Radiance Mp

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Radiance2000 MP

Operating Manual


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Radiance Operating Manual

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Table of Contents

1. Preface and Warnings.................................................................................................6

1.1 LaserSharp2000 Software - limited use licence conditions.......................................61.2 Software copyright...................................................................................................61.3 Software use ...........................................................................................................6

1.4 Unauthorized use of software ..................................................................................61.5 Software support limitations.....................................................................................61.6 Other products referred to in this manual.................................................................61.7 Manual part number ................................................................................................61.8 Laser Safety............................................................................................................7

1.8.1 Laser warnings .................................................................................................71.8.2 Laser cautions..................................................................................................71.8.3 Laser safety labels............................................................................................81.8.3.1 Identification and Certification Labels...........................................................81.8.3.2 Warning Logotype Label...............................................................................91.8.3.3 Aperture Label............................................................................................101.8.3.4 Labels for Non-interlocked Housing............................................................ 101.8.4 Microscope Interface Specification .................................................................11

1.9 Electrical safety..................................................................................................... 121.9.1 Electrical warnings.......................................................................................... 121.9.2 Electrical cautions .......................................................................................... 12

1.10 System Cooling.....................................................................................................121.10.1 Cooling warnings............................................................................................12

1.11 System Management............................................................................................. 121.12 Comments............................................................................................................. 121.13 Symbols and conventions...................................................................................... 12

2. The system at a glance.............................................................................................. 142.1.1 Scan head ......................................................................................................152.1.2 Instrument Control Unit...................................................................................162.1.3 Beam Conditioning unit .................................................................................. 172.1.4 Direct detectors ..............................................................................................18

2.1.5 Computer and software .................................................................................. 182.1.6 Z-drive............................................................................................................182.1.7 Transmission detector .................................................................................... 18

3. Getting started........................................................................................................... 193.1 Switching on ancilliary equipment..........................................................................193.2 Switching on the femtosecond pulsed laser ...........................................................193.3 Switching on the system ........................................................................................ 193.4 Starting the LaserSharp2000 software...................................................................233.5 Switching off the system........................................................................................ 233.6 A brief introduction to the software ........................................................................ 24

4. Factors Which Effect Optical Performance................................................................ 304.1 Objective lenses.................................................................................................... 30

4.1.1 Care and maintenance of objective lenses .....................................................33

4.2 Confocal aperture size........................................................................................... 344.3 Telecentric adjustment .......................................................................................... 354.4 Visible laser considerations ................................................................................... 364.5 Femtosecond pulsed laser considerations .............................................................364.6 Alignment of the Beam Conditioning Unit .............................................................. 374.7 Filters.................................................................................................................... 39

4.7.1 Filter configuration For Radiance2000 MP AG-2.............................................394.7.2 Filter configuration For Radiance2000 MP K-2 ............................................... 404.7.3 Filter configuration For Radiance2000 MP AGR-3.......................................... 414.7.4 Filter configuration For Radiance2000 MP KR-3.............................................424.7.5 Filter configuration For Radiance2000 MP AGR-3 (Q) .................................... 434.7.6 Inserting custom emission filters.....................................................................444.7.6.1 Physical insertion of filters.......................................................................... 444.7.6.2 Updating the firmware................................................................................ 46

4.8 Direct detectors ..................................................................................................... 47


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4.8.1 Introduction .................................................................................................... 47Direct detectors coupled to an Olympus BX50WI upright microscope.................... 48Direct detectors coupled to a Nikon TE300 inverted microscope............................ 48

4.8.2 Optical configuration.......................................................................................494.8.3 Filter configurations........................................................................................504.8.3.1 Bi-Alkali/Bi-Alkali Configuration (BB) .......................................................... 50

4.8.3.2 Bi-Alkali/Multi-Alkali Configuration (BM) ..................................................... 504.8.4 Selecting filter cubes ......................................................................................514.8.5 Changing filter cubes...................................................................................... 514.8.6 Using the Direct Detectors..............................................................................524.8.6.1 Nikon E600FN............................................................................................ 524.8.6.2 Olympus BX50WI....................................................................................... 554.8.6.3 Nikon TE300 .............................................................................................. 56

4.9 Fluorophores......................................................................................................... 574.9.1 Fluorophores excited by 1-photon excitation...................................................574.9.2 Fluorophores excited by 2-photon excitation...................................................61

4.10 Dynamic range ......................................................................................................624.11 Monitor adjustment................................................................................................634.12 Sampling resolution...............................................................................................64

4.13 Signal strength and noise ......................................................................................655. Sample preparation suggestions for confocal and muti-photon imaging..................... 66

5.1 Fixatives for biological tissue................................................................................. 665.2 Bleaching and anti-fade agents ............................................................................. 665.3 Mounting media..................................................................................................... 665.4 Autofluorescence...................................................................................................67

6. Software reference .................................................................................................... 686.1 Menu bar............................................................................................................... 68

6.1.1 File Menu ....................................................................................................... 68

6.1.1.1 Create a New Experiment ................................................................ 68

6.1.1.2 Open an existing Experiment ........................................................... 68

6.1.1.3 Close a currently Open Experiment .................................................. 696.1.1.4 Save an Experiment................................................................................... 69

6.1.1.5 Print an image .................................................................................. 696.1.1.6 Log Out ...................................................................................................... 696.1.1.7 A list of recently opened Experiments.........................................................696.1.1.8 Exit the application..................................................................................... 696.1.2 Edit menu....................................................................................................... 696.1.3 Methods menu................................................................................................ 69

6.1.3.1 Save Method ..................................................................................... 70

6.1.3.2 Edit... (Method) .......................................................................................... 706.1.4 Method Wizard...............................................................................................716.1.5 Acquire menu.................................................................................................76

6.1.5.1 Live Scan ....................................................................................... 76

6.1.5.2 Sequential live scan ........................................................................ 76

6.1.5.3 XY (Z-Series) collection ................................................................... 77

6.1.5.4 X-Z (vertical section) collection ........................................................ 78

6.1.5.5 X-T (line scan) collection ..................................................................796.1.5.6 Pixel Size (depth)....................................................................................... 80


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6.1.6 Image menu ................................................................................................... 806.1.6.1 Adjust contrast ........................................................................................... 816.1.7 Tools menu .................................................................................................... 826.1.7.1 System Setup............................................................................................. 826.1.7.2 User set up................................................................................................. 846.1.7.3 Objective lens set up.................................................................................. 85

6.1.8 Script Menu....................................................................................................876.2 Image display window............................................................................................886.3 System control panels ........................................................................................... 90

6.3.1 Microscope control .........................................................................................906.3.2 Channels control.............................................................................................946.3.3 Focus motor control........................................................................................ 956.3.4 Optics panel ...................................................................................................976.3.5 Mixer control panel .........................................................................................98

6.4 Image operators .................................................................................................. 1006.4.1 Arithmetic..................................................................................................... 1016.4.2 Histogram..................................................................................................... 1026.4.3 Line Profile................................................................................................... 1036.4.4 Merge........................................................................................................... 104

6.4.5 Projection.....................................................................................................1056.4.5.1 Single view............................................................................................... 1056.4.5.2 Multiple Views.......................................................................................... 1066.4.5.3 Projection method (type) .......................................................................... 1076.4.5.4 Source data.............................................................................................. 1086.4.5.5 Two Pass Projections ............................................................................... 1096.4.6 Seed Fill ....................................................................................................... 1106.4.7 Importing and exporting files ........................................................................ 111

7. Technical details...................................................................................................... 1127.1 Filter specifications.............................................................................................. 112

7.1.1 Radiance2000 MP Dichroic filters................................................................. 1127.1.2 Radiance2000 MP Emission filters ...............................................................1157.1.3 Radiance2000 MP Blocking filters ................................................................121

7.2 System calibration............................................................................................... 1227.3 Switching the scanhead between microscopes .................................................... 123


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1. PREFACE AND WARNINGS

Bio-Rad Microscopy Division

April 1999

Radiance2000 MP Operating manual, Issue 2

Copyright 1999 Bio-Rad Microscience Ltd

All rights reserved. Customers may only copy this manual for their own use.

1.1 LaserSharp2000 Software - limited use licence conditions

The software described in this manual is supplied under a limited use licence agreement.

1.2 Software copyright

The LaserSharp2000 software is the sole and exclusive property of Bio-Rad. The customerundertakes not to copy any part of the software without written permission from Bio-Radexcept for a back-up copy for security purposes.

1.3 Software use

The software may only be used on the machine for which it was originally supplied.

1.4 Unauthorized use of software

The customer agrees to protect the software from unauthorized use.

1.5 Software support limitations

The LaserSharp2000 software will only be supported when run on a Bio-Rad recommendedcomputer (at the time of purchase).

1.6 Other products referred to in this manual

Windows NT and Windows 2000 are registered trademarks of Microsoft Corporation.

The Trademarks of all fluorochromes and probes are recognised.

1.7 Manual part number

Further copies of this manual may be obtained by quoting Manual Part Number9MRC60UM04 issue 2.


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1.8 Laser Safety

Lasers are potentially hazardous to the eyes and skin and you should, for your own safetyand that of your co-workers, read and comply with the following warnings and cautions.

1.8.1 LASER WARNINGS

WARNING: VISIBLE AND INVISIBLE LASER RADIATION IS EMITTED FROM THE

MICROSCOPE OBJECTIVE. AVOID EXPOSURE TO DIRECT OR

SPECULARLY REFLECTED RADIATION.

WARNING: ENSURE THAT EACH POSITION OF THE MICROSCOPE TURRET

CONTAINS AN OBJECTIVE LENS, AN ALIGNMENT PRISM OR A

BLANKING COVER. NO PORTS MAY BE LEFT OPEN.

During operation it is possible to have access to laser radiation at the objective

which can pose a skin hazard. This is only true when the system is scanning.

WARNING: AVOID EYE OR SKIN EXPOSURE WHEN PLACING OR REMOVING

SAMPLES FROM THE MICROSCOPE STAGE.

The white BCU LED is il luminated when laser radiation is present.

WARNING: VIEWING OF THE MICROSCOPE OBJECTIVE WITH EXTERNAL

OPTICAL INSTRUMENTS COULD BE HAZARDOUS WHEN THE

SYSTEM IS SCANNING.

A special laser blocking filter can be obtained which fits over the optical instrument

(for example, a microscope for observing patch clamps and micro-manipulators).Contact your local Bio-Rad office for details.

WARNING: USE OF CONTROLS OR ADJUSTMENTS OR PERFORMANCE OF

PROCEDURES OTHER THAN THOSE SPECIFIED HEREIN MAY RESULT

IN HAZARDOUS VISIBLE AND/OR INVISIBLE RADIATION EXPOSURE.

WARNING: EYE PROTECTION MUST BE WORN WHEN THE SYSTEM IS

SCANNING.

1.8.2 LASER CAUTIONS

Improper use of this equipment could damage the equipment.

The system carries BRH recommended interlocks and warning labels. Ensure that

all personnel read and follow these warnings.

The Scan Head cover, Instrument Control Unit covers and BCU cover should only

be removed by Bio-Rad authorised technicians and staff.


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1.8.3 LASER SAFETY LABELS

1.8.3.1 Identi f icat ion and Cert i f icat ion Labels

Primary Identification and Certification Label (fitted at rear of Instrument Control Unit)

Secondary Identification and Certification Label(fitted inside front door of Instrument Control Unit)

Identification label for Ion Secondary Laser

MODEL #: IS19BR12SERIAL #:DATE MFD:VOLTAGE:CURRENT:

REV:Hz:

AMERICAN LASER CORPORATION

1832 SOUTH 3850 WESTSALT LAKE CITY, UTAH 84104 USA


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Identification label for Ion Secondary Laser

1.8.3.2 Warning Logoty pe Label

Warning Logotype Label(fitted to the BCU)

THIS LASER PRODUCT ISDESIGNATED FOR USE SOLELY

AS A COMPONENT (ORREPLACEMENT) IN AN

ELECTRONIC PRODUCT ANDTHEREFORE DOES NOT COMPLY

WITH THE APPROPRIATEREQUIREMENTS OR DHHS

REGULATIONS No 21 CFR1040.10 AND 1040.11

FOR EXPORT OR OEM

USE ONLY


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1.8.3.3 Ap ertur e Label

Aperture Label(fitted to the scan head focus ring)

1.8.3.4 Labels for Non-interlocked Housing

(Fitted to base of Scan Head adj. to cover screws and to ICU on l/hand side of electronicsunit towards rear of ICU)fitted to Tube supports (2off), periscope mirror covers (2off), power

meter pocket, BCU connector panel and laser connecting tubes (2off).)

(Fitted to the ICU on both sides of the 4-axis laser launcher, to the fibre interface ateach detector module and also to the Scan Head chassis beneath the cover.)

(Fitted to the ICU on the l/hand side of the electronics unit and on theright-hand side of the secondary gas laser module.)

VISIBLE AND INVISIBLELASER RADIATION WHEN OPEN

AVOID EYE OR SKIN EXPOSURE TODIRECT OR SCATTERED RADIATION

DANGER

For SERVICE ACCESS only by BIO-RADtrained personnel.

In case of instrument malfunction contactBIO-RAD or an authorised representative.

DO NOT OPEN


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1.8.4 MICROSCOPE INTERFACE SPECIFICATION

The Radiance scan heads attach to the photoport of a conventional optical microscope. Theoptical microscope must have been originally designed, or modified by Bio-Rad, so that it isimpossible for laser light to be directed into the binocular viewer.

Bio-Rad staff are instructed not to proceed with the installation if this requirement is not met.


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1.9 Electrical safety

Electricity is potentially hazardous and you should, for your own safety and that of your co-workers, read and comply with the following warnings and cautions.

1.9.1 ELECTRICAL WARNINGS

POTENTIALLY LETHAL VOLTAGES ARE PRESENT IN THE INSTRUMENT CONTROL

UNIT WHEN EITHEROF THE MAINS SUPPLIES ARE PLUGGED IN.

1.9.2 ELECTRICAL CAUTIONS

Ensure that the electrical system earth (ground) is connected at all times during

operation.

Ensure that the mains supplies (outlets) are sufficiently rated and correctly fused.

1.10 System Cooling

Effective cooling (air flow) is essential for the correct operation of the equipment and toensure the expected operating lifetime of lasers in particular.

1.10.1 COOLING WARNINGS

ENSURE THAT THE INSTRUMENT CONTROL UNIT AND THE LASER COOLING FANS

ARE POSITIONED SO THAT ADEQUATE AIR FLOW IS ENSURED.

WHEN TURNING OFF THE SYSTEM ENSURE THAT THE SHUT-DOWN PROCEDURE,

PARTICULARLY WITH REGARD TO COOLING THE GAS LASER HEAD, IS

RIGOROUSLY FOLLOWED.

1.11 System Management

It is recommended that one person is given the job of System Manager. The SystemManager should be responsible for understanding how to use and align the system. TheSystem Manager should control access to the system, and should also keep alignment toolssafely out of the reach of unqualified personnel.

1.12 Comments

Written comments on the contents of this manual should be addressed to:

Marketing GroupBio-Rad Microscience LtdBio-Rad HouseMaylands AvenueHEMEL HEMPSTEADHerts HP2 7TDUK

Fax: +44 (0)181 3282500email: [email protected]

1.13 Symbols and conventions


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indicates the identification of a specific key on the computer keyboard.

File|Open... indicates a menu title on the menu bar followed by a menu item choice withinthat menu.

Drop-down menus: When you click on a menu title a drop-down menu appears.

Dropdowns: Click on the down arrow to select an option from a dropdown menu. This canalso be an option in a drop-down menu, which produces a second menu.

Pop-up menus: These context sensitive menus offer functionality relevant to the mouse

position. They are accessed by right-clicking.

Spin box : Click on the left or right arrow to move the slider left or right. Alternatively, youcan enter a value directly in the box provided alongside the slider or grab the slider with themouse.


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2. THE SYSTEM AT A GLANCE

Figure 1 Schematic view of system

The system consists of six main components:

- Femtosecond pulsed laser

- Beam Conditioning Unit

- Scan Head

- Instument Control Unit

- Direct detectors

- Computer and software

and two optional ancilliary components:

- Z-drive

- Transmission detector


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2.1.1 SCAN HEAD

The miniaturised scan head is permanently connected to the Instrument Control Unit via asingle umbilical conduit (bottom left). This conduit carries electrical control signals to themotorised devices inside the scan head e.g. the dichroic f ilter wheels (B), the visible laserillumination input f ibre (A) and the output signal fibres (C) which route the optical signal to the

remote detector modules (Section 2.1.2).

Additionally, the pulsed laser is directly coupled into the scan head (D) where it is thencombined with the visible beam using a polarisation and chromatically dependent beamsplitter (E). An optional Multi-photon Demagnification Lens (MDL) (F) can be switched intothe emission beam path to enhance collection of scattered light when imaging in multi-photonmode.

Figure 2 Schematic internal view of scan head

A

C

B

D

E

F


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Each collector module has its own switchable polarising analyser (F) and continuouslyvariable confocal aperture (G).

Figure 3 Schematic view of collector module

2.1.2 INSTRUMENT CONTROL UNIT

The Instrument Control Unit houses either a combination of an Argon laser (A), green HeliumNeon laser (B) and the red laser diode (C), or a 2-line Krypton/Argon laser (A) plus the redlaser diode (C). All lasers are combined, passed through an AOTF (D) and then into thesame single mode fibre as shown in the scan head diagram above (section 2.1.1)

Figure 4 Internal view of Instrument Control Unit

A

B

F

G

C

D


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On the opposite side of the unit an elctronics enclosure houses the remote detector modules(E) and system control electronics.

Figure 5 Electronics enclosure internal view

2.1.3 BEAM CONDITIONING UNIT

The Beam Conditioning Unit (BCU) contains all the components to control the infra-redbeam:

1. Beam steering (alignment) mirrors2. Beam collimator module (manual or motorised)3. Diagnostic pick-off beamsplitter

4. 50:50 beam splitter5. Spectrum analyser6. Near-point alignment window

E


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7. Far-point alignment window8. Pockels cell (alternatively Neutral density filters)9. Power meter pick-off beam splitter10. Power meter11. Safety shutter

2.1.4 DIRECT DETECTORS

Direct detectors (also known as external or non-descanned (NDS) detectors) for theRadiance2000 MP system are available in three basic models:

Two channel manually operated

Two channel motorised (software controlled)

Four channel motorised (software controlled)

These detectors are designed to collect scattered light (typically from deep within tissue)highly efficiently. They do this by picking-off the emitted fluorescence from immediatelybehind the objetive lens in the microscope and diverting it to one or more photo-multiplier

tubes. Because the 2-photon excitation process inherently generates an optical section thereis no need to pass the signal through a (confocal) detection aperture and hence the beamdoesnt need to be descanned. This optically simple method of detection has the addedadvantage that shorter wavelength light 350 < ? > 700 can also be detected.

2.1.5 COMPUTER AND SOFTWARE

The system computer is a PC with a powerful processor, large hard disk, integratednetworking hardware and a CD-ROM drive. Optional data storage devices such asrecordable CD-ROM drives (CD-R) and Jaz

TMor Zip

TMdrives from Iomega are available.

The operating system is MS Windows NT4.0TM

.

2.1.6 Z-DRIVE

Z-drives are available for most microscopes from the key microscope manufacturers; Zeiss,Nikon and Olympus. In all cases a 2000 half-step stepping motor is used to drive themicroscopes focus mechanism. For the Nikon and Olympus microscopes the fine focus knobis driven directly giving a focusing resolution of 50nm (0.05 microns). For the Zeissmicroscopes the stepper motor output is geared down to drive the coarse focus knob, alsoresulting in a resolution of 50nm.

2.1.7 TRANSMISSION DETECTOR

As above, transmission detectors are available for most microscopes from the keymicroscope manufacturers; Zeiss, Nikon and Olympus. The detector is situated between thebody of the microscope and the trans-illumination lamp housing. In operation a motorisedand computer controlled mirror is translated into the path of the transmitted scanned laserlight and is focused into a multi-mode optical fibre. The optical signal is relayed to either aphotodiode detector module or a PMT detector module housed in the Instrument Control Unit(Section 2.1.2)


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3. GETTING STARTED

3.1 Switching on ancilliary equipment

If you have a conventional epi-fluorescence lamp attached to the host microscope, switchthis on before anything else.

Refer to the manufacturers documentation regarding the switch-on sequence for devicessuch as external disk drives and printers.

3.2 Switching on the femtosecond pulsed laser

Refer to the laser manufacturers documentation regarding turning on and off thefemtosecond pulsed laser. It will generally be necessary to allow the pulsed laser severaltens of minutes to warm up and reach stable operating conditions, so it will usually beadvisable to turn this laser on before any other parts of the system. However, the pulsedlaser can be turned on or off at any stage.

3.3 Switching on the system

1. Make sure that the mains supplies are connected to the ICU, the PC, the monitor and

any other ancilliary equipment.

WARNING

AVOID ELECTRICAL SURGES FROM OTHER EQUIPMENT, EITHER ATTACHED TO

THE SYSTEM OR IN THE VICINITY. IF POSSIBLE, USE A CLEAN ISOLATED

MAINS SUPPLY TO AVOID SURGES AND EARTH LOOPS.

IF YOU HAVE A CONVENTIONAL EPI-FLUORESCENCE LAMP ATTACHED TO THE

HOST MICROSCOPE, SWITCH THIS ON BEFORE ANYTHING ELSE.

2. Turn ON the laser mains breaker switch (A) on the rear of the Instrument Control Unit(Figure 6 Rear panel of ICU). If desired (and if this does not contravene localregulations), this mains breaker can be left turned ON, but it should be noted that mainsvoltage will be present in the Instrument Control Unit whenever the breaker is ON.


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Figure 6 Rear panel of ICU

3. Switch on the computer and monitor. The computer will boot directly into Windows NTand you will be presented with the desktop. An icon named LaserSharp2000 will befound on the desktop. Do not start the LaserSharp software until all steps in this sectionhave been completed. It is important that the ICU is not powered on when the PC bootsup because spurious serial commands are sent out during boot up which can corrupt theICUs firmware status.

4. Turn ON the mains switch (A) on the Instrument Control Unit switch panel (Figure 7 ICU

switch panel). The adjacent green LED power indicator (B) will then be lit.

A


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Figure 7 ICU switch panel

5. Check that the green LED power indicators on the scan head (A, Figure 8) and on theBCU (Figure 9 - A) are lit.

6. Turn the laser key switch (C) clockwise 90. This switch enables all three visible lasers.7. Press the button(s) (D, E & F) for the required visible laser(s).The Argon or Argon/Krypton laser has two associated LED indicators; the blue LED (G)indicates that the laser has been turned on and the orange LED (H) indicates that the

external cooling fan is running. As soon as the button is pressed an intermittent beeping,lasting approximately thirty seconds, will be heard from the ICU. When the beeping stopsthe Argon laser will be emitting. To reach full stability the Argon laser should be allowed torun for thirty minutes, but in practice image acquisition can commence immediately.The green Helium Neon laser and the red laser diode each have a single LED indicator toshow that the lasers have been turned on. There will be a brief delay of a few seconds (lessthan ten) before these lasers emit.

A

B

C

D

F

G

H

E


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Figure 8 Scan head LED indicators

Figure 9 BCU LED indicators

BA

A

B


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3.4 Starting the LaserSharp2000 software

To start the software double click the LaserSharp2000 icon:

The software will initially prompt you to Login. The system will be supplied with the Defaultusers password set to 1.

Once you have successfully logged in, the system will proceed with an initialisation phaseduring which firmware is downloaded to microprocessors distributed around the system andsystem communication protocols are checked.

3.5 Switching off the system

It is important to turn off the computer and the Instrument Control Unit in the prescribedmanner. Failure to shut down the computer properly may cause disruption to the operatingsystem. Failure to turn off the gas laser properly may cause its lifetime to be shortened.

1. Exit LaserSharp2000. Either use the single-click exit icon at the top right of theapplication or select Exit from the File menu.2. Shut down Windows NT. Select Shut down from the Start button.

3. Turn the laser keyswitch anti-clockwise 90. All lasers will immediately cease to emit.The Green Helium Neon laser and the red laser diode indicator LEDs will immediatelyextinguish. The blue gas laser indicator LED will immediately extinguish but the orangecooling fan running indicator LED will remain lit until the gas laser tube has beensufficiently cooled. Once the orange indicator LED is extinguished the mains switch onthe Instrument Control Unit switch panel can be switched OFF.

4. If desired, or if required by local regulations, the mains breaker on the rear of the

Instrument Control Panel can be switched OFF.5. Turn off the computer and monitor.


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3.6 A brief introduction to the software

The LaserSharp software is run under Windows NT4.0TM

operating system. It is a unifiedapplication which controls acquisition and provides functionality for processing and analysisof images and data. The following text should be read by al lusers before attempting to usethe system.

The user interface has been designed to be used with a minimum screen resolution of 1280 x1024 pixels. The main menu bar and tool bars will initially appear in the top left hand corneras below:

But they can be repositioned in almost any way desired by clicking the double vertical bar atthe left of each bar:


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On the raghthand side of the screen the instrument control panel will be displayed:

As can be seen, this panel controls the scanning, the detectors and the focus motor.


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One or more image display windows can be open at any one time:

Each image display window has its own display controls in the toolbar and an animationslider control below the image.

An experiment browser for the management of images is provided:

This will show all your open experiments. You will notice that within each experiment therecan be one Raw Data set, there could be several imported data sets, and for any of thosedata sets there can be further data resulting from operations upon them. All of this data isarranged in a hierarchy with just one experiment name e.g. C:\Experiments\Neurons.


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Control of the optical system set up is achieved through the Optics/Filter setup configurationdialog:

To show this dialog press the Optic button in the Microscope control panel:

Optic button

Mix button


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To display the Mixer control dialog press the Mix button.

The Mixer controls enable the additive combination of multiple detectors into one datachannel.

To show the Direct Detector setup dialog press the Direct Detectors button in the Opticspanel.


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Image processing and analysis functions are accessed by right clicking on the image to showa pop-up menu:


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4. FACTORS WHICH EFFECT OPTICAL PERFORMANCE

4.1 Objective lenses

There are five key properties to consider when choosing a lens for use on a confocal or multi-photon microscope:

Magnification

The magnification of the objective lens determines the maximum field of view.

Numerical aperture

The numerical aperture of an objective lens is the refractive index of the (immersion)medium times the sine of the semi-angle of the included cone. The latter means the angle tothe optical axis of the extreme rays, those which only just get into the lens.

The expression is normally written:

N.A. = n sin

The N.A. of a lens is a measure of both its light collecting and resolving capabilities.

As is well known, the performance of any imaging system is dependent on the numericalaperture (NA) of the objective lens - this particularly so for confocal imaging systems. Thelarger the numerical aperture, the better the spatial resolution of the system. A generally

accepted expression for the lateral resolution (Rlat ) in a confocal microscope given by:

RAlat

=0 61.

. .

in multi-photon mode the lateral resolution is given by:

2

1

..

61.0=

ANRlat

where is the wavelength of the light and N.A. is the numerical aperture of the lens.

At first sight it may appear that the lateral resolution in multi-photon mode is better than forconfocal, but one should remember that the multi-photon wavelength will be approximately 2times greater than that used in confocal (1-photon) excitation.

Note: The effective wavelength in fluorescence imaging is a function of the excitationwavelength and the emission wavelengths - a practical approach is to use the mid value ofthe emission band pass filter or the peak emission wavelength of the fluorophore.

So it is seen that the lateral resolution is inversely proportional to the N.A. and the axialresolution is inversely proportional to the square of the N.A.

Immersion medium


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If the lens immersion medium and the samples mountant have different refractive indices,the light will be brought to a focus at a point displaced from the expected position.

Medium Refractive Index

Air 1.00

Water 1.333Glycerol 1.473Oil 1.515 to 1.517Glass 1.525

The result of this unexpected refraction will be to distort the images aspect ratio whenviewed either in the XZ or YZ orientation. The distortion will be seen either as an elongationor a compression depending upon the refractive indices of the media.

The LaserSharp software applies a correction factor to the calibration of the Z-axis using analgorithm devloped by White et al described in the publication:

Aberration control in quantitative imaging of botanical specimens by

multidimensional fluorescence microscopyN.S. White, R.J. Errington, M.D. Fricker & J.L. Wood.Journal of Microscopy, Vol. 181, Pt 2, February 1996, pp. 99-116

The table below shows a selection of correction factors for various immersion media andmountant combinations for a few different N.A.s, but the algorithm works for all lenses andrefractive index values. It can be seen that for very low N.A.s the correction factor is closelyrelated to the ratio of the refractive idices of the mountant and the immersion medium (M/I),but that this changes somewhat with higher N.A.

It will also be noticed that when the immersion medium and the mountant have identicalrefractive indices the correction factor is unity. This is the ideal arrangement as mismatchedrefractive indices introduce spherical abberation which can adversely effect resolution and

sensitivity. This spherical aberration can also be seen in the point spread funcion of thesystem and is an important consideration where image deblurring (or deconvolution) isplanned.

N.A. of objective lens

Immersion Mountant Ratio M/I 0.2 0.5 0.75 1 1.4

Air Glycerol 1.473 1.49 1.6 1.685 1.685 -

Air Water 1.333 1.34 1.425 1.49 1.49 -

Equal 1 1 1 1 1 1

Oil Glycerol 0.972277 0.97 0.97 0.965 0.96 0.96

Glycerol Water 0.904956 0.905 0.89 0.87 0.855 0.86

Oil Water 0.879868 0.88 0.865 0.835 0.82 0.82

This correction factor is automatically written into the file notes and applied to the z-axiscalibration value. The correction factor can be viewed by selecting View|Notes to display thedialog box below:

Chromatic and field corrections

To achieve results with the highest possible spatial integrity planapochromatic lenses with ahigh numerical aperture should be used. These are highly chromatically corrected across thevisible spectrum, and have a very flat field. It should be remembered that in a confocalmicroscope chromatic correction between the illumination wavelength and the emissionwavelength is very important - if these two wavelengths are not brought to the same focus by


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the lens then there will be significant signal loss. Chromatic correction between differentemission wavelengths is also very important when imaging multiple labelled specimens -particularly if accurate 3D colocalisation measurements are to be made.

However, there may be applications where colour correction and flatness of f ield are not ofsuch great importance and in these cases lenses optimised for light throughput (e.g. Fluor or

Fluar) should be considered. The user should be aware that the highly corrected lensestypically contain many lens elements held together with optical cement - this inevitably leadsto transmission losses. The fluor/fluar type lenses are often very much simpler constructionswith accordingly higher throughput.

Transmission charactersitics of infra red light

To achieve deep penetration in scattering samples it is often necessary to deliver relativelyhigh power to the sample. This clearly makes it important that the objective lens transmitswell in the infra red part of the spectrum this is not true for many lenses.

Below is some transmission information for a limited number of objectives from different

manufacturers:

Nikon

CApo40x/WI, greater than 65% from 700 to 1000nm.

PlanApo60x/WI, greater than 60% from 700 to 1000nm.

PlanApo60x/oil, greater than 60% from 700-800nm, less than 50% from 900-1000nm.

Olympus

LUMPLFL40xIR-2, dipping lens, good to 1000nm, greater than 65%.

LUMPLFL60xIR-2, dipping lens, good to 1000nm, greater than 65%.

PlanApo40x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm.

UPlanfl100x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm.

PlanApo100x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm.

PlanApo60x/oil, greater than 70% to 800nm, between 30-40% between 900-1000nm.

Zeiss

PlanApo63x, linear drop from 80% at 700nm to 30% at 1000nm.

PlanApo100x, linear drop from 80% at 700nm to 30% at 1000nm.

CApo63x/WI, greater than 60% at 800nm, no information above.

CApo40x/WI, linear drop from 80% at 700nm to 40% at 1000nm.

Achroplan40x/WI, dipping, 90% at 700nm, no information above.

Currently, our strongest recommendations are for the following lenses:


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Nikon CApo40x/WI and PlanApo60x/WIOlympus LUMPLFLIR-2, 40x and 60x, dipping and PlanApo60x/oilZeiss CApo40x/WI

Summary of objective lens specifications

In summary, lenses should generally have the following properties:

High N.A./magnification ratio

Flat field (Plan)

Good chromatic correction (Achromat or Apochromat) for confocal imaging

High working distance/N.A. ratio

High transmission at required wavelengths particularly for IR illumination in MPimaging

4.1.1 CARE AND MAINTENANCE OF OBJECTIVE LENSES

It is certainly the case that the objective lenses on the microscope are the most vulnerableaccessible components on the system - they are also expensive!

The following points should be carefully borne in mind:

Microscope objective lenses should be kept dust and grease-free and should be kept in theirprotective cases when not in use.

Objective lenses often become accidentally contaminated with inappropriate immersion oil ormounting medium. Should this occur, refer to the manufactures literature or contact theobjective lens manufacturer for advice about cleaning with solvents. This is important,because the glue or mountant which holds the lens in place may dissolve in some solvents.Oil should be removed from oil immersion lenses after use. This is for two reasons:

1. Oil remaining on the lens can accumulate dust particles which reduce image quality.2. Oil can harden on to the objective lens after a while which also reduces the imaging

performance.

Once most of the oil has been removed with clean tissue, a piece of lens tissue should beplaced over the immersion end of the lens. A drop of recommended solvent should beadministered, and the tissue gently drawn across the lens surface. This should be repeated(with a clean piece of lens tissue each time) as often as is necessary to attain totalcleanliness. A magnifiying lens or a dissecting microscope is useful for assessing this.Coverslips on top of specimens should also be kept dust and grease-free. Water orcondensation should never be allowed to remain on the surface of the coverslip duringimaging. In general, ethanol or acetone can be used to clean the surface of a coverslip oncemost of the water or oil has been removed with a tissue. Care should be taken however toensure that the cleaning solvent does not contact any mounting medium or sealant whichmay dissolve in it. The lens tissue should be placed over the coverslip and a small drop ofsolvent placed on top. The lens tissue should be slowly drawn across the coverslip. Thisshould be repeated (with clean lens tissue each time) until the coverslip is clean.


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4.2 Confocal aperture size

Confocal aperture size is, of course, only relevant when using the system in confocal mode.

In multi-photon mode the confocal aperture should be opened to its full extent.

In common with the MRC series of instruments the Radiance systems employ macroscopic(large), continuously variable, circular apertures. The aperture should be set to Airy diskdiameter to achieve theoretically optimal sectioning performance.

To calculate the Airy disk diameters in the Radiance systems the following formula can beused:

Diametermag

N A

obj=

73 2.

. .

where is the wavelength (in fluorescence the mid value of the band pass emission filtershould be used)

magobj is the magnification of the objective

and N.A.is the numerical aperture of the objective lens

There is a button in the User Interface which ca be used to set the aperture to its optimal sizeas defined by the equation above. When an aperture is set to its optimal size the opticalsection thickness will be reported in the Z Info box.

Z Info box


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4.3 Telecentric adjustment

The Radiance systems are designed to work optimally with a wide range of differentmicroscopes and objective lenses. To achieve this a small degree of adjustment in therelative position of the eyepoint and the final scanning mirror in the system is provided.This variation in eyepoint position occurs in conventional microscopes but most users are

oblivious to the small change caused when low power objective lenses are used - we simplymove our head to the correct position relative to the eyepiece on the microscope. In theRadiance systems it is important that this adjustment is checked and corrected, if necessary,when using low power objective lenses (< 20X). Failure to make this adjustment can result inreduced field uniformity.

The system will have been set optimally for lenses of magnification 20 X and higher asdescribed in section 7.3. Before making any adjustment you should familiarise youself withthis procedure so that you are confident in your abili ty to return the system to its normalconfiguration.

To adjust the system specifically for a low power objective lens follow the steps below:

1. Select the chosen objective2. Image a uniform sample - for example a piece of fluorescent plastic3. Release the focus locking screw4. Rotate the focussing ring until the most even field illumination can be achieved5. Tighten the focus locking screw6. After imaging has been completed return the system to its normal configuration


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4.4 Visible laser considerations

The three visible lasers used in the Radiance systems have outputs as shown below.

Laser Wavelength & power

Argon ion 488 nm (14 mW) 514 nm (11 mW)Argon/Krypton 488 nm (5 mW) 568 nm (5 mW)Green HeNe 543 nm (1.5 mW)Red laser diode 638 nm (5 mW)

It is immediately obvious that there is less power in the green HeNe lasers single line thanthere is in any of the lines from the other lasers. For this reason it is quite normal to use amuch higher percentage of the available power from the HeNe laser.

One should always use the minimum possible power to minimise the illumination dose to thesample - in the case of fixed samples this will minimise bleaching and in the case of live cellsit will prolong their life.

4.5 Femtosecond pulsed laser considerations

The Radiance2000 MP can be configured with one of any of the following lasers:

Coherent/Microlase Bio Light DPM1000 1047 nm

Coherent Vitesse 800 nm

Coherent Vitesse XT 700-760 nm or 760-860 nm

Spectra Physics Mai Tai 750-850 nm

Spectra Physics Tsunami/Millenia 690-1050 nm

Coherent Mira/Verdi 690-1050 nm

As with lasers for confocal imaging, one should always use the minimum possible power toimage the sample. However, particularly when imaging deep (several hundred microns) itwill be necessary to use comparatively high laser powers.

It has been suggested by several researchers that there is often a well defined damagethreshold for a particular set of circumstances. This threshold will be heavily dependent uponthe sample and laser wavelength and will be found experimentally for different samples.


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4.6 Alignment of the Beam Conditioning Unit

The beam conditioning unit has four alignment screws that should be used in conjunctionwith the near-point and far-point alignment indicators to re-align the beam following tuning ofthe IR laser. The extent to which the beam needs to be re-aligned will be dependent uponthe particular laser being used.

To re-align the beam follow these steps.

1. Open the shutter to allow the IR beam to hit the alignment targets - turn the knob so thatit is aligned aong the Y axis of the BCU.

2. Adjust the near-point X and near-point Y adjusters so that the IR beam is centred in thenear-point indicator window.

3. Adjust the far-point X and far-point Y adjusters so that the IR beam is centred in the far-point indicator window.

4. Iterate around steps 2 and 3 until the beam is accurately centred in both indicatorwindows.

Y

X

Far-point X

Far-point Y

Near-point X

Near-point Y

Side view

Plan view

Shutter

Near pointindicator

Far pointindicator


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5. Close the shutter.

Following these simple steps it should be possible to quickly re-align the IR beam onto themicroscope axis. It is important that the beam is aligned on the microscope axis so that:

The intensity of the beam through the lens is maximised

The field illumination is even The MP image is co-aligned with the confocal image


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4.7 Filters

Filters for the Radiance2000 MP systems are fitted as shown in the following schematics:

4.7.1 FILTER CONFIGURATION FOR RADIANCE2000 MP AG-2

Dichroic 1

Pos 1 Plane mirror

Pos 2 440DCLPXR

Pos 3 500DCLPXR

Pos 4 560DCLPXR

Pos 5 Open window

Pos 6 -

Dichroic 2

Pos 1 Plane mirror Pos 2 560DCLPXR

Pos 3 650DCLPXR

Pos 4 Open window

Pos 5 -

Pos 6 -

Det 1 Emission filters Blocking filters

Pos 1 Open Open

Pos 2 HQ390/70 BGG22

Pos 3 HQ450/80 E625SP

Pos 4 D488/10 -

Pos 5 HQ500LP -

Pos 6 HQ515/30 -

Pos 7 HQ528/50 -

Pos 8 - -

Det 2

Pos 1 Open Open

Pos 2 E460LP E525/150

Pos 3 HQ515/30 HQ575/150

Pos 4 HQ528/50 E625SP

Pos 5 E570LP -

Pos 6 HQ590/70 -

Pos 7 HQ600/50 -

Pos 8 E600LP -


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4.7.2 FILTER CONFIGURATION FOR RADIANCE2000 MP K-2

Dichroic 1

Pos 1 Plane mirror

Pos 2 440DCLPXRPos 3 500DCLPXR

Pos 4 560DCLPXR

Pos 5 Open window

Pos 6 -

Dichroic 2

Pos 1 Plane mirror

Pos 2 560DCLPXR

Pos 3 650DCLPXR

Pos 4 Open window

Pos 5 -

Pos 6 -


Pos 1 Open Open


Pos 3 HQ450/80 E625SP

Pos 4 D488/10 -

Pos 5 HQ500LP -

Pos 6 HQ515/30 -

Pos 7 HQ528/50 -

Pos 8 - -

Det 2

Pos 1 Open Open

Pos 2 E460LP E525/150

Pos 3 HQ515/30 HQ575/150

Pos 4 HQ528/50 E625SP

Pos 5 E570LP -

Pos 6 HQ590/70 -

Pos 7 HQ600/50 -

Pos 8 E600LP -


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4.7.3 FILTER CONFIGURATION FOR RADIANCE2000 MP AGR-3

Dichroic 1

Pos 1 Plane mirror

Pos 2 440DCLPXR

Pos 3 500DCLPXR

Pos 4 560DCLPXR

Pos 5 Open window

Pos 6 -

Dichroic 2

Pos 1 Plane mirror

Pos 2 560DCLPXR

Pos 3 650DCLPXR

Pos 4 Open window

Pos 5 -

Pos 6 -


Pos 1 Open Open


Pos 3 HQ450/80 E625SP

Pos 4 D488/10 -

Pos 5 HQ500LP -

Pos 6 HQ515/30 -

Pos 7 HQ528/50 -

Pos 8 - -

Det 2Pos 1 Open Open

Pos 2 E460LP E525/150

Pos 3 HQ515/30 HQ575/150

Pos 4 HQ528/50 E625SP

Pos 5 E570LP -

Pos 6 HQ590/70 -

Pos 7 HQ600/50 -

Pos 8 E600LP -

Det 3

Pos 1 Open Open

Pos 2 E570LP HQ575/150Pos 3 HQ590/70 -

Pos 4 HQ600/50 -

Pos 5 E600LP -

Pos 6 HQ660LP -

Pos 7 - -

Pos 8 - -


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4.7.4 FILTER CONFIGURATION FOR RADIANCE2000 MP KR-3

Dichroic 1

Pos 1 Plane mirror

Pos 2 440DCLPXR

Pos 3 500DCLPXR

Pos 4 560DCLPXR

Pos 5 Open window

Pos 6 -

Dichroic 2

Pos 1 Plane mirror

Pos 2 560DCLPXR

Pos 3 650DCLPXR

Pos 4 Open window

Pos 5 -

Pos 6 -


Pos 1 Open Open


Pos 3 HQ450/80 E625SP

Pos 4 D488/10 -

Pos 5 HQ500LP -

Pos 6 HQ515/30 -

Pos 7 HQ528/50 -

Pos 8 - -

Det 2

Pos 1 Open Open

Pos 2 E460LP E525/150Pos 3 HQ515/30 HQ575/150

Pos 4 HQ528/50 E625SP

Pos 5 E570LP -

Pos 6 HQ590/70 -

Pos 7 HQ600/50 -

Pos 8 E600LP -

Det 3

Pos 1 Open Open

Pos 2 E570LP HQ575/150

Pos 3 HQ590/70 -

Pos 4 HQ600/50 -Pos 5 E600LP -

Pos 6 HQ660LP -

Pos 7 - -

Pos 8 - -


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4.7.5 FILTER CONFIGURATION FOR RADIANCE2000 MP AGR-3 (Q)

Dichroic 1

Pos 1 Plane mirror

Pos 2 440DCLPXR

Pos 3 500DCLPXR

Pos 4 560DCLPXR

Pos 5 Open window

Pos 6 -

Dichroic 2

Pos 1 Plane mirror

Pos 2 560DCLPXR

Pos 3 650DCLPXR

Pos 4 Open window

Pos 5 -

Pos 6 -


Pos 1 Open Open


Pos 3 HQ450/80 E625SP

Pos 4 HQ485/30 -

Pos 5 D488/10 -

Pos 6 HQ500LP -

Pos 7 HQ515/30 -

Pos 8 HQ528/50 -

Det 2

Pos 1 Open OpenPos 2 E460LP E525/150

Pos 3 HQ515/30 HQ575/150

Pos 4 HQ528/50 E625SP

Pos 5 HQ545/40 -

Pos 6 E570LP -

Pos 7 HQ590/70 -

Pos 8 HQ600/50 -

Det 3

Pos 1 Open Open

Pos 2 E570LP HQ575/150

Pos 3 HQ590/70 -Pos 4 HQ600/50 -

Pos 5 E600LP -

Pos 6 HQ660LP -

Pos 7 - -

Pos 8 - -


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4.7.6 INSERTING CUSTOM EMISSION FILTERS

The Radiance detector modules are designed so that users can insert their own emissionfilters. These standard size emission filters are available from Chroma and are simplyrefered to as 12.5 mm diameter filters. The filter thickness is 5.0 mm. These filters arenormally supplied housed in a metal f ilter ring which has the Chroma Part Number 10-12.5-

5.0.

There are two steps involved in inserting a custom emission filter :

Physical insertion of the filter

Update of firmware using a proprietary download utility

Warning: Unless you feel confident in both of these steps you should not attempt to insert,

remove or replace filters in the detector modules.

4.7.6.1 Physic al insert ion of f i l ters

The procedure for inserting a filter is dependent upon the number of fi lters already in place inthe filter wheel. If there are three or more filters (equivalent to four or more options in theEmission Filter drop down) follow Procedure 1 then the General Procedure, if there are two

or less filters f ilters (equivalent to three or less options in the Emission Filter drop down) thenfollow Procedure 2 then the General Procedure.

Procedure 1

Warning: Do not open the filter access cover whilst the system is running - this is likely tocause permanent damage to the PMT.

1. With the system running select the filter position according to the table below:

Number of filters currently in place

(Not including OPEN position)

Set filter wheel position to -

3 OPEN

4 Second position

5 Third position

6 Fourth position

2. This will have positioned the first free fi lter wheel space in line with the access port.

3. Turn the system off in the normal way.

4. Open the ICU door and using a 3.0 mm allen key (wrench) open the appropriate filteraccess cover by unscrewing (counter clockwise) the stainless steel screw marked A in thefigure below. The cover will slowly rise and then turn to reveal the filter wheel. Check thatthe filter position corresponds to the required position - the number is marked in white in theposition shown by the black spot B in the following figure.

Procedure 2

1. Turn off the system in the normal way.


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2. Open the ICU door and open the appropriate fi lter access cover by unscrewing (counterclockwise) the stainless steel screw marked A in the figure below. The cover will slowly riseand then turn to reveal the filter wheel.

3. Rotate the filter wheel to the required position (marked on the wheel at B) using a finetool, such as a small screwdriver blade, by pushing gently either clockwise or counterclockwise in the slot marked C in the f igure above.

Warning: The filter wheel is not free to rotate continuously. It can only move as follows:

5 6 7 8 1 2 3 4

Any attempt to move the filter wheel directly between position 4 and 5 may cause damage to

or misalignment of the filter wheel assembly.


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Page 46

General procedure

1. Firmly grasp the filter top-to-bottom with the tweezers at right angles to the face of the

filter using the tweezers supplied. The filter will probably be marked on its edge with anarrow - the filter must be inserted with this arrow pointing out from the system (towards you).If the filter is not marked with an arrow then its orientation is not important.

2. From above, push the filter into the vacant slot until it clicks firmly home. Check that thefilter is properly seated before closing the fi lter access cover by turning the screw Aclockwise until the cover is completely closed and light-tight.

4.7.6.2 Updatin g the firmwar e

Information to follow.


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4.8 Direct detectors

4.8.1 INTRODUCTION

Multi-photon excitation only occurs at the focal plane thereby generating optical sectioning

without the need for an emission aperture (pinhole) as used in confocal imaging. Thereforeas much of the emitted fluorescence as possible must be collected. Some of this emittedlight will travel directly back to the objective lens, but (increasingly so with depth) aproportion will be scattered on its return path. As it emerges from the back of the objectivelens the scattered light will not be in a parallel beam as shown below:

In order to collect as much of this scattered light as possible the direct detectors are designedwith a large collection lens placed close to the pick-off beam splitter (chromatic reflector).

Objective lens

Scattering event

Sample


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Direct detectors coupled to an Olympus BX50WI upright microscope

Direct detectors coupled to a Nikon TE300 inverted microscope


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4.8.2 OPTICAL CONFIGURATION

The 2 channel direct detectors have a very simple optical configuration. The emitted light issplit by a single fi lter cube and the light is sent into one or both of the PMTs.

The shortest wavelength light is reflected into PMT 1and the longer wavelength light passesthrough the long pass (LP) dichroic into PMT 2. The filter cube containing the dichroic andthe emission filters is a standard Olympus block. There are two different types of 2 channeldirect detectors one has two bi-alkali PMTs (BB) and the other has one bi-alkali and onemulti-alkali PMT.

PMT 1PMT 2

Emission 1

Emission 2


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4.8.3 FILTER CONFIGURATIONS

4.8.3.1 Bi-Alkal i /Bi-Alkal i Configurat ion (BB)

Olympus filter cubeEmission 1 Dichroic Emission 2

Blue/Green(DAPI/Fluorescein)

HQ450/80 DC500LP HQ515/30

UV/Visible(Serotonin/

Fluorescein)

UG11/IR UV400DCLP HQ575/150

UV(Serotonin)

Open Open UG11/IR

Indo-1 HQ390/70 440DCLPXR HQ495/20

4.8.3.2 Bi-Alkal i /Mult i -Alkal i Configurat ion (BM)

Olympus filter cube

Emission 1 Dichroic Emission 2

Blue/Green(DAPI/Fluorescein)

HQ450/80 DC500LP HQ515/30

Blue/Red(DAPI/Rhodamine)

HQ450/80 DC500LP HQ620/100

Green/Red(Fluorescein/

Rhodamine)

HQ515/30 DC560LP HQ620/100

UV/Visible

(Serotonin/Fluorescein)

UG11/IR UV400DCLP HQ575/150

UV

(Serotonin)

UG11/IR 670UVDCLP Open

Indo-1 HQ390/70 440DCLPXR HQ495/20


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4.8.4 SELECTING FILTER CUBES

To select a f ilter cube the following procedure should be used:

a. Remove the filter carousel wheel coverb. Rotate the carousel wheel to the desired filter position.

c. Replace the filter carousel wheel cover.

4.8.5 CHANGING FILTER CUBES

To change a filter cube the following procedure should be used:

a. Remove the filter carousel coverb. Slide the window to the left to reveal the filter blocksc. Loosen locking screw (maximum 2 turns) and slide the filter block toward you and out of

the unit.d. Insert the new filter block (check correctly orientated) and secure locking screw.e. Slide back window and replace cover.

Filter carousel wheel coverFilter carousel wheel


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4.8.6 USING THE DIRECT DETECTORS

4.8.6.1 Niko n E600FN

a. Depending on filters supplied, there will be a number of filter options entered into the

software.

b. To enter further filter options go to the Control Panel ToolBox - Optic Filter Setup andselect Direct Detectors to reveal the Carousel Setup:


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c. Under Filter Blocks, click on Add to enter a new filter block name (i.e. green/red). Pleasenote that the filter selection in the direct detector unit must be visibly verified.

d. Under Filter Block Components, select Emission filter A, click on add and enter the filter(i.e. HG515/30). Note that the position of the filter is displayed on the adjacent diagram.Repeat with the Dichroic and Emission filter B. Please note that Blocking fi lter are nowincluded in the emission f ilters and do not need to be added.

e. Drag and drop the filter block diagram in Filter Blocks into the desired position in theoptical diagram. (Please note that manually changing the position of the fi lters blocks inthe unit will therefore invalidate the optical layout).

f. Create a method with direct detectors selected. From the Optic Configuration diagram,click on which direct detectors are required. The fi lter block selection entered here will besaved in the method which is recorded in the experiment properties.

g. Select filter position 4 on the microscope epislider unit to divert the fluorescence from thesample to the direct detectors:


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The Epi-slider positions are as follows:

1 DDS - Direct Detectors (multi-photon only), 670UVDCLP2 INT - Internal Detectors (multi-photon, confocal and transmission), glass window3 EF1 - Fluorescence cube4 EF2 - Fluorescence cube

1 3 4

Filter cubepositions

Aperture to scanhead and objectivelens

21

Position 4


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Page 55

4.8.6.2 Olym pus BX50WI

a f. As described above for Nikon E600FN

h. Select filter position 4 on the microscope epislider carousel unit to divert the fluorescencefrom the sample to the direct detectors.

1 EF1 - Fluorescence cube2 EF2 - Fluorescence cube3 INT - Internal Detectors (multi-photon, confocal and transmission), glass window4 DDS - Direct Detectors (multi-photon only), 670UVDCLP

4

1

2

3

To Direct Detectors


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Page 56

4.8.6.3 Niko n TE300

a f. As described above for Nikon E600FN

g. Select filter position 4 on the microscope epislider unit to divert the fluorescence from thesample to the direct detectors.

Position 4 for directdetectors


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4.9 Fluorophores

The selection of appropriate fluorophores is clearly very important - the use of aninappropriate fluorophore, or combination of fluorophores, could seriously impair key imagingperformance measures such as sensitivity and specificity.

The choice of fluorophores for 1-photon imaging and multi-photon imaging should beconsidered separately.

4.9.1 FLUOROPHORES EXCITED BY 1-PHOTON EXCITATION

To judge whether a particular fluorophore is suitable for use with the system is fairlystraightforward - one simply looks at excitation curve of the fluorophore. These are usuallypublished by the manufacturers. If the peak of the excitation curve is close to one of theavailable laser lines then it should be possible to image it successfully - however, even if thepeak is not close to an available line then it may stil l be possible to image the fluorophorewell. This is particularly true when the laser line falls below the excitation peak - as in thecase of Cy3.

It can be seen that the peak excitation occurs at 554 nm, but that 15% excitation is achieved

at 488nm, 49% 514 nm and 73% at 543 nm. It is important to remember that the excitationwavelength affects the amount of emitted light which can be collected; if the excitation peakand the emission peaks are close together then using a laser line very close to (or at a longerwavelength than) the peak excitation wavelength will result in loss of emitted signal becausein blocking the reflected laser light the dichroic amd emission filters will necessarily cut offpart of the emission.

So, for single labeling one should choose a fluorophore with its excitation peak close to butpreferably slightly longer than an available laser line.

For double or triple labeled preparations there are slightly more complex considerationswhich are dependent upon the configuration of the system, the required data acquisition rateand the intended use of the resultant data.

For the best separation of the dif ferent fluorophores (e.g. FITC Cy3 as shown below) oneshould collect the data sequentially.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650 660 670 680 690

Wavelength (nm)

Excitation

Emission

Laser lines


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This approach ensures that there is almost negligible excitation of the green emittingfluorophore (FITC) when the red emitter (Cy3) is being excited at 543 nm - so even thoughFITC has an extended emission spectrum there will be v irtually no bleed through into the reddetector.

In a multiple detector system it is possible to acquire two or three channels simultaneously.However, it is important to remember that sequential acquisition will stil l generate data inwhich the two channels are most clearly separated.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600

Wavelength (nm)

CY3 Ex

FITC Ex

Lines


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The graph above clearly shows that although the green channel (515/30) exclusively detectssignal from the emission of FITC the red channel (570LP) detects signal from Cy3 and asignificant amount of signal from FITC. However, it should be noted that illuminationintensity balancing with the AOTF can reduce bleedthrough very significantly.

This bleed through can also be reduced by using Cy2 or Alexa 488 which both have shorter

emission tails than does FITC.

The following table gives the excitation and emission maxima for a wide range offluorochromes and probes which might be used with the Radiance2000:

Green emitting fluorochromes

and stains

Excitation

maximum

Emission

maximum

Acridine Orange (DNA) 487 520

BOBO-1 462 481

Bodipy-fl 505 513

CY2 489 506

Dabsyl chloride 335 536

DiOC6(3) 484 501DiOC7(3) 482 504

DTAF 493 518

ELF-97 345 530

Eosin 524 544

Eosin F3S 535 542

FITC 494 518

GFP 395/470 509

Hydroxystilbamidine (fluorogold) 385 536

IANBD amide 478 541

Lucifer yellow 428 533

MitoFluor green 489 517

MitoTracker green 490 516

Oregon green 488 493 520



Rhodamine green 502 527

Rhodol green 499 525

SYTO 16 488 518

TOPRO-1 515 531

TOTO-1 514 533

YOPRO-1 491 509

YOYO-1 491 509

Green emitting fluorescent probes

BCECF 439/490 530

Bilirubin 452 525

Bodipy FL brefeldin A 503 510

Calcein 494 517

Calcium green 506 533

Carboxyfluorescein 492 517

CellTracker Green Bodipy 522 529

CFDA 494 517

Cl-NERF 518 544

DiOC5(3) 484 500

DiOC16(3) 484 501

DM-NERF 515 542

Fluo-3 506 526

5 hexadecanoyl fluorescein 497 519

JC-1 (low) 514 529/590


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LysoSensor Green DND-153 442 505

LysoTracker green DND-26 504 511

NBD-C6 ceramide 466 536

Rhodamine -123 505 533

Stachyose fluorescein 491 516

Orange emitting fluorochromes

and stains

Bodipy R6G 528 550

Bodipy 530/550 534 554

Bodipy TMR 542 574

Bodipy 558/568 558 569

Carboxyrhodamine 6G 520 546

CY3 550 565

DiA 491 590

Dichloro-dimethoxyfluorescein 522 550

DiI 547 565

JOE 525 555

B-Phycoerythrin 546/565 575

R-Phycoerythrin 480/546/565 578POPO-3 534 570

POPRO-3 539 567

Rhodamine 6G 525 555

SYTO 25 521 556

TAMRA (carboxytetramethylrhodamine)

TRITC 544 572

Orange emitting fluorescent probes

Calcium orange

DiC18(3) 549 565

DiC16(3) 549 565

LysoTracker Yellow DND-68 534 551

MitoTracker Orange 551 576Octadecyl Rhodamine B 556 578

Rhodamine B Hexyl ester 556 578

SNARF (Acid) 490 580

Tetramethylrosamine 550 574

Red emitting fluorochromes and stains

BoBo-3 570 604

Bodipy 576/589 576 590

Bodipy 589/617 589 617

BOPRO-3 575 599

Chromomycin A3 458 590

Cy3.5 581 596

Dihydroethidium 518 605DIOC2(5) 579 601

DiQ 562 >= 600

Ethidium Bromide 510 595

Ethidium homodimer-2 528 617

Hexidium iodide 518 600

Lissamine rhodamine B 570 590

MitoTracker red 578 599

Propidium iodide 536 617

Radiant redTM 500 610

Rhodamine Red 570 590

Texas red 587 602

Texas red-X succinimidyl ester 583 603XRITC 572 596


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Red emitting fluorescent probes

Bodipy TR ceramide 589 617

DASPMI 475 605

DiA 491 590

DiD 644 665

DiQ 562 681

FM1-43 510 626

LysoTRacker Red DND-99 577 590

SNARF (Base) 490 630

Sulforhodamine 101 586 605

Sulforhodamine B 565 586

Sulforhodamine G 529 548

Sulfonerhodamine Bis-(PEG 2000) 581

Far red emitting fluorochromes and

stains

Acridine orange (RNA) 487 650

Allophycocyanin 650 661

7-aminoactinomycin D 555 655

Bodipy 630/650 630 650

CY5 650 670

LDS 751 543 712

Napthofluorescein 598 668

TOPRO-3 642 661

TOTO-3 642 660

Far red emitting fluorescent probes

Fura red 436/500 640

Napthofluorescein 605 675

RH-414 532 716

4.9.2 FLUOROPHORES EXCITED BY 2-PHOTON EXCITATION


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4.10 Dynamic range

In order to correctly set the dynamic range it is helpful to understand a little about signaldigitisation in the Radiance systems.

Light detection is achieved when one or more photons strike the photocathode of the

photomultiplier giving rise to photoelectrons. The resulting current is converted to a voltage,amplified and fed to an analog-to-digital converter (A/D), where it is sampled and thenconverted to a digital number (see figure below).

In order to realise the full 12 bit resolution of the ADC it is important to set the gain and offsetof the detectors correctly. To achieve this reliably a false colour look up table (LUT) calledsetcol.lut is supplied and can be quickly loaded from the pop-up menu. ThisLUT sets thelowest few grey levels to green and highest few grey levels to red. In direct scanning modethe gain and offset (black level) should be set so that a few green and few red pixels arevisible in the image. When a frame averaging filter is invoked (e.g. Kalman) you willprobably notice that the green and red pixels disappear or reduce in number (due to theimprovement in S/N) - this is to be expected and will result in an image with a perfectdynamic range.


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4.11 Monitor adjustment

Clearly, the adjustment of the monitor doesnt have a direct effect upon the quality of theimages one collects. However, one should be aware that the monitor settings together withthe ambient lighting can influence your judgement of the dynamic range in the image (if oneis not using the setcol LUT). If you are working in a darkened room then it is very easy to

misjudge the dynamic range in the image and to consequently collect images in which thebrightest pixels fall far below a grey level of 255.


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4.12 Sampling resolution

One must consider sampling resolution both in the XY plane and the Z axis.

XY Plane

Ideally (according to Nyquist) one should strive to set the pixel size to be half of the opticallateral resolution. This resolution is given by:

RA

lat =0 61.

. .

as discussed in section 4.1.

However, one should be aware that this may well result in the collection of very large files sothe sampling resolution could be reduced so that the pixel size is equivalent to the opticalresolution.

Z axis

Again, the ideal sampling resolution in the Z axis (the Z step) should be half the axialresolution which is given by:

( ) ( )( )

Rn n N A n

ax = =

2

2

2

21

sin sin sin . .

as discussed in section 4.1.


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4.13 Signal strength and noise

It is well understood that that in confocal f luorescence imaging the range of light levels l ikelyto be encountered will result in a photon flux of between 10 and 1,000 photons per pixel at apixel dwell time of 2 microseconds. (Reference: The Pixelated Image, R. H. Webb & C. K.Dorey - Chapter 4, Handbook of Biological Confocal Microscopy second edition - Ed. J

Pawley) This range extends from extremely low signal levels in live cell applications throughto the brightest possible fluorescence. For the purposes of the following discussion a flux of200 photons per second, relating to bright immunofluorescence, is used.

Poisson statistics tells us that the signal to noise ratio is equal to the square root of thenumber of detected photons. So, for a signal of 200 photons the noise will be 14 photons or7% of the total signal.

An 8 bit ADC has a digitisation resolution of 1/255 or < 0.4% so it is immediately obvious thatit has much more than sufficient resolution to faithfully sample signals from typicalfluorescently labelled specimens. Indeed, it is worth noting that one would need to detectmore than 65,000 photons per pixel to fully util ise the sampling resolution of an 8 bit ADC.

It is clear from the figures above that most confocal fluorescence images will require adegree of averaging to improve the S/N level to an acceptable level. This can be achievedeither by scanning more slowly (intra-pixel averaging) or by frame averaging (e.g. kalmanfilter). The slow scan speed on the Radiance2000 increases the pixel dwell time by a factorof three and will increase the S/N by approximately 1.7 (square root of 3). The frameaveraging filters offer a very flexible method of increasing the S/N until it is judged to besatisfactory. Again, the S/N will improve proportionally to the number of photons detected (orframes scanned). Caution should be taken when averaging for a large number of frames (>10) that the sample is not photo-bleaching as this will result in the S/N rising to a peak valueand then falling off with further scanning.


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5. SAMPLE PREPARATION SUGGESTIONS FOR CONFOCAL AND MUTI-PHOTON

IMAGING

5.1 Fixatives for biological tissue

There are basically two types of fixative:

The cross-linking fixatives such as paraformaldehyde and Glutaraldehyde are normally usedto preserve morphological structure and localisation of antigenic sites. However, they renderthe cells/tissues fairly Impermeable to fluorescent antibody molecules, and detergents suchas Triton X or DMSO are required to make the membranes more permeable.

The second type of f ixative is known as 'precipitating', and includes the methanol/acetic acidand acetone groups of compounds. Whilst these render the tissue permeable to antibodies,they will precipitate the proteins, thus endangering the retention of antigentic site localisation.

Sometimes a crosslinking fixative is applied first, in order to preserve the structure, and aprecipitating fixative used afterwards to render the membranes permeable prior to staining.

Note that Glutaraldehyde is highly f luorescent and may obscure fluorochrome staining. Theuse of NaBH4 can quench this fluorescence if required.

5.2 Bleaching and anti-fade agents

Light-induced damage to f luorochromes can occur largely (though not entirely) due to thepresence of molecular oxygen. The total extent of bleaching of the fluorochrome is ingeneral less than in conventional microscopy. However the addition of an anti-fade agent

(antioxidant) to the mounting medium appears to be even more important for laser scanningfluoroscence than for conventional f luorescence.

Various anti-fade reagents are available (e.g. p-phenylene-diamine, DABCO [Diazabicy clo-octane], Propylgallate Hydroquinone and Citif luor), but the most recently introduced reagentis called Fluoroguard

TM.

FluoroguardTM

is the first of many fluorescence-based reagents from Bio-Rad.

Quantitative results show that Fluoroguard reduces photo-bleaching in an aqueous sodium

fluorescein solution by greater than 95% i.e. it prolongs fluorescence by >20 fold and reduces

photo-bleaching in a solution of DAPI stained DNA by greater than 85% i.e. prolongs

fluorescence by greater than 6 fold compared to identical solutions substituting buffer foranti-fade. Rhodamine derivatives Texas Red, AMCA, propidium iodide and other dyes

appear to be protected from photo-bleaching.

Further information can be received by requesting Bulletin 2047 from your local Bio-Radrepresentative.

5.3 Mounting media

Canada Balsam and certain epoxy resins fluoresce strongly. This defect may be put toadvantage in confocal imaging of non-fluorescent specimens such as silicaceous shells and

mineral grains, which appear in negative contrast. The same technique can be used inmeasurement of the thickness of live cells. Surounding the cells in a weakly fluorescentmedium permits x-z sectioning through the cell thickness with the cell section appearing dark


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against a bright background. Fluorochromes attached to large molcules like dextran are veryuseful for this purpose.

In the vast majority of situations, a non-fluorescing mounting medium is desirable. Thesemay range from glycerol based media to PVA based media. The type of medium used willlargely depend on whether the sample is aqueous or solvent based following fixation/clearing

etc.

Fluid mounts which are made with anti-fade solutions should be sealed with, for example,nail varnish. Media which solidify seem to have poor anti-fade properties, but are convenientto use with specimens resistant to photo-bleaching, e.g. those stained with acridine orange orLucifer Yellow. Non-fluorescent media are available, e.g. Fluoromount (British Drug Houses)or Citifluor (Agar Scientific Ltd.)

Using anti-fade with living specimens is much more of a problem because of theconcommitant removal of oxygen. However, attempts are being made to use vitaminderivatives as live cell anti-fade agents. The success of these remains to be seen.

5.4 Autofluorescence

Autofluorescence can be useful for some forms of imaging e.g. the 488 nm line excites s

Radiance Mp

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