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Page 1: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

Microscopy and Laser Imaging

602

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

www.thorlabs.com

Video-Rate Confocal Camera

Confocal Camera Features ■ Confocal Imaging at Video Rate■ Attaches to Camera Ports of Lab Microscopes■ Diffraction-Limited Resolution Over a Large Field of View■ Compatible with Thorlabs’

Cage and Lens Tube Systems■ Fiber or Free-Space Coupled Input

and Output■ Numerous Filter and Detector

Configurations; FluorescenceImaging Ready

■ Cost Effective Confocal ImagingSolution

Thorlabs is pleased to announce the Video-Rate Confocal CameraSystem. This is a full-featured confocal microscopy system, yet theoptical head is packaged in a compact ready-to-use module thatconnects directly to the camera port of any standard microscope.

This powerfulcombination ofproven Thorlabsimaging technologyenables a trueconfocal solution at afraction of the cost ofcompeting systems.

Thorlabs’ Confocal Camera offers real-time confocal imaging ina customizable open platform. The fiber coupled design of thissystem ensures a spatially filtered input beam, which is essentiallya perfect Gaussian. In addition, the two single mode fibers thatare used to deliver the illuminating light as well as collect thebackscattered signal replace the pinhole that is used in traditionalconfocal systems. The confocal arrangement of the fiber positionrejects out-of-focus light thus creating a true confocal image.All the necessary components such as a laser diode source,photodetector, associated controlelectronics, computer, and softwareare included.

Confocal Camera SpecificationsImaging:■ Resolution:1 1 µm■ Frame Rate: 23 fps @ 800 (X) x 640 (Y) Pixels

(2-Channel Detection)■ 3D Acquisition:2 800 x 640 x 256 (X, Y, Z) Pixels for 11 sElectronics:■ Analog Input: 2 Channels, 14 Bit, 100 MS/s*■ Analog Output: 4 Channels, 12 Bit, 1 MS/s*, ±10 V■ Digital I/O: 8 PortsOptical:3

■ Output Power: ~2 mW■ Beam Diameter: ~ 4.5 mm

Hardware:■ CPU:

Intel® Processor■ Memory: 2 GB Memory■ Operating System: Windows® XP Professional, SP2■ Hard Drive: 250 GB SATA■ Optical Drive: 16X DVD ± RW■ Monitor: 19" LCD 1280 x 10241 Resolution specified using a RMS40X objective and 660 nm diode. Actual resolution will

vary with objective used.2 When a piezo stage is used.3 Output power and beam diameter measured at exit port of VCM101H with no objective using

a 660 nm laser diode light source (included).

*MS/s= Megasamples per Second

(5.94")150.8 mm

(3.11")79.1 mm

(26.32")668.6 mm

(1.14")29.1 mm

(7.38")187.3 mm

(11.71")297.3 mm

(2.86")72.7 mm

VCM101HVideo-Rate Confocal Camera Schematic

ITEM# $ £ € RMB DESCRIPTION

VCM101H CALL CALL CALL CALL Video-Rate Laser Scanning Confocal Camera

Thorlabs’ newVCM101H Video-RateConfocal CameraMounted on an OlympusBX41 Microscope.

Page 2: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

Microscopy and Laser Imaging

603www.thorlabs.com

Combined OCT and Confocal Microscope Solution:Multi-Modality Imaging on a Single Platform

OCT and Confocal SynergiesShared Strengths: Video Rates

In-Vivo Imaging3D Imaging

Complementary Strengths: OCT: ■ Large Field of View■ Greater Depth Scanning Profiles

Confocal Imaging:■ Very High Resolution (Submicron)■ Subcellular Imaging Capabilities

The substantial line of OCT systems and solutions offered byThorlabs demonstrates our commitment to developing OCT-basedsolutions to challenging imaging problems (see pages 2-15). In anongoing effort to make this technology widely available, Thorlabshas recently developed an OCT Microscope Module that can beeasily integrated (as an add-on component) with laboratoryresearch microscopes (see page 9). That module is shown heremounted between the eyepiece and the objective turret of anOlympus BX41 microscope. For even greater versatility, this newsystem can be combined with the Thorlabs VCM101H ConfocalCamera. By simply sliding a channel selector bar (See Fig. 1), thismicroscope system can switch among OCT, confocal, and standardmicroscopy configurations.

Never before has such a powerful tool been combined soeffortlessly into a commercially available laboratory microscope.

OCT and confocal imaging are ideally suited to be combinedinto a single imaging system. Each imaging method brings uniquestrengths to challenging imaging applications. Since they sharea number of features, it is possible for the two methods to worktogether seamlessly. Each technique has the ability to image atvideo rates, to test in-vivo sampling, and to obtain 3D images;these similarities allow the two methods to be used together to takeadvantage of their unique strengths. OCT systems allow for large-depth probing into samples and create images characterized bylarge fields of view (FOV). In particular, swept source OCTsystems have a FOV up to 10 mm and can probe sample depths

to 3 mm. In contrast, confocal imaging isbeneficial because it produces very highresolution (submicron) images even thoughthe FOV for an image is typically ~1 µm.By combining these two techniques, it ispossible to obtain wide fields of view andresolutions capable of probing insidebiological cells.

Fig. 1 Swept Source OCT Microscope Module

Combined OCT and Confocal Microscope■ Thorlabs VCM101H Confocal Camera■ Thorlabs Swept Source OCT Microscope Probe■ Olympus BX41 Microscope

Features■ Enables Full-Featured High-Speed

and High-Resolution 3D OCTImaging for Research Microscopes

■ Compact Design for an IntegratedSolution

■ Multiple Imaging ModalitiesCombined into One Microscope

■ Easy Switching Among Modalitiesfor Imaging the Same Sample

■ Highly Reconfigurable Design■ Supports Future Integration of

Multiphoton and FluorescenceImaging Channels

Page 3: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

Microscopy and Laser Imaging

604

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

www.thorlabs.com

Reflection Confocal Imaging Using the VCM101

Microelectronics

Confocal scattering images of a memory chip taken withVCM101H using a 660 nm incoming light source.The backscattered light is coupled into the single modefiber and then detected with a photodiode.

75 µm55 µm

20 µm

2.

1. Confocal scattering image ofa memory chip with XZ and YZcross-sectional images taken witha 60X objective. The total imagesize is 75 x 55 µm.

2. Confocal scattering image of amemory chip taken using a 100Xobjective with 3D projection. Eachmicrocircuit is 2.5 µm wide. Totalimage size is 55 x 35 µm.

3. Reconstructed 3D projectionmodel of the confocal backscatteringsignal from a portion of the circuit

of a microchip, obtained using a 100X objective.

Figure 1

+200

-20

1.

The VCM101H is a highly versatile imaging system. The standard reflection mode allows surface imaging of reflective opaque material formicroelectronics, material science, and surface studies (see images below) and can also provide optical slicing of semitransparent scatteringsamples, as demonstrated by the image to the right.

Pseudo-color 3D projection and cross-sectional confocal scattering image of a greenleaf obtained with VCM101H using a 60Xobjective. The image was taken using a fibercoupled 660 nm laser diode. (250 x 185 µm,Z-depth is 80 µm)

250 µm

185 µm

80 µm

Leaf

3.

110 µm

150 µm

Page 4: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

Microscopy and Laser Imaging

605www.thorlabs.com

Confocal Fluorescence Imaging Using the VCM101

(optional)

Figure 3 shows theVCM101H reconfiguredfor fluorescence imaging.By changing thebeamsplitter to a dichroicfilter in the standardVCM101H setup andadding the appropriateexcitation and emissionfilters, the system becomesa powerful fluorescenceimaging tool. In mostsituations, a PMT shouldalso replace the fastphotodetector.

Figure 3

Peach WormThese images represent pseudo-colorconfocal fluorescence images of a peachworm taken with the VCM101H using a60X water immersion objective. A total of256 Z-slices (0.3 µm step-size) were used tocreate the pseudo-3D projection shownbelow. The total 80 µm Z-scan isrepresented by the selection of data imagesshown in Figs. 1 – 8, which are individualZ-slices at 10 µm increments. Eachindividual image is 440 µm x 430 µm.

1

7

65

43

2

8

The true power of confocal laser scanning microscopy is most often demonstratedin fluorescent samples where the confocal technique spatially separates the desiredfluorescent signal from the out-of-focus background fluorescence, allowing opticalsectioning along the Z-plane. Combining this with image processing software allowscross-sectional imaging, 2D projections, and pseudo-3D rendering of the opticallysectioned sample as demonstrated by the data taken with a VCM101H that hasbeen reconfigured for fluorescence collection (See Fig. 3).

The figures shown below are pseudo-color 2D projections and 3D confocal fluorescent images ofpollen taken with VCM101H using a 60X objective. Pollen grains were mounted on a standardmicroscope slide and excited with 405 nm light from a laser diode (DL5146-152). The emission signalwas selected using a dichroic mirror (FD05D) with a cutoff wavelength of 505 ±15 nm. The signal wascollected through a single mode fiber (P1-460A-FC-5) and directed to a PMT for detection.

(Image size: 150 µm x 110 µm,Z-scan depth: 80 µm)

Pollen

The VCM101H is Easily Reconfigured

for Florescence Imaging

Page 5: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

Microscopy and Laser Imaging

606

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

www.thorlabs.com

Confocal Fluorescence Imaging Using the VCM101

Multichannel Fluorescence Imaging Using the VCM101

Human Skin and Sweat Gland Rabbit ArteryThis image shows the top view projection of a slice of rabbitartery mounted on a standard microscope side. Here, apsuedo-color confocal fluorescent image was taken with theVCM101H in fluorescence mode using a 60X infinity-corrected objective. The total image size is 250 µm x 210 µm.

Plant SectioningBelow is a pseudo-3D image of an optically sliced fluorescent plant sample, excited at 405 nm. Channel 1 (shown in red)was collected from 505 nm to 555 nm, while channel 2 (shown in green) represents light collected from 569 nm to 600 nm.

VCM101H withMultichannel ConfigurationFigure 4

For most fluorescence applications, multiple dyesare used to distinguish visually and chemicallyspecific cell structures or metabolic processes.Whether the protocol uses a combination ofexternal fluorophores like DAPI, Alexa Fluor 488,and Rhodamine or multiple fluorescent proteins,it is useful to have mulitchannel detection, andthe VCM101H can easily be reconfigured to meetexperimental requirements. A VCM101H wasreconfigured as depicted in the schematic layoutshown in Fig. 4. This system was used to obtainthe two-color images shown below. Although thisexample utilizes two-channel detection, simplyadding a third PMT allows the VCM101H toproduce three-color images, making this systemcomparable to a large commercial confocalfluorescent microscopy system but with a smallerfootprint and a fraction of the cost.

This figure shows a view of a sample slice of human skin andsweat gland that was mounted on a standard microscope slideand observed with the VCM101H using an infinity-corrected60X water immersion objective. Here, a psuedo-color projectionwas created from a series of individual Z-slices.

Page 6: Microscopy and Laser Imaging Video-Rate Confocal Camera · 2008. 7. 14. · Microscopy and Laser Imaging 604 ASOM Spectral Radar OCT Swept Source OCT Video-Rate Laser Scanning Microscope

ASOM

Spectral Radar OCT

Swept Source OCT

Video-Rate LaserScanning Microscope

Swept Source Lasers

OCT Components

LaserMicroscopy Optics

Microscopy Tools

Microscopy and Laser Imaging

607www.thorlabs.com

Many new techniques have been developed toenhance imaging contrast and biological andchemical specificity. With the advent of turnkeyultrafast lasers, nonlinear imaging methods suchas multiphoton fluorescence imaging (e.g. 2P or3P Microscopy), second harmonic imaging(SHG), and resonant Raman techniques (e.g.CARS) are increasingly being adopted bymicroscopists to expand their biochemicalunderstanding of natural processes. Often, morethan one of these techniques is usedsimultaneously, which necessitates a customsystem or a modification to a standard confocallaser scanning microscope. As is the case withother Thorlabs’ products, we have designed theVCM101H to provide you with the tools neededto create your own photonics solutions.

The reconfigurable VCM101H module is ideallysuited for nonlinear microscopy setups. In manycases, it is cheaper and more convenient toconvert an existing confocal laser scanningmicroscope system into a multiphoton systemrather than purchasing a commercial version; in particular, for SHG and CARS applications, a customized system is the only solutiondue to the lack of industry options. In the VCM101H, internal tap holes are provided for changing optical components, and thecompact footprint allows it to be adapted to any available microscope port with minimal mounting hardware.

Multiphoton Fluorescence MicroscopyMultiphoton microscopy combines fluorescent dyes with an ultrafast laserto allow fluorescence imaging using less damaging IR radiation. With theincreased penetration depth and the inherently localized nature of theultrafast pulse, multiphoton microscopy provides noninvasive sub-cellularimaging of living systems. The VCM101H can be reconfigured formultiphoton microscopy or SHG imaging simply by replacing the singlemode fiber before the detector with the appropriate bandpass and emissionsfilters, as depicted in Fig. 5. This setup was used to image a slice of ratkidney using the VCM101H, which was reconfigured for two photonfluorescence imaging using a TC780-150 femtosecond laser (MenloGmbH) for excitation.

Like mulitiphoton microscopy, SHG uses an ultrafast laser for signalgeneration, but in this case, no fluorophore is used; instead, the signal isgenerated from noncentrosymmetric features within the sample, making thistechnique advantageous in many material science applications. In addition,SHG is increasingly being used in biological applications, includingmammalian studies. For example, the large SHG signal created by myelinand collagen yields excellent cellular structure determination without theneed for either an intrinsic or externally applied fluorophore.

The VCM101H can also be used for SHG imaging when reconfigured withan optical configuration similar to that shown in Fig. 5. The only differenceis that detection of the SHG signal requires that the filters placed in frontof the detector be chosen so as to select light at half the excitationwavelength. (Note: It is important to use an appropriately high NAobjective for collection of the SHG signal since the signal is not ubiquitousbut instead is generated only at discrete angles.)

31 2

6

4

5 7 8

Emission Filter, Laser Blocking 750-1000 nm

Dichroic Filter, 750 nm Long Pass

Focus Lens

Figure 5

Nonlinear Imaging Using the VCM101