy applications with low magnification factor and large field of view, thereby reducing processing times and increasing throughput. The figure compares the potential of the new field of view of the pco.edge to the 1.3 Mpixel image resolution which is widely used in microscopy applications for scientific cameras high speed recording and data streaming. The new pco.edge offers in fast mode a frame rate of 100 frames/s (fps) at full resolution of 2048 x 2048 pixel as a full download stream to the PC. Therefore the recording time is just limited by either the amount of RAM in the PC or, in case of a RAID system, by the capacity and number of hard disks. As in many CMOS based cameras the frame rate increases significantly if smaller regions of



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interest (ROI) are used. The reduction of the image area works as well in favour of the frame rate of CCD sensors, but here unwanted regions still need to be read out at the expense of the total readout speed. The typical frame rate for a 1.3 Mpixel scientific CCD camera (6 e-read out noise) is 10 fps. The new pco.edge camera provides at 1.3 Mpixel resolution (< 1.0 e-readout noise) a frame rate of 200 fps in comparison. Specification: frame rate: 100 fps @ 2048 x 2048 pixel, fast scan exposure / shutter time 500 �s .. 10 s dynamic range A/D 16 bit A/D conversion factor 0.46 e-/count pixel scan rate: 272.3 MHz fast scan 95.3 MHz slow scan pixel data rate: 544.6 Mpixel/s 190.7 Mpixel/s binning horizontal x1, x2, x4 binning vertical x1, x2, x4 region of interest (ROI) horizontal: steps of 1 pixel vertical: steps of 2 pixels non linearity < 1 % cooling method: + 5 °C stabilized, peltier with forced air (fan) / water cooling (up to 30°C ambient) trigger input signals frame trigger, sequence trigger, programmable input (SMA connectors) trigger output signals exposure, busy, line, programmable output (SMA connectors) data interface: Camera Link Full (10 taps, 85 MHz)



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Safety Instructions Never operate the camera in humid or dusty environments or in places with high amounts of X-ray radiation. Humidity, dust or X-rays could damage the camera. To avoid the risk of water condensation, protect the camera against extreme changes of ambient temperature. If condensation enters the camera, there is the risk of electric shock. To prevent damage to the camera, the system must be kept stable and protected against strong jolts or vibrations. The socket al the bottom of the camera is 10 be used for mounting purposes only. The slits In the camera ease (side & back planes) are designed for heat dissipation by the camera fan. To prevent overheating of the camera, do not block these slits. Do not leave the camera system in direct sunlight to avoid the risk of overheating. Electric shock warning - Never slide any items through the slits into the camera because of the risk of electric shock if the voltage parts inside are touched. Each time the camera is used, check the power cable for any damage. Never position the cable in a way that It could become a tripping hazard. Do not force the lens onto the camera. To protect the lens connector thread from damage, use minimal force when attaching a lens to the camera. If any of the following conditions apply, immediately switch off the camera, separate it from the power line and contact our customer support:

� If the power cable or the power plug seems to be worn or damaged . � If liquids have penetrated the device . � If, after thoroughly reviewing the instruction manual, the device Is still not

operating properly . � If the camera has been dropped or the casing is damaged.



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LED Indicates camera status

• green continues: camera is booting green blinking: Camera is ready for operation • yellow blinking: recording 00 • red blinking: error Input/Output 4x SMA connectors 2x input - 2x output Interlace Dual Carreralink 'full'

Installation

You will find all necessary files on the accompanying CD. You may also download the newest versions of our software, camera drivers. and third party software drivers from the Visitron website. Minimum computer system requirements:

• Clock speed > 1.6GHz • RAM> 512MB • Windows XP (Service Pack 2) or Windows 7 • 1280 x 1024 pixel resolution display • nVIDIA CUDA GPU



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Rolling Shutter Timing

In rolling shutter mode the pixel reset and exposure start is carried out row by row. Each row has the same exposure time, but a different start of exposure. The pco.edge image sensor consists of two discrete halves,which are exposed and read out simultaneously, i.e. from the outside to the center. Within one row the exposure starts simultaneously for all pixels.



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General Timing Diagram The exposure time of each row starts with the corresponding reset of the row. Then after a predefined time, exposure is stopped. The light induced accumulated charge carriers of the pixels in a row are recorded into memory in a low noise fashion (readout). This results in the total image appearing in memory corresponding to the row readout.



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Exposure time > Sensor frame readout time (Auto Sequence) In case the required exposure is longer than the frame readout time, the image sensor is completely exposed to light for some time (tglobal). In case of a triggered flash illumination, this would be the best moment to illuminate the image sensor.

The hardware signal for the time tglobalis available on connector #4 (Global OUT). Setting can be made through SDK (not available in Camware). Obviously, if during exposure and readout, parts of the viewed image are moving horizontally, this would result in image distortion. This is why the global shutter mode may be a prerequisite for some applications. However, as most dynamic events can be captured in 1ms, as is evident with the use of SLR cameras set at 1/1000 exposure, maximum readout time for the sCMOS image sensor of approximately 10ms is sufficient. The 10ms is also faster than the image shift process of most frame transfer emCCD image sensors previously used for low light applications. If this does not influence the image recording and processing, then rolling shutter mode will not affect it either.



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Global Shutter Timing First, all pixels are globally reset and these reset values are shifted into so-called diffusion nodes. From there, they are non-destructively read out into memory as reset dark images. The exposure starts after transfer of the reset dark image to the diffusion nodes, where they are stored on the chip. The exposure is stopped by global charge transfer to the diffusion nodes. Then, the exposure image is read out to the memory, where the former reset dark image is subtracted to perform an external correlated double sampling, which reduces the noise. Since two images have to be read out to receive one resulting image, the sCMOS image sensor’s global shutter mode has only half of the frame rate of the rolling shutter mode.



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5. Confocal and Laser Systems 5.1 CSU-W1 Spinning Disk Confocal The basic principle of the CSU-W1 is to achieve parallel multi-points scanning to acquire confocal images by rotating the pinhole array disk (Nipkow disk), which contains numerous pinholes arranged in a special spiral pattern, as the point light source for confocal microscope. Another disk containing numerous microlens arranged in the same pattern with the pinholes is connected above the pinhole array disk at the focal length distance of the microlens, and the two disks rotate together. Parallel illumination lights from above the disks gets focused on each pinhole to become point light source. The point light out of each pinhole gets focused by the objective lens on the specimen to illuminate a point in the specimen. The emitted light from the specimen returns along the same path through each pinhole, being reflected by the beam splitter and focused on the camera by a relay lens.

Under this confocal principle, two-dimentional position information is reserved and can be directly captured by a camera. Data (image) are acquired by multiple scanning by the disk rotation and is restricted by the camera exposure time. Under the multi-point scanning mode, image acquisition speed can be much faster than that of single point scanning, but multi-points’ division of illumination light means much weaker illumination at each focal point. Thus, it is necessary to extend camera exposure time to accumulate multiple scanned images to obtain brighter images,, or strengthen the illumination light in case temporal resolution is important.



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For live cell imaging, weak illumination by multi-point scanning is effective to prevent photo bleaching and/or photo damages caused by strong illumination light. Description of CSU-W1 CSU-W1 was developed as an advanced model of our conventional CSU series which have been widely used as a standard tool for live cell imaging. As enlisted below, CSU-W1 is significantly improved in its basic optical features. Moreover, various optional systems are available to allow flexible design of total imaging system as you need. Major product types: T1 model : basic model for one camera T2 model : for two cameras T3 model : for split-view with one camera. For each model, options such as NIR port or external light path (EXP) can be attached to allow versatile applications. � Newly developed large diameter disks offers wide FOV, in combination with much reduced cross talk to achieve clearer images. Most suitable to image large specimen at high resolution. � In addition to the conventional 50um pinhole disk, 25um pinhole is available, and either one or the both disks are selectable. � Bright field light path without passing pinhole disk is available as default, and confocal and non-confocal imaging can be automatically selectable. � Offers various options to meet versatile application requirements. � T2 Model is to allow simultaneous dual color imaging by two cameras. � T3 Model offers image splitter for simultaneous dual color imaging by one camera, while non-split imaging is also possible. � Optional NIR port offers deep imaging at near IR range with minimal photo toxicity. � Optional external light path offers easy incorporation of external beams for such applications as photo-activation.



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T1 Model

A : VIS Port B : Shutter C-1 : Confocal path C-2 : BF path D : Dichroic mirror (Refer to the specification sheet F : EM Filter (Refer to the specification sheet G : Camera port Selected dichroic mirror and EM filters are installed at factory T2 Model

A : VIS port B: Shutter C-1: Confocal path C-2: BF path



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D: Dichroic mirror -Refer to the specification sheet F: EM Filter -Refer to the specification sheet G: Camera port H: Light path exchange unit (Either a dichroic mirror or a 100% mirror) Selected dichroic mirror and EM filters are installed at factory T3 Model

A : VIS port B: Shutter C-1: Confocal path C-2: BF path D: Dichroic mirror -Refer to the specification sheet I: Variable aperture J: Light path exchange unit (Dichroic mirror) K: EM filter Refer to the specification sheet L: Camera port Selected dichroic mirror and EM filters are installed at factory



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Options (Example: T1 Model with full options)

M : NIR Port N: Shutter for NIR Port O: Dichroic mirror for NIR Port (Refer to specification sheet) P: ariable aperture Q: Dichroic mirror for External Light Path (Refer to specification sheet) Selected dichroic mirrors are installed at factory



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Name and Function T1 Model

(1) Key Switch Main switch for the scanner unit. Put ON the Power unit first, and then turn the key switch from OFF position to ON position. To reset the CSU-W1 after initialization, turn the key switch to OFF, and then to ON. Key switch cannot be removed at the ON position. (2) POWER Indicator When power is ON, green LED flickers during initialization process, and then lights when being ready to use. Green LED gets off when the power is off. (3) Shutter button Get ON/OFF at each push. LED light shows ON/OFF status. Do not keep pushing the button longer than 5 seconds. If more than 2 shutters are installed for options, one push closes all shutters when any shutter is open, and one push opens all shutters when all shutters were closed. (4) EMISSION Indicator Red LED lights when shutter is open, and turns off when shutter is closed. If more than two shutters are installed for option, red LED lights when more than 1 shutter is open.



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(5) Fiber guard To protect optical fiber. WARNING The visible laser fiber is connected with the CSU-W1 after removing this cover, which should be done by a qualified service engineer. (6) VIS (Visible) port Fiber connector port to induce excitation laser into the CSU-W1. Match the guide key of the fiber connector with the FC connector, insert fiber and fix with the screw. Please keep attached fiber cap for dust protection when keeping the fiber. (7) Front panel (8) 1st Camera Port (T1 T2 Models) Port for a camera. Attach supplied microscope adaptor to the camera to install. (9) Camera adaptor Adaptor to install the camera to the camera port. Screw in the adaptor to the C mount of the camera, insert into the camera port and fix. (10) Level adjuster Assists adjusting the height of the scanner unit with the microscope port. There are a total of 4 adjusters. Please adjust the height by using supplied adjuster wrench. (11) Handle hole cover plate Plates to cover the handle holes. (12) Dichroic mirror exchange cover panel



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(13) Bonnet (14) Microscope port Specific for each microscope type (15) Right side panel

(16) Top Panel (17) Left side panel For T1 and T2 Models (18) Main cable connector To connect Power unit and scanner unit. (19) Air ventilation for cooling fan Be careful to leave enough space for air ventilation to prevent overheating inside the scanner unit. (20) Rear panel For T1 Model (21) Name plate Model name (MS code), product number, manufacturing number and made-in Japan are described.



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T2 Model

(22) 2nd Camera port (23) Rear panel For T2 Model T3 Model



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(24) Left side panel For T3 Model (25) 1st Camera port For T3 Model (26) Rear panel For T3 Model Power Unit

(27) POWER Indicator Green LED lights when power is ON, and turns down when power is OFF. (28) EMISSION Indicator Red LED lights when the shutter of the scanner is open, and turns off when the shutter is closed.



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(29) PC Port Connect to PC with supplied RS232C cable. (30) External control/Connector for Interlock Release Connector Shutter operation enables by inserting the Interlock Release Connector here. In emergency, laser emission could be stopped by either removing the Interlock Release Connector or shutting down the connection. In such case all actuators including spinning disk motor will stop. It also has functions such as pulse control of motor rotation, shutter and speed. (Refer to 7.8: External control) (31) Breaker (32) Power switch (33) Main cable connector Connect the main cable here. (34) Cooling fan Please keep enough space for ventilation. (35) Power code port (36) EXT Port CAUTION - Do not connect anything here. Microscope Adapter Attachment Firmly connect the adaptor to the side panel with screws.

Microscope adaptors for each microscope model: (From left to right) Zeiss Axio Observer, OLYMPUS IX81/IX71, and Nikon ECLIPSE Ti.

Microscope adaptors (for external light pass option) (From left to right) Zeiss Axio Observer, OLYMPUS IX81/IX71, and Nikon ECLIPSE Ti.



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EM Filter Installation Please refer to EM filters are installed at factory following your requested specification. CAUTION Please confirm both the CSU-W1 and laser are turned off before starting EM filter installation. CAUTION Please do not forget to insert the filter wheel cable and set the top cover after EM filter installation. Otherwise, the CSU-W1 may not work properly. Dichroic Mirrors The dichroic mirror is installed at factory following your requested specification. Connect with the Microscope A total of 4 level adjusters at each corner are provided on the scanner unit. Please adjust the height of the scanner to match the light path of the side port of the microscope by using supplied adjuster wrench. Do not firmly fix the adjuster rings at this adjustment stage.



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Wiring Diagram T1 Model:

T2 Model:



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T3 Model:

Necessary preparations: � PC � Appropriate imaging software installation � Laser unit or system (such as 405nm, 488nm, 561nm or 640nm) � Appropriate EM filters matching the laser lines. � Camera



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5.2 VS-Laser Power Control Typical Configuration with Laser Merge System and VS-AOTF System The VS Laser Control unit is typically used in combination with an AOTF module for intensity control and the VS-LMS Laser Merge System for merging of two or more laser lines. Other configurations are also possible. Safety and Precaution Instructions The VS Laser Control unit controls connected lasers of the Class 3B. The laser(s) is (are) compliant with IEC 60825-1. Each laser line emits up to 100 mW of laser radiation between 488nm and 642nm. Eye and skin exposure to direct or reflected laser light is hazardous and may be extremely harmful. Always wear eye protection appropriate to the beam wavelength and intensity. The device must be handled by personnel with experience of lasers in laboratory environment and with access to adequate laser safety equipment. You are obliged by law to obey the regulations for laser class 3B products.



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Precautions

� If you are using high-voltage equipment (such as an arc lamp) with your unit, be sure to turn the high-voltage equipment power on first and off last.

� Never connect or disconnect any cable while the unit is powered on. � Do not power on the unit with the laser cables disconnected. � Use caution when triggering high-current switching devices (such as an

arc lamp) near your system. The unit can be permanently damaged by transient voltage spikes. If electrically noisy devices are present, an isolated, conditioned power line or dedicated isolation transformer is highly recommended.

� Always leave at least 5 cm of space around the unit for air flow. � Never open the unit. Opening the unit voids the warranty. � Use only original cables. Using a different cable may result in permanent

damage to your system. Environmental Requirements The unit should be operated in a clean, non-condensing environment. The unit’s ambient operating temperature is 15°C to 30°C. Input Voltage 230-240 VAC / 50 Hz Fuse 2x 2 A T Power dissipation 60W Setting up

Connect the cables from the Laser Merge Module with the corresponding connectors at the rear panel of the Laser Control unit. Be sure that on the Laser Merge Module the light guide(s) is (are) connected and the other end(s) of the light guide(s) is connected to the appropriate unit. On the unit, the appropriate precautions must be taken so that no laser light is emitted to the ambient. Connect the mains cable with the unit. Operation

The unit has a power switch at the rear left side of the housing. For each supported laser line there is a key switch on the front panel. Be sure that all key switches are in the Off (vertical) position before switching the power on. The power-on condition is indicated by the green Power LED on the front panel.



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When power is supplied to the unit, the temperature control of the lasers start operating to reach set point values. It is recommended to let the system temperature stabilize during ~1 min before further actions. Make sure that laser safety precautions are taken so that a laser emission cannot harm people and environment. You may then switch on the required laser lines by turning the appropriate key switch in the on (horizontal) position. The corresponding laser warning light will go on immediately. However, the laser will actually go on only some seconds later for safety reasons. Key Switch and laser emission warning lamp Note! The ON-position of the turn-key is the horizontal position. If power is supplied to the unit with the turn-key in its ON-position it is needed to turn the turn-key to its OFF-position and then again to its ON-position in order to start the laser. To switch the unit off, turn all key switches to the off (vertical), then switch the unit off with the power switch at the rear panel. Optional connections The Laser Control unit may have one or more additional connectors at the rear panel for intensity control (RS232 or USB) and for external interlock.



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5.3 VS Laser-Merge-System Visitron System is offering several laser merge systems for realtime confocals, Photoactivation, FRAP and TIRF technology. The latest Visitron Systems development is a compact and easy to use laser merge system including an AOTF. All common solid state lasers or laser diodes can be coupled and aligned to one fiber output. Also future extensions with additional lasers are easily possible. Overview of configuration Alignment of dual channel laser merge module

1. Remove the cover of the VS-LMS4 Laser-Merge-System by 4 screws at the corner of the unit.

2. Using only the laser 1 that points directly at the AOTF. Aligning the micro lens FC adapter to get optimum laser power at the end of the fiber.



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3. There are 3 adjusters on the back of each of the dichroic and the mirror mount. These are arranged in an L shaped configuration. Use the adjusters at the end of the L not the one at the junction.

4. Using the second laser, position the mirror at 45° to the beam and adjust the mirror position so that the beam from this laser is directed to superimpose on the position of the first laser beam at the output of the dichroic position 2.

5. Adjust the dichroic position 2. so that the two laser beams become co-linear and booth pass correctly into and through the AOTF.

6. Measure the power of the laser 2 at the end of the fiber and optimize the power by careful adjustment of the adjustable dichroic. Do not change the adjustment of the FC micro lens adapter.

7. Fit the cover to totally enclose the laser beam and check the co-linearity of booth beams.

AOTF Frequency adjustment

The AOTF crystal is heated to guarantee a highly stable control of the laser power. The heating control closed loop circuit requires about 10..15 minutes settling time before any adjustment of the system should be done.

1. Connect the USB port at the back plane of the VS-AOTF controller to the

PC. 2. Switch on the VS-AOTF controller 3. Start the AOTF setting (MDS.exe) Software 4. Select Interface USB and IMode – internal 5. Use Query all button 6. Select line 1 e.g. for 491nm laser line. Use laser power meter at the end

of the fiber and measure the power. By changing the frequency and dbm attenuation optimize the maximum laser power. The same has to be done for each laser line

7. store the settings 8. Switch off the VS-AOTF controller, remove the USB cable and switch on

the controller again.



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Safety and Precaution Instructions

VS-LMS4 is a Class 3B laser product. The laser is compliant with IEC 60825-1.

Each laser line emits up to 100 mW of laser radiation between 488nm and 642nm.

Eye and skin exposure to direct or reflected laser light is hazardous and may be extremely harmful. Always wear eye protection appropriate to the beam wavelength and intensity. The device must be handled by personnel with experience of lasers in laboratory environment and with access to adequate laser safety equipment. The VS-LMS4 clearly displays a yellow warning label that shows the location of the laser beam aperture. This label must be visible unless the laser beam is totally enclosed. Connecting the Cables Overview of the cables required VS-laser power control and VS-AOTF



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VS Laser-Merge-System LMS4 Connectors Connector for mechanical Shutter control Sh1 and Sh2 for laser beam blocking, TTL control for fast switching of diode laser and AOTF. Front panel with laser output micro lens FC adapter for confocal and FRAP



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Laser configuration:

Overview of up to 4 laser combination 405, 445, 473, 488, 561 and 640nm with dial output by mirror system for simultaneous observation confocal / FRAP. For solid state lasers the integrated AOTF (Acousto Optical Tunable Filter) module permits the control of laser line selection, individual line intensity control and high speed switching in about 3ms. Additional mechanical shutters at e.g. 405 laser allows the selection, combination and blocking of laser lines depending of the application configuration. VS-LMS System with 405, 488 and 561nm laser with AOTF VS-LMS System with 405, 488 and 561nm laser based on fast VS-Shutter



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5.3.1 VS Laser-Merge-System LMS6 with Dual Output Option

The unique design of VS-LMS6 system combines the beams of up to six solid state or laser diodes to a single collinear laser beam. This beam can be channelled to two different outputs. This allows one laser merge module to be used for two different laser application like confocal combined with FRAP or FRAP combined with TIRF.

VS-Laser Power Control for LMS-6 Operation

The unit has a power switch at the rear left side of the housing. For each supported laser line there is a key switch on the front panel. Be sure that all key switches are in the Off (vertical) position before switching the power on. The power-on condition is indicated by the green Power LED on the front panel. When power is supplied to the unit, the temperature control of the lasers start operating to reach set point values. It is recommended to let the system temperature stabilize during ~1 min before further actions. Make sure that laser safety precautions are taken so that a laser emission cannot harm people and environment.



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You may then switch on the required laser lines by turning the appropriate key switch in the on (horizontal) position. The corresponding laser warning light will go on immediately. However, the laser will actually go on only some seconds later for safety reasons. Key Switch and laser emission warning lamp Note! The ON-position of the turn-key is the horizontal position. If power is supplied to the unit with the turn-key in its ON-position it is needed to turn the turn-key to its OFF-position and then again to its ON-position in order to start the laser. To switch the unit off, turn all key switches to the off (vertical), then switch the unit off with the power switch at the rear panel.



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Connecting the Cables Overview of the cables required VS-laser power control and VS-AOTF VS Laser-Merge-System LMS6 Connectors Connector for laser power, computer control by USB / RS232 configuration, mechanical Shutter control Sh1 and Sh2 for laser beam blocking, TTL control for fast switching of diode laser and AOTF.



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Front panel with laser output micro lens FC adapter for confocal and FRAP Block diagram of Laser combinations



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Overview of 6 laser combination 405, 445, 473, 488, 561 and 640nm with dial output by mirror system for simultaneous observation confocal / FRAP. Output for confocal 405,445,488,561,640nm and for second output for FRAP 405 and 473nm. For solid state lasers the integrated AOTF (Acousto Optical Tunable Filter) module permits the control of laser line selection, individual line intensity control and high speed switching in about 3ms. Additional mechanical shutters at e.g. 405 and 473nm laser allows the selection, combination and blocking of laser lines depending of the application configuration.



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Connection of CSU-X1 fiber cable It is important to connect the fiber cable with the correct input and output . For the CSU fiber cable the ladled INPUT must be connected to the VS-Laser Merge System. For the blue OZ-fiber, the green input must be connected to the VS-Laser Merge System.



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5.3.2 Additional Laser Output with Galvo

The VS-DOL dual output laser option can be configured by using dichroic beamsplitter with e.g. 50/50 %, 30/70% or 80/20% for separation of the laser into two simultaneous laser outputs. The VS-DOL high speed galvanometer scanner option allows the laser separation with full 100% laser power to up to three laser outputs. The switching between the laser outputs are in a few milliseconds. VS-LMS-3 Laser Merge System can be also used with single or multiple laser configurations.



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VS-LMS-6 Laser Merge System Galvo The VS-DOL dual output laser option is controlled easily by TTL signals for the different laser output selection. The switching of the channels are in millisecond range. The illumination dialog of the VisiView software will setup the correct condition for FRAP or Confocal output. FRAP and Confocal output Internal view to Galvo-Scanner and the optics for the two laser outputs



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5.4 VS AOTF The VS-AOTF controller allows manual or programmable selection of the single laser lines. For manual intensity control of laser power, potentiometer are available as a option. A new “SBT - smear buster technology has been developed by Visitron Systems GmbH for blanking the laser e.g. between the frame transfer of scientific grade CCD cameras. Overview front panel Overview back plane Optional: Intensity control potentiometer if no computer control is selected Overview rear panel



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Features

� Control of up to four AOTF channels � Selection of active AOTF channel(s) via PC(TTL) or manually � Control of AOTF laser power via PC(D/A board, optional) or manually via

front panel pots � Blanking of laser during frame transfer shift of camera CCD (see list of

compatible cameras) Setting up

� Make sure the short cable connection on the Modulation input is seated fast.

� Connect the RF Out and Temp. Stab. Cables with the AOTF � Connect the PC control cable with your PC (optional) � Connect the Analog Control cable with the PCI-DDA08/12 board in your

PC (optional) � Apply the interlock connector � Connect the mains cable � If you are using a piezo focus device or a polychromator lamp controlled

by the D/A board, then connect these devices on the Focus or Lamp BNC connector

Important: The RF Out, Temp. Stab. cable MUST be connected with the AOTF. Switching on the controller without these connections may lead to damage.



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Usage

Switch the power on The green LED on the front panel indicates that the interlock connector is on. Manual control

� Front panel switch in setting Man. � Push the button of the desired laser channel so that it is lit. � Adjust the laser power with the appropriate front panel pot. (Versions

without pot: 100% power output) Computer Control

� Front panel switch in setting PC � The channels can be selected by TTL signals, see ‘Cables’ � The laser power can be controlled by D/A board, see description of the

Laser Power Control utility program.

Aux Laser

An additional laser (e.g. 405nm) can be controlled (On/Off) in PC mode, see ‘Cables’ CCD Shift Blanking (‘Smear Buster’) Images of frame transfer CCD cameras can be improved when there is no light during the transfer of the image within the CCD chip (FT Shift). Some cameras supply a TTL signal indicating the FT Shift. This signal can be used to blank the AOTF channel. (See ‘Cables’) Cables

Available cables for the AOTF controller � AOTF PC control cable

Connects to PC LPT port. Control by software � AOTF D/A box

Connects to a PCI-DDA0x/12 D/A board � Camera/Aux Laser cable

Versions for 1 or 2 Photometrics Cascade II cameras and/or TTL controlled Diode Laser.

Other types on request



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Pin Assignments AOTF Rear Panel

“Blanking” D-Sub9m 1 /Cam1Shift 2 /Cam1Sense (to Cam1 GND) 3 /Cam2Shift 4 /Cam2Sense (to Cam2 GND) 5 CamShift 6 /Laser1Blank 7 /Laser2Blank 8 GND 9 GND

“PC ”

D-Sub15f 1 AOTF Chan0 HIGH enables Channel 2 AOTF Chan1 HIGH enables Channel 3 AOTF Chan2 HIGH enables Channel 4 AOTF Chan3 HIGH enables Channel 5 AOTF Chan4 HIGH enables Channel 6 AOTF Chan5 HIGH enables Channel 7 AOTF Chan6 HIGH enables Channel 8 AOTF Chan7 HIGH enables Channel 9 Aux Laser1 10 Aux Laser2 11 12 13 /Disable SmearBuster 14 /Unlock HIGH disables all Channels* 15 GND

* This safety feature can be disabled on request.



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AOTF Frequency adjustment The AOTF crystal is heated to guarantee a highly stable control of the laser power. The heating control closed loop circuit requires about 10..15 minutes settling time before any adjustment of the system should be done.

1. Connect the USB port at the back plane of the VS-AOTF controller to the PC.

2. Switch on the VS-AOTF controller 3. Start the AOTF setting (MDS.exe) Software 4. Select Interface USB and IMode – internal 5. Use Query all button 6. Select line 1 e.g. for 491nm laser line. Use laser power meter at the

end of the fiber and measure the power. By changing the frequency and dbm attenuation optimize the maximum laser power. The same has to be done for each laser line

7. store the settings 8. Switch off the VS-AOTF controller, remove the USB cable and switch

on the controller again. Troubleshooting No Laser light

� Laser switched on? � Laser mechanical shutter open? � AOTF Controller switched on? � AOTF Interlock Plug connected (green LED on)? Serially controlled laser only: � Serial cable connected? � TTL cable connected? � Laser Power Program started? � Appropriate COM port selected in Laser Power Program? � Laser Power Program slider set bigger than 0? � Laser Power Program appropriate enable checkbox checked? � PC LPT – AOTF PC cable connected?



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Analog controlled lasers: AOTF mode switch in Manual position:

� Push button on (LED is lit)? � Power pot setting bigger than 0? � Camera blanking connector on? In this case, laser is only on when

camera acquisition running or AOTF Channel 8 is set HIGH AOTF mode switch in PC position:

� Software appropriate AOTF channel is switched on? � Camera blanking connector on? In this case, laser is only on when

camera acquisition running or Software AOTF Channel 8 is set HIGH

Precautions � Never power on the unit with the AOTF cables disconnected! � If you are using high-voltage equipment (such as an arc lamp) with

your unit, be sure to turn the high-voltage equipment power on first and off last.

� Never connect or disconnect any cable while the unit is powered on. � Use caution when triggering high-current switching devices (such as

an arc lamp) near your system. The unit can be permanently damaged by transient voltage spikes. If electrically noisy devices are present, an isolated, conditioned power line or dedicated isolation transformer is highly recommended.

� Always leave at least 5 cm of space around the unit for air flow. � Never open the unit. Opening the unit voids the warranty. � Use only original cables. Using a different cable may result in

permanent damage to your system. � Laser light may be emitted when powering on the AOTF!

Environmental Requirements The unit should be operated in a clean, non-condensing environment. The unit’s ambient operating temperature is 15°C to 30°C. Ratings Input Voltage 230-240 VAC / 50 Hz Fuse 2x 1 A T Power dissipation 40W



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5.5 VS-AOM - Acousto Optical Modulator Control - High Speed Optical Shutter for Solid State Laser Systems An acousto-optic-modulator (AOM) is a device which can be used for controlling the power, frequency or spatial direction of a laser beam with an electrical drive signal. It is based on the acousto-optic effect, i.e. the modification of the refractive index by the oscillating mechanical pressure of a sound wave. The AOM will be used at the VS-LMS for high speed switching and intensity control if only one solid state laser is used. VS-LMS3 Laser-Merge-System with VS-Laser Control and AOM Control Unit



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VS-AOM Control Unit Front-Panel Control

Power On LED Interlock LED Laser On Switch with LED indication Mode Switch must be selected for local mode

Mode Switch for remote control if PC and VisiView software is used. Local mode selection if manual control of laser is required



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VS-AOM Control Unit Back-Panel Control TTL Input from

VS-110 DAC-Trigger Box Controlled by VisiView software

Laser TTL Control connected to VS Laser Control Unit

Interlock Laser “Smear-Buster” control for Frame-Transfer CCD cameras

Intensity control for AOM laser system. Connected to Analog Output of VS-110 DAC Trigger Box



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Back-Panel Control with used cables and VS-Laser Control


Overview of up to 3 laser combination with single state laser e.g. 561nm and AOM for laser intensity control and fast switching.



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5.5.1 VS-110 DAC-Trigger Box The VS-110 DAC-Trigger Box is used for application synchronization of the experiment with confocal, laser systems, digital slow scan CCD cameras etc. typically for Trigger TTL signals. The additional DAC computer controlled analog voltages are used for intensity control of laser systems or focus control of Piezo focus devices of the microscope. 6.0 Fiber Coupling Adapter 6.1 OZ Coupler



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5.6 Fiber Coupling Adapter 5.6 OZ Coupler 1.1 Operation principle

Figure 1 shows the major components of the laser to fiber coupler. Take a moment to compare the item you received with the one illustrated. While the detailed appearance of the coupler will depend on the exact model ordered, the key components indicated on the drawing are the same for all adjustable focus couplers. In particular it is important to identify the three tilt adjustment screws, and the three locking screws. The tilt adjustment screws have washers and/or large diameter heads, while the locking screws do not. Warning: Some screws on the coupler are fixed at the factory and ARE NOT to be adjusted. These screws are coated with a red varnish. Removing or damaging this coating may void your warranty. Figure 1: Laser to Fiber Coupler Construction OZ Optics' couplers consist of two baseplates, separated by a resilient O-ring. One plate, the laser head adapter, is designed to attach directly onto the laser. The other plate contains the focusing optics and the connector for the input fiber. The O-ring acts as a pivot allowing the focusing optics to be tilted with respect to the laser head adaptor. The two baseplates are held together with the three tilt adjustment screws. These screws pass through three clear holes in the coupler flange. These holes are about 1.5mm larger than the diameter of the screws. This allows one to adjust the centering between the laser beam and the focusing optics. About ±O.8mm of travel in any direction in the X-Y plane is available. One advantage of OZ couplers is that only coarse centering of the optics is required. Fine adjustment of the focused beam is achieved using the three tilt adjustment screws.



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By adjusting the tilt adjustment screws, one can adjust the tilt angle between the focusing optics and the incident laser beam. This in turn moves the point at which the focused beam strikes the fiber. Thus by adjusting the tilt angle, one can align the focused rays to strike the fiber core. 1.2 Initial Installation

Important: Before using OZ Optics' laser to fiber couplers with your equipment, make sure that you are familiar with all operating and safety instructions provide with the laser. OZ Optics is not liable for any damage or harm caused by misuse of either the laser or of OZ Optics devices. 1. To avoid damage to the fibers being used, perform the initial alignment at low power «250mW). Only after the optics are fully aligned and locked should the laser power be increased. This is explained further in the section titled FINAL LOCKING AND OPERATION 2. (For systems supplied with a laser to C-Mount Adapter) With the laser OFF, attach the laser to C-mount adaptor ring to the laser using four of the provided M3 screws. Attach it so that you have easy access to the two 4-40 set screws located in the side of the C-mount ring. Ensure that all screws fit snugly, with no binding. 3. With the laser OFF, thread the laser to fiber coupling optics into the C-mount ring by hand until the coupler stops tuning by hand. Remove any cap covering the fiber optic connector. Check that the locking screws on the coupler are not pushing against the laser head adaptor. This would prevent the tilt adjustment from working properly. 4. Turn on the laser and operate it at the lowest recommended operating power. Examine the output on a white opaque screen, located about 50cm from the coupler. If the laser being used does not emit light at visible wavelengths, then an infrared or ultraviolet viewer or screen can be used to study the beam. 5. The image on the screen should appear to be centered, as shown in figure 2. If it does not, then adjust the lateral position of the focusing optics with respect to the laser beam. To do this, loosen the tilt adjustment screws each by about half a revolution, and apply lateral pressure to the side of the coupler flange. This will shift the lateral position of the coupler flange with respect to the laser head adaptor. Once the image has been centered, tighten the tilt adjustment screws again to their original position. Ensure that the image remains centered. This step is important to minimize losses caused by numerical error mismatch between the focused laser rays and the fiber.



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Figure 2: Centering the Coupling with Respect to the Laser Beam 6. (Users who are not using polarization maintaining fiber or do not have the polarization alignment tool, please proceed to the next section, Multimode alignment) Insert the polarization alignment tool into the FC connector receptacle. The alignment tool contains a polarizer with its transmission axis aligned 90 degrees to the key on the connector. When the laser to fiber coupler is aligned with the key on the connector aligned to the polarization of the laser light, then the alignment tool will block as much light as possible. 7. Slowly rotate the entire coupler counter clockwise within the C-mount ring while observing the output on the screen. When the light is as dark as possible, tighten the two set screws in the C-mount Ring against the laser head adaptor on the coupler. (Warming: Tightening the set screws too much can potentially damage the laser head adaptor.) Verify that the polarization tool is still fully blocking the light. Remove the polarization alignment tool. NOTE: If you are using a laser head adapter that consists of a baseplate and threaded adapter (such as #14, #15, #17, #22, or #23), it is important that the two set screws in the base plate be tightened against the threaded adapter, to ensure that the adapter is secure and does not rock.



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SINGLEMODE ALlGNMENT I. Remove the multimode fiber jumper assembly, and replace it with the single mode or polarization maintaining jumper to be used with the coupler. Again, ensure that the fiber ends are clean be fore attaching them to the laser. If the previous section was completed properly, a small amount of light should come from the fiber. 2. Adjust tilt screw I again to find the tilt angle that gives the most light from the fiber. Do this fairly slowly, as a small adjustment significantly affects the measured output power. Repeat this process for tilt screws 2 and 3. 3. Because of diffraction effects, the focused light rays from the laser may form an Airy disk pattern on the plane where the fiber is located. The small core size of single mode fiber makes it possible to distinguish local maxima within the Airy disk pattern. If proper care is not taken, the wrong maxima may be focused onto the fiber, resulting in an unacceptably low output intensity. If this appears to be the case, readjust the tilt screws again until the central peak can be located. 4. Once the optimum coupling efficiency has been achieved, tighten the three tilt adjustment screws evenly, one quarter turn at a time, while maintaining the tilt orientation of the optics with respect to the laser head adaptor. This will improve the stability of the coupler against vibration. The tighter the screws are, the better the resolution and stability of the couple will be. The coupler can be considered sufficiently tightened if the 0.050" ball driver cannot be inserted between the laser head adaptor and the coupler flange. 5. (Optional) Recheck the centering between the coupler and the laser beam, by applying lateral pressure to the coupler flange to shift it slightly. The coupling efficiency may improve when the coupler is pushed in one direction, indicating that the centering is slightly off. If necessary readjust the tilt adjustment to optimize the coupling efficiency.



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FINAL LOCKING AND OPERATION. 1. Tighten the three locking screws (a, b, and c) until each just make contact with the laser head adaptor. The coupling efficiency should remain unchanged. If the coupling efficiency changes by more than 10%, then loosen each locking screw slightly, until the efficiency is restored. 2. Tighten each locking screw by an additional quarter turn. This should lock the alignment sufficiently for most applications. 3. If the coupling efficiency has dropped significantly, adjust each locking screw slightly, in a similar manner to that used to adjust the tilt screws, until optimum coupling is restored. The coupler is now adjusted for optimum operation. 4. Note to high power laser users: At this point the output power from your laser can be increased safely, without damaging the fiber. It is recommended that the laser power be increased by only 0.25 Watts every fifteen minutes, and that the output power from the fiber should be monitored, to ensure that the coupling efficiency is not changing with power.



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5.6.1 KineFlex Coupler Operating Instructions Laser/Fiber Manipulator Adjustment The kineFLEX features a high precision ultra-stable manipulator for coupling the fiber to the laser of your choice. Because the fiber has pre-focussed optic assemblies, tilt of the assembly becomes translation in the focal plane and translation becomes tilt. Refer to figure 1. Because the fiber has a relatively large acceptance angle the system is less sensitive to translational motion provided by the manipulator. The manipulator thus requires only 2 degrees of freedom enabled by using a 4 point cradle design that is kinematic.

Figure 1.: Principles of operation The manipulator is pre-aligned using an alignment tool with a pinhole aperture. The input fiber coupler and manipulator are keyway aligned such that the polarization alignment is instantaneous. Once optimized for coupling efficiency, locking mechanisms on the adjustment screws ensure long term stability of launch conditions. The kinematic design ensures maintenance of polarity on repeat connections. Points of Note During adjustment, the tension provided by screw D on the fiber coupler will change dependent on the direction of travel provided by the adjustment. Therefore additional tensioning or release of screw D may be required during the launch optimization process.



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The purpose of tension screw C is to locate the carrier in the mount such that the pads will be correctly aligned with the adjustment screws. Screw C should not be tensioned fully as it is not intended to increase the level of bias against the adjustment screws. If excessive tension is provided on screw C, then the translational movement range of the fiber coupler will be limited. To avoid damage to the end face of the fiber, the fiber manipulator alignment process must be performed at low powers, ideally less than 30mW C.W. Ensure that adequate laser safety precautions are taken before proceeding. Mounting 1. Drop the carrier into the body of the mount.Kinematic operation is achieved by contact between the adjustment screws and the hardened pads on the fiber carrier. 2. Increase tension on screw C to engage into notch H on the underside of the carrier. Limited tension is required. See points of Note regarding screw C. 3. Loosen tension screw D and ensure there is no protrusion into the bore of the carrier. 4. Mount the manipulator to the laser or a mounting bracket using 4 M3 mounting screws. The manipulator must be aligned vertical to the input plane of polarization. The polarization keyway on the carrier must be positioned at the bottom of the mount as shown in figure 2. Pre-alignment Pre-alignment can be performed visually and without the aid of a power meter. 5. Insert Alignment Tool with the pinhole aperture G nearest to the laser and, increase the tension on screw D. The alignment tool is used to pre-position the adjusting screws relative to the position of the laser beam. 6. Increase tension on screw D. This increases bias on the tool against the carrier and creates kinematic contact between the pads and the adjustment screws 7. Align the Tool by adjusting screws A1 & B1, such that the pinhole aperture is causing minimal clipping the beam. Adjust each screw in turn to maximize the light transmitted through the pinhole aperture. Release tension on screw D. 8. Reverse the Tool and align screws A2 & B2. Repeat step 5 for screws above. 9. Reverse the Tool once more and repeat step 5 for A1 & B1. The mount is now pre-aligned and ready for the insertion of the fiber delivery system.



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Launch Optimization You will be required to monitor the transmitted power through the fiber. For this you will require a power meter preferably with an audible output or analogue display. Normalize the power reading by taking a direct reading from the laser. You are now ready to optimize the launch of the fiber and laser. 10. Insert the fiber input coupler assembly into the carrier and increase tension on screw D. Ensure the polarizing key E locates into the keyway F on the fiber carrier. Monitor the transmitted light on the power meter. 11. Rapid adjustment is provided using screws A1 & B1. Tilt the fiber coupler using screws A1 & B1 in turn to achieve the highest transmission efficiency. 12. Optimize the launch by ultra-fine adjustment using horizontal translation provided by screws A1 & A2. Alternately turn screws A1 & A2 in the same direction in small steps whilst monitoring the output power. The fiber coupler can be translated in one direction axis by using this ‘walking’ motion and the point of maximum coupling efficiency detected. 13. Adjust screws B1 & B2 by vertical translation for final optimization. Repeat step 8 for screws B1 & B2. Fixing of Launch 14. Lock down all adjustment screws J. Tighten locking nuts J whilst holding the adjustment screw in position using Allen wrench provided. Monitor the transmitted power whilst performing the lock-down process. It is now safe to fully tension screw D (see points of note). 15. Insert retaining screw K. The regulations governing laser safety dictate that the laser beam is prevented from free space propagation if the fiber is accidentally removed. Once the fiber launch has been optimized the retaining key L on the fiber coupler will locate in the open section of the mount. A retaining screw K is provided to ensure that the fiber is not accidentally removed from the mount.



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Repeat Coupling If the fiber is required to be repeatedly coupled and uncoupled from the laser, then only screw D should used to release the bias of the fiber coupler in the mount.

Figure 2: 3D exploded view of manipulator and fiber



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Care and Maintenance Output optic couplers During the lifetime of the product it will undoubtedly be exposed to environments with air-borne contaminants. To avoid contamination of the lenses & fiber ends the unit must operate in the conditions as specified in the datasheets namely: Operating Pressure: Atmospheric Operating Temperature: 10 to 40 °C Operating Humidity: Non-condensing When the system is not in operation the dust caps provided should be used. Connectors Careless handling and poor maintenance can have a detrimental effect on the connector’s performance. The following guidelines should help maintain the connector’s integrity. · Do not touch or expose the fiber end to any contaminants. · Use the protective dust cap provided when not in use or connectorised. · Adhere to strict cleaning procedures. If the fiber connector end is contaminated, it is essential that the contaminant is removed as quickly as possible. If you are unsure whether the fiber has been exposed, it is preferable to clean the fiber. The contaminant could otherwise cause irreparable damage to the core of the fiber, which would require the connector be replaced. Cleaning Procedure · Ensure no light is emitted from the fiber. · Point Source recommends cleaning connectors with an ‘automatic connector cleaner’, which incorporates a dry, woven polyester cloth as the cleaning medium. These units are commercially available on request from Point Source or from fiber termination houses. · Alternatively moisten a cotton bud with acetone and wipe the face of the connector. · Inspect the emitted beam for circularity and flare.



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Connector Adapters For kineFLEX systems that contain in-line FC/PC polarization maintaining connectors, the connector adapter will influence the performance of the system. A good adapter should provide a lifetime of around 2500 couplings. Point Source adapters contain only zirconia insert sleeves for extended lifetime performance. However, repeated coupling/uncoupling of connectors can result in a build up of dirt and contaminants and system performance degradation. This may be detected if system transmission decreases with an adapter that has been used several times. Point Source recommends cleaning the adapter every 25 connector insertions. Cleaning should be performed with a clean cotton bud and alcohol.



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6.0 VS-Laser Safety 6.1 VS- Laser Safety for Eye Piece on Microscope Removable safety cap for microscope eye piece if removed, automatic switch off of laser system Laser Safety control box for TTL switch of for laser interlock Figure show UV Laser 355 control unit with VS laser safety unit



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VS- Laser Safety for TIRF sample plate cover

VS- Laser Safety for movement of microscope illumination arm



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7. Imaging Software 7.1 VisiView® Software Installation To run the VisiView Software please perform the following installation steps:

1. Check the needed Requirements for the VisiView installation.

2. If you use a camera, install the Camera Driver.

3. If you use a microscope, install the Microscope Driver.

4. If you use an on USB connected Ludl Controller, install the Ludl USB Driver.

5. Install the VisiView main software.

6. Install the SafeNet / Sentinel Driver.

1. The following requirements are needed for installation of the VisiView software:

1. PC-computer with Windows XP-pro or Windows 7-pro operating system with 32 or 64bit

2. Microsoft .NET 2.x Framework installed

3. Minimum of 1 GByte RAM memory

4. Minimum of 2 GByte free disk space on drive C:

5. At least one free USB slot for VisiView software key / dongle

6. One available PCI slot or one available Fire Wire Interface depending on the type of camera

If one of the above listed requirements is not matched, first configure your computer to meet the requirements and then start the installation.



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2. Camera driver The VisiView Software comes with full support for Photometrics PVCam cameras, PCO cameras, Diagnostic Instruments or Hamamatsu cameras. Depending on your type of camera you need to install the drivers from Diagnostic Instruments, PCO , PVCam driver from Photometrics or Hamamatsu DCam. Example of Photometrics PVCAM:

a. On the VisiView Installation CD, go to Photometrics PVCam driver start the PVCam Installer PVCamSetup.exe. If you do not use the Installation CD, please make sure, that you are using at a minimum the Version 2.7.7.3.

b. Follow the menu of the installer.

c. After the installation of the driver had been successfully, the PVCam camera driver needs to be configured using the tool RSConfig.

d. Make sure that the planned camera is connected to the computer and switched on.

e. To start RSConfig go to Windows Start, All Programs, Photometrics, PVCam, Launch RSConfig.exe.

f. Please check if the Camera 1: shows a name for the camera. If this first line is empty, there are problems with the camera. Check the cables for data and power supply. Make sure that the camera is switched on. Redo from step 4 until the camera name comes up.

g. If at least in the first line Camera 1: the name of the camera is shown, please hit Done. The installation of the camera's driver is finished.

In case of continuous problems please contact our Support.

Example Diagnostic Instruments SPOT:

On the VisiView Install-CD, go to the folder Diagnostic Instruments Camera driver, start the installer SPOT_Camera_Driver_Installer4.x.x.x.exe and follow the installation routine.



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Example PCO camera:

1. Depending of your camera model you will have to install the framegrabber board driver or start USB/Firewire driver installer, which you can find in the “PCO Camera Drivers” folder of the VisiView Install-CD. If you are unsure what the right driver is contact our Support.

2. After this you can install the PCI(e) frame grabber of your camera, connect the camera to the computer and switch on both.

3. Wait until the camera is recognized and the camera driver is installed properly (You can check this in the Windows Device Manager)



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3. Microscope driver If you use an automated microscope, you need to install a microscope driver. Currently in VisiView the Zeiss microscopes are fully supported. 1. To install the Zeiss microscope drivers, go in the VisiView Install-CD to the

folder Zeiss and unpack and run the setup MTB2004_1-5-0-7wo_net_runtime.zip for new Zeiss microscopes and MTB_2_60_100.zip for older ones.

2. To install the Leica microscope driver, the Leica LAS software has to be installed to setup the microscope configuration correctly. Then the com port has correctly selected in the VisiView software. 4. Ludl USB driver 1. You can simply connect the MAC 6000 with a null modem serial cable to

a COM port of your computer. Then you can directly choose the COM Port in the VisiView Device Configuration menu.

2. If you want to connect the Ludl Controller by USB, you need to install the Virtual COM Driver, which you can find in the Ludl folder of your VisiView CD (or Updates and Drivers CD). Follow the instructions in the “Virtual_COM Install File_revB.pdf” to activate the Virtual COM Port of the MAC6000 controller. Afterwards you can install the VCP_V1.3.1_Setup.exe

5. For the VisiView installation please perform the following steps:

1. Insert the VisiView CD in the CD/DVD drive of your computer.

2. If the auto start mechanism of the CD does not start the installation routine by itself, please locate the installation file Setup.exe on the CD and double click on it.

3. If the Microsoft .NET 2.x Framework is not installed, it will be automatically installed by the Setup. If not, go to the folder DotNetFX on the VisiView Install-CD and click on dotnetfx.exe to install the Microsoft .NET 2.x Framework.

4. Follow the menu of the installer, but do not forget to place a valid Name and Institute in the installation dialog when requested. They are needed for correct registration.



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5. After installing the VisiView Software, you need to install the Microsoft .Net 2.0 Service Pack 1. Go to the folder "DotNetFX" on the Install-CD and execute the NetFx20SP1_x86.exe file.

6. Finally, you need to install the SafeNet / Sentinel Driver.

6. Sentinal driver The VisiView software needs a dongle/key for licence check. The driver for this dongle needs to be installed for correct detection of the licence key. Warning: When installing the SafeNet Sentinel driver you must remove your USB dongle from the computer! Please perform the following steps:

1. On the VisiView Installation CD, go to SafeNet Sentinel driver start the installer file Sentinel Protection Installer 7.3.3.exe.

2. Follow the menu of the installer.

3. After the installation of the drivers had been successfully please connect your Software Key / dongle which represents the licence of the VisiView into a free USB slot. After the software has located the key the end of the dongle should glow green.

4. Now you are ready to start the VisiView software.



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7.2 How to Update VisiView® Software Update to a higher version

In case Visitron recommends updating your VisiView version, please follow these steps:

� Download the folder in ftp://ftp.visitron.de/visitron/Pub/Support/VisiView/ to a temporary folder on your hard disk

This folder contains the files of the latest installation CD. The folder name represents the version numer, e.g. V174.

� We recommend you burn a backup CD � Run SETUP.EXE

Please note:

The installation disk also contains all Windows drivers required for the hardware supported by VisiView. In case of an update it will not be necessary to install these drivers again, unless otherwise instructed by Visitron. Bug fixes of the current version (Fast Fix)

Visitron’s aim is to react fast on bugs reported. That’s why we publish patch files for the current version. In case Visitron recommends to load a Fast Fix for the current VisiView version, please proceed as follows:

� Download the folder ftp://ftp.visitron.de/visitron/Pub/Support/VisiView/<your version number>/Patches � Copy the files from the Patches folder to your VisiView program folder

(Default: C:\Program Files\Visitron Systems\VisiView), replacing the original files.



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Activating additional program options (dongle update)

In case you order an additional program option, your VisiView dongle must be updated accordingly.

� You will receive a dongle update file from Visitron. � Copy this file to your Visitron program folder � Ensure that your VisiView dongle is connected � Run the Secure Update Utility (Start->All Programs->Visitron Systems-> Secure Update Utility) � Select the dongle update filename and click Apply Code:



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7.3 VisiView® Software The VisiVIEW® is a high performance imaging software from Visitron Systems GmbH for BioImaging application. It is specially designed to meet the needs for high-speed image acquisition and processing with ease of use. The full multi- tasking software supports parallel use of e.g. image acquisition and analysis at the same time. This is an important feature for Life Cell Imaging application as used with the VisiScope system. Between the data acquisition of e.g. several minutes or hours the PC is not blocked, the processing and analysis of already stored or acquired images can be done in the same time. Control of VisiScope Acquisition and Analysis

Flexible Acquisition and Camera Support: A well arranged acquisition menu allows easy selection of camera parameter like binning, exposure time, live mode etc. Currently we support a range of scientific grade cameras. Illumination Control: Control of various Illumination devices including Shutter, filter-wheels, filter-cubes, light-path, mirrors etc.. It allows full automated and easy to use multi-wavelength acquisition for nearly simultaneous observation of up to 8 different dyes or illumination setups. The illumination will be combined with the image acquisition sequence in the MDA - Multi Dimensional Acquisition Mode.



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Overlay Multichannel Fluorescence: The overlay module allows to combine up to 7 different images. Luminescence and fluorescence channels can be merged with brightfield light images. Automatic colour identification makes this tool very effective in easy usage. If a luminescence or fluorescence channel is optically shifted, the xy pixel alignment compensates the shift.

Measurement Option: offers comfortable evaluation of image data - Statistic processing - Line scan graph - Intensity over time graph - Histogram display Processing Option: offers comfortable processing of image data - addtion, subtraction, multiplication, division - with constants or between images - processing of single images or image sequences - operation in background without blocking of the entire computer



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7.4 VisiView® Hot-Key Function Hot-Key Function: function key combination -------------------------------------------------------------------------------------------------- open file ctrl O ( drag&drop from file explorer) save file ctrl S duplicate image ctrl D duplicate stack ctrl shift D region load ctrl R region save ctrl shift R region delete backspace file information dialog ctrl I add panel entry ctrl + drag/drop illumination entry into panel (left mouse key) remove panel entry delete edit toolbar entry shift + left mouse key link/unlink images ctrl + left click into images time lapse sequence select right / left z plane select up / down multi stage position select page up / down open display dialog right-click into image zoom image/window coarse scroll mouse zoom image fine left mouse key to drag window border zoom image/ fixed window mouse over + shift + scroll mouse pan image (zoomed image) shift + any mouse key zoom graph coarse scroll mouse zoom graph fine left mouse key to drag window border zoom graph/ fixed window (mouse over +) shift + scroll mouse zoom graph x-axis (mouse over +) shift + ctrl + scroll mouse zoom graph y-axis (mouse over +) shift + alt + scroll mouse



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8. Microscope Peripherals 8.1 MAC6000 Filter wheel System When you analyze dynamic processes by using multiple stained preps or by examining the changes of spectral properties of fluorochromes you require the appropriate change of the excitation and/or emission filter. The MAC6000 filter wheel system is designed for such applications. The filter wheel has been designed to easily integrate with various imaging systems between the lamp housing and a detector e.g. CCD camera. The hinged construction of the filter wheel housing facilitates easy filter interchange without removal of the filter wheel and realignment of the light source. General Description The stepper motor filter wheel/shutter is a microprocessor half step motor driver capable of operating up to two six position filter wheels and three high speed shutters.



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The stepper filter wheels are driven by stepper motors via a timing belt. An integral home position (Hall Effect) sensor is utilized for automatic initialization. The maximum speed is 100ms between adjacent filter positions. A filter wheel can have either a 25mm / 32mm or 50mm filter. Filters can be easily inserted and removed from the filter wheel while the entire assembly is mounted due to the hinged construction of the filter wheel. Up to three high speed shutters may be controlled using the same module. The drive electronics utilize a bi-level drive, supplying an initial high opening voltage for high speed action (<15ms to open or close) followed by a lower holding voltage which allows shutters to be held open indefinitely without damage. Manufactured specifically for high temperature applications, these shutters have proven to be extremely durable under heavy usage due to their simple design.



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8.2 XY- Motorized Stage High Precision XY-Stage Systems

High resolution XY-table getting more and more important in the growing market of microscopy applications for histology- and fluorescence analysis. The high accuracy of these stages allows the repetitive measurement of different positions of specimens. With the Joystick several regions of interest can be selected easily. These coordinates can be stored and recalled, e.g. by the Software, during analysis and experiment control. The scan of sample slides or microtiter plates with different sizes and up to 1536 wells can be scanned without any problems. The control unit offers an RS-232 serial or USB Interface. Additional modules for Z-axis and filter wheel control are available. XY stages are perfect components for multiple dimension acquisition. Perfusion and incubation chambers for Live Cell Imaging can be combined with the stage setup.



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Specifications:

- flat, ergonomic table design - travel range 120 mm x 100 mm - resolution: 0,05 μm - repeatability: 0,75 μm - accuracy: 6 μm - speed: 60mm/sec

Option:

- linear Encoder Option LE for XY motorized stage for microscope - improvement of accuracy - resolution: 0,1 μm - repeatability: 0,25 μm - accuracy: 2 μm

Insert:

- Inverted stage adjustable insert - Slide or Petri Dish

MAC6000 XYZ-stage control unit

- including Joystick - small housing - serial interface, USB or Ethernet to computer



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8.2.1 Z- Piezo Insert for XY-Stage

The NanoPrecision Piezo Z-stages feature the speed, resolution and accuracy of Piezo nano positioning. Each NanoPrecision device is specifically designed to suit a particular application.

Piezo Z-microscope stage insert specification :

- Piezo Focus / Z-axis - travel range 350um - resolution about 3nm - repeatability 1nm High speed streaming control by analog voltage / DAC board



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8.3 Filterset Understanding Filter Spectra

More and more demanding fluorecence applications in the field of biology and medicine require better optical filter systems. Increased brightness without a corresponding increase in noise or undesired background is impossible without optimized filtersets. Filters with steep pass bands are essential for new fluorochroms with very short Stoke´s shift. New automatic image acquisition and screening systems need this filter quality for separation of signals from multi-stained samples without loss of throughput. Visitron Systems GmbH offers a large variety of filtersets for nearly all established applications. On demand we provide the complete mounted filter cube or slider for all major brands of microscopes and filter wheels (Zeiss, Leica, Nikon, Olympus, Ludl, Sutter, Cairn ) A fluorescence microscope usually contain three parts in a filter system. An excitation filter, an emission filter and a dichroic beamsplitter. The dichroic beamsplitter is a coated optics that reflects excitation light and transmit emission light.

Correct mounting of filterset into filter cube: Orientation: Proper orientation of the filter is necessary in order to minimize autofluorescence and maximize performance. Please take a moment to locate the caret (arrow) located on the edge of each filter. Excitation (x) filters should be positioned with the caret pointing toward the specimen and the inside of the cube, and away from the light source.



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Emission (m) filters should be positioned with the caret pointing toward the specimen and inside of the cube, and AWAY from the detector / eye. Dichroic mirrow (bs) should be mounted with the coating surface TOWARD the light source, excitation filters and specimen. The coating side will be identified with a caret pointing to it, or a bevel. The beveled side is the smaller surface. Cleaning and Handling of filters

Handle coated pieces by the edges only. Clean gently only if necessary. Loose particle should be removed with bulb puffer filtered pressurized air cleaner. If necessary, gently wipe surface using anhydrous alcohol and lint free lab towels. Use a new surface of towel with each wipe. Avoid touching or wiping A/R coated or metal mirror surfaces ! Avoid handling exposed coatings with bare fingers ! ..............................................................................................................................



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Optical Filter with different quality / transmission – from standard, HQ- quality to hardcoated.

Typical Filter curve for Excitation and Emission Spectra High-Q – Filterset

� with steepest passband � recommended for dyes with

short Stoke´s shift Example: Yellow GFP Hardcoated Highest Transmission

� optical filters with highest spectral performance

� very steep edges, highest transmission, high blocking

� no pixelshift within one filterseries � high thermal and mechanical

resistance Example: GFP

Dualband – Filterset � simultaneous measurement � of two dyes

Example: FITC and Cy3 Tripleband – Filterset

� simultaneous measurement of three dyes

� minimum overlap Example: DAPI, FITC and Texas Red



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8.4 LED Illumination PE-300 White

CoolLED’s pE-300-White light source is designed to offer broad spectrum LED illumination for general use in fluorescence microscopy applications. It can be fitted directly to the microscope as a better and safer alternative to high pressure mercury or metal halide light sources. Spectral coverage is from the UV (DAPI excitation) to the Red region (Cy5 excitation). It will excite common fluorophores used in hospital and research environments. With a comprehensive range of microscope adaptors, the pE-300-White can be fitted to most current and older microscopes. The result is a safe, convenient light source which will last for many years without any additional operating costs. This manual should give you all the information required to install and operate your new light source.



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Safety Precautions While LEDs are a much safer light source than the mercury and metal halide lamps that they replace in the application of fluorescence microscopy, precautions should still be taken with this product. When operating or maintaining this product, please observe the following safety precautions at all times. Failure to do so may result in personal injury or damage to other items. 2.1 UV light is emitted from this product. Avoid eye and skin exposure. Never look directly into the light output beam from the LED head. The emissions could damage the cornea and retina of the eye if the light is observed directly. 2.2 Always ensure that the LED head is securely attached to the microscope prior to turning on the power to the unit. This will minimise the risk of injury and damage. 2.3 If for any reason the unit is to be operated when not attached to a microscope, all personnel should wear eye shielding and clothing to protect the exposed skin. 2.4 Disconnecting the mains supply is achieved by unplugging the power cord from the power supply block. Only plug in the power cable, once the unit is attached to the microscope. 2.5 There are no serviceable parts within the unit. Removing any of the screws and covers will result in the safety of the unit being impaired. 2.6 Any electronic equipment connected to this product must comply with the requirements of EN/IEC 60950. 2.7 To clean the exterior of the unit, use a slightly dampened cloth with a simple water/detergent solution only. Avoid the optical surfaces and lenses. Cleaning of optics should only be carried out using optical wipes and fluids.



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Getting Started – System Components The CoolLED pE-300-White Excitation Source is supplied with the following components: 1. LED Head; 2. Manual Control Pod; 3. Microscope adaptor for specific microscope model (supplied already attached to LED head); 4. DC Power Supply Type GS90 A12; 5. IEC Power Cable (not shown); 6. User Guide (not shown).

If any components are missing or appear damaged, please contact CoolLED immediately.



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4.0 Installation and setup 4.1 Carefully unpack the components from the shipping cartons. 4.2 Remove the protective cap from the end of the pod cable connector. 4.3 Insert the pod cable into the LED head using the red dots as a guide for orientation of the plug.

4.4 Connect the power connector from the DC power supply as shown. At this stage do not connect the mains power lead to the DC power supply.



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4.5 Attach the LED head to the epi-fluorescence port on your microscope. Your pE-300-White will have been supplied with a compatible fitting for the microscope you specified at order. Attach the head ensuring that it is secure and true/flush with the microscope. 4.6 Ensure that there is free airflow around the LED head so that the cooling system is not impaired. A gap of 200mm on either side is sufficient. The diagram shows the unit in the preferred orientation. However it may be set with the cables at the top or at either side. 4.7 With the LED head now attached to the microscope it is safe to connect the mains power. Connect the mains lead supplied to a convenient socket, plug in the IEC connector into the DC power supply and switch the power on at the socket. 5.0 Configuration of LEDs as a White Light Source 5.1 Conventional ‘white light’ sources used for fluorescence microscopy (e.g. mercury lamps), have a single element which emits light in a series of peaks across the spectrum, giving the effect of white light. LEDs are different in that a single LED element will emit light in a particular colour. To create a white light source, LEDs of different wavelengths have to be combined together. Using a pumped phosphor, a broader peak covering green, yellow and red emissions can also be created. In the pE-300-White, LEDs emitting in the UV and blue regions are combined with a pumped phosphor to create a white light source covering all the commonly used fluorescence stains.



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5.2 The pE-300-White has independent circuits giving the user control of the three main peaks of emissions. On the standard configuration, these are referred to as UV, BLU (blue) and GYR (green, yellow, red).

5.3 There is a special variant of the pE-300-White which has been configured for use with multi-band filter sets where the first peak has been shifted from the UV region to the VIO (violet). See Appendix 1 for more information.



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6.5 Intensity Control. The control pod enables the user to control the intensity of the LEDs that are exciting different stains. This helps to balance the emissions so that one stain does not dominate another. This feature is very useful in multi-band work (see application note in Appendix 1). 6.6 Reduce intensity of one stain by pressing the down intensity button

6.7 Individual bands can be switched off (de-selected) by pressing the ‘select’ button. Light is then only generated where it is required to excite the stains in use. This has many attractive benefits with improvements in contrast, cell viability and savings in energy. Switching off UV will help to reduce damage to cells through photobleaching.



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6.8 Switch off selected bands by pressing the ‘on/off’ button.

6.9 Switch selected bands back on by pressing ‘on/off’ button again.



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7.0 Operation - TTL Control 7.1 The pE-300-White can be controlled remotely via a TTL signal. The input uses the single BNC socket on the reverse of the head.

7.2 The TTL signal controls the on/off function of the unit. A TTL ‘high’ will cause the LEDs to be on, independent of the state of the on/off button. Only those bands which have been manually selected on the Pod will be switched by the TTL signal. The intensities of the selected bands are manually set on the pod. 7.3 The TTL input circuit has been designed to maximise the switching speed of the LEDs to give the user precise control of the excitation light reaching the sample.



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8.0 Optical setup 8.1 The pE-300-White has been designed to work on the majority of fluorescence microscopes, both new and old. As would be expected, there is some variation in the optical path and elements within every microscope. In order to accommodate these variations, the pE-300-White is supplied with a small adjustment which allows the user to optimise the performance of the light source when it is first fitted. This is a one-time adjustment. No further adjustment will be required during the life of the product unless changes are made to the microscope or the light source is fitted to a different microscope.

8.2 To make the adjustment, set up a typical sample on the microscope that gives an image over the whole field of view. Loosen the thumbscrew and slide the post back and forth until you achieve the maximum brightness with an even field of view. Tighten the thumbscrew to prevent the setting from changing.



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Product specifications 14.1 Power requirements 110-240Va.c 50/60Hz 2A 14.2 Power consumption Standby mode max 2W White (all 3 bands at 100% intensity) max 80W Two bands at 100% max 63W Single band at 100% max 38W 14.3 Dimensions Main Head Unit 77mm (w) x 186mm (d) x 162mm (h) -weight 1.40kg Control Pod 88mm (w) x 125mm (d) x 37mm (h) - weight 0.32kg Power Supply 140mm (w) x 56mm (d) x 32mm (h) - weight 0.43kg 14.4 Environmental Operating Conditions Operating 5 – 35 deg C



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8.5 LED Illumination PE-100 Single wavelength excitation source

This source offers intense, stable excitation at a single wavelength. The user can select extensive range of LED wavelengths. It is designed to fit on all current microscopes. A simple once-ever adjustment will allow optimisation to the optical path of your microscope. The pE-100 is supplied as a complete working package with power supply and control pod. Manual operation offers instant on/off and 0-100% intensity control. The control pod accepts a BNC-type external TTL trigger.

Setup Procedure



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Fit to the Microscope

Connect control panel and power supply

� Connect the cable with D-type connector from control pod to the PE100 head

� Secure screws using a flat screwdriver � Connect power supply to control pod � Connect power cable to power supply and finally connect to an

electronical outlet Control Pod Operation



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Adjustment of PE100 To ensure that the PE100 is optimized for optics of your microscope, a simple adjustment needs to be made. No future adjustment will be required during the life of the product unless changes are made the microscope.

Optional TTL Trigger An optional BNC cable is available for connection to the control pod from an external TTL source will allow fast switching On/Off of the LED excitation.



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9. Incubation of the VisiScope System 9.1 okolab - Incubation Chamber



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10. EC Declaration of Conformity



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EG Konformitätserklärung

EC Declaration of Conformity

Visitron Systems GmbH · 82178 Puchheim · Germany

Wir erklären hiermit die Übereinstimmung des genannten Gerätes mit der EG-Richtlinie 2006/95/EG - Niederspannungsrichtlinie und mit der Richtlinie 89/336/EWG über die elektromagnetische Verträglichkeit

einschließlich der Änderungen 92/31/EWG und 93/68/EWG.

Bei Änderungen am Produkt, die nicht von uns autorisiert wurden, verliert diese Erklärung ihre Gültigkeit. We declare the compliance of the device with the requirements of the Council Directive 2006/95/EG – Low

Voltage Directive and with the Council Directive 89/336/EWG about the Electromagnetic Compatibility including modifications 92/31/EWG and 93/68/EWG.

Any modification of the product, not authorized by us, will invalidate this declaration.

Gerätebezeichnung/Device Name:

VS-Laser Control VS-Laser-Merge

Normen/Standards: EN 61010-1, amendment A1,A2 EN 55011, class B EN 50082-1 EN 6100-3-2 EN 61000-3-3 EN 61326-1 EN 60825-1 Das Gerät ist gekennzeichnet mit / The device is marked with Puchheim, 11.01.2010 Visitron Systems GmbH

Helmut Wurm

Die Erklärung bescheinigt die Übereinstimmung mit den Richtlinien und mit dem Gesetz. Gewährleistung und Haftung sind in unseren allgemeinen Lieferbedingungen geregelt. The declaration certifies the compliance with the directives and the law. Conditions of guarantee and liability are regulated in our General Conditions of Sale.



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Visitron Systems GmbH � 82178 Puchheim � Germany

Wir erklären hiermit die Übereinstimmung des genannten Gerätes mit der EG-Richtlinie 2006/95/EG -Niederspannungsrichtlinie und mit der Richtlinie 89/336/EWG über die elektromagnetische Verträglichkeit

einschließlich der Änderungen 92/31/EWG und 93/68/EWG. Bei Änderungen am Produkt, die nicht von uns autorisiert wurden, verliert diese Erklärung ihre Gültigkeit.

We declare the compliance of the device with the requirements of the Council Directive 2006/95/EG – Low




VS-AOM

Normen/Standards: EN 61010-1, amendment A1, A2 EN 55011, class B EN 50082-1 EN 6100-3-2 EN 61000-3-3 EN 61326-1

Das Gerät ist gekennzeichnet mit / The device is marked with Puchheim, 11.01.2010 Visitron Systems GmbH

Helmut Wurm




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Visitron Systems GmbH � 82178 Puchheim � Germany

Wir erklären hiermit die Übereinstimmung des genannten Gerätes mit der EG-Richtlinie 2006/95/EG -Niederspannungsrichtlinie und mit der Richtlinie 89/336/EWG über die elektromagnetische Verträglichkeit

einschließlich der Änderungen 92/31/EWG und 93/68/EWG. Bei Änderungen am Produkt, die nicht von uns autorisiert wurden, verliert diese Erklärung ihre Gültigkeit.

We declare the compliance of the device with the requirements of the Council Directive 2006/95/EG – Low




VS-2D FRAP Scanner

Normen/Standards: EN 61010-1, amendment A1, A2 EN 55011, class B EN 50082-1 EN 6100-3-2 EN 61000-3-3 EN 61326-1

Das Gerät ist gekennzeichnet mit / The device is marked with Puchheim, 01.07.2011 Visitron Systems GmbH




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