Real-time Video-rate Laser Imaging

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Real-Time Video-Rate Laser Imaging www.thorlabs.com Real-Time Video-Rate Laser Imaging Video-Rate Confocal Camera Confocal Camera Features ■ ■ ■ ■ Confocal Imaging at Video Rates Mounts onto Camera Port of Optical Microscope Diffraction-Limited Resolution Over a Large Field of View Convenient Access to Optics ■ ■ ■ Compatible with Thorlabs' Lens Tube and Cage Systems Fiber or Free-Space Coupled Input and Output Numerous Filter and Detector Configurations Thorlabs’ VCM405 Video-Rate Confocal Camera Head Shown on an Olympus BX41 Microscope Confocal scanning optical microscopy is a high-resolution optical imaging technique that has recently gained wide-spread popularity in the industrial and scientific communities. Unlike wide-field fluorescence microscopy, 3D images are reconstructed on a point-by-point basis, leading to higher resolution and the ability to produce infocus images of relatively thick samples. Thorlabs offers full-featured, video-rate Confocal Camera Systems with laser diode sources centered at 405 nm, 635 nm, or both wavelengths. In either case, you receive the optical head, which is packaged in a compact ready-to-use module that connects directly to the camera port of any standard microscope via the appropriate microscope Thorlabs offers preconfigured Confocal Camera adapter, the confocal control Systems with source wavelengths centered at 405 nm, module, imaging software, and a 635 nm, or both wavelengths; we can also fabricate custom confocal heads that utilize other diodes to computer. This powerful provide alternative source wavelengths. Please contact combination of proven Thorlabs imaging us to discuss your specific needs. technology enables a true confocal solution at a fraction of the cost of competing systems. For those preferring open access to the microscope, Thorlabs also provides a Confocal Translation Stand (see page 30), which includes a mechanism for coarse/fine vertical camera translation, an objective turret, and a sample platform. (5.94") 150.8 mm Mouse Kidney A 16 µm cryostat section of a mouse kidney simultaneously excited with 403 nm and 471 nm light. The glomeruli and convoluted tubules are stained with Alexa Fluor 488 wheat germ agglutinin (green) while the nuclei are stained with DAPI (blue). This pseudo-colored image was obtained by modifying a VCM405 Video-Rate Confocal Camera to provide excitation at 403 nm and 471 nm and then mounting it on a Nikon TE2000 microscope equipped with a 40X Olympus Objective (NA = 0.75). 28 www.thorlabs.com (2.86") 72.7 mm (1.14") 29.1 mm (7.38") 187.3 mm (11.71") 297.3 mm (3.11") 79.1 mm (26.32") 668.6 mm VCM101H Video-Rate Confocal Camera Schematic Real-Time Video-Rate Laser Imaging Confocal Camera Input Thorlabs' Confocal Camera offers real-time confocal imaging in a customizable open platform. The laser source can be free space or fiber coupled into the scan head; the fibercoupled option ensures a spatially filtered input beam that is essentially a perfect Gaussian. In addition, the two single mode fibers used to deliver the illuminating light and collect the backscattered signal replace the pinhole that is used in traditional confocal systems. The confocal arrangement of the fiber position rejects out-offocus light thus creating a true confocal image. Output Confocal Head: Constituent Parts 1. 2. 3. 4. 5. 6. 7. To Sample 8. 9. 10. The camera head contains the counter rotation scanner (resonant scanner), galvo scanner, turning mirrors, beamsplitter, fiber collimators, and kinematic mounts, all of which can be accessed by removing the cover plate (see photo to the right). The supplied BSW07 50:50 Ø1" Broadband Beamsplitter for the 400 - 700 nm range, which fits into the mount labelled as #5 in the photo, can be easily swapped out for any user-supplied Ø1" round or 36 mm x 25 mm x 1 mm rectangular beamsplitter or dichroic mirror. In addition, an SM1 Lens Tube has been supplied adjacent to the entrance port (#7 in the photo) as well as the exit port (#8 in the photo), thereby enabling the user to insert the Ø1" longpass, shortpass, bandpass, or notch filter appropriate for the user's fluroescence imaging application. The electronic controller module is included in all the system options we offer. This module drives the resonant scanner, galvo scanner, and laser diodes; it also interfaces the computer with the scan head. Resonant Scanner Galvo Scanner Scan Lens (f = 80.4 mm) Kinematic Mirror Mount for Ø1" Optics Kinematic Mount for Ø1" Beamsplitter or 25 mm x 36 mm x 1 mm Dichroic Filter Turning Mirror Mount for Ø1/2" Optics Kinematic Mount with SM1 Thread and Fiber Collimator Adapter Kinematic Mount with SM1 Thread and Fiber Collimator Adapter SM1 Lens Tube for Ø1" Filter SM1 Lens Tube for Ø1" Filter The green lines and arrows in the photo indicate the path that the excitation light follows. Fluorescence from the sample (when in the fluorescence imaging mode) or backscattered light (when in the backscattering configuration) will then following the light path indicated with red lines and arrows. Confocal Camera Specifications Optical and Imaging: ■ Central Wavelength: 405 nm and/or 635 nm ■ Bandwidth: ±5 nm ■ Output Power:* ~2 mW ■ Resolution:† 1 µm ■ Imaging Speed: 23 fps @ 800 (X) x 640 (Y) Pixels DAQ Electronics: ■ Analog Input:‡ 2 Channels, 14 Bits, 125 MS/s ■ Analog Output:‡ 4 Channels, 16 Bits, 1 MS/s, ±10 V ■ Digital I/O: 8 Ports BPAE Cells Computer: Supply Voltage: 100/240 VAC, 50-60 Hz ■ CPU/Memory: Intel® Processor ■ Memory: 2 GB ■ Hard Drive: 250 GB SATA ■ Optical Drive: 16X DVD ±RW ■ Monitor: 19" LCD (1280 x 1024 Pixels) ■ Operating System: Windows® XP Professional, SP2 ■ Confocal Controller: Supply Voltage:** 100 V - 240 VAC, 50 - 60 Hz ■ Storage/Operating Temperature: 15 - 40 °C ■ Relative Humidity: <85% Non-Condensing ■ Weight of Control Unit: 2.26 kg (5 lbs) ■ Dimensions of Control Unit (L x W x H): 305 mm x 254 mm x 127 mm (12" x 10" x 5") ■ *Output power was measured at output port of VCM101H with no objective using a 635 nm laser diode light source ‡ Ms/s = Megasamples per second † Resolution specified using RMS40X objective and 635 nm diode. Actual resolution will vary based on objective used. **Control Unit has universal AC input. F-actin (filamentous actin) and nuclei distribution in bovine pulmonary artery endothelial (BPAE) cells obtained using a modified Thorlabs’ videorate confocal camera mounted on a Nikon TE2000 microscope. The pseudo-colored green fluorescence indicates F-actin, which was stained with BODIPY FL phallacidin while blue fluorescence labels nuclei, which were stained with DAPI. The image was recorded using simultaneous scanning of both 403 nm and 471 nm laser sources and a Nikon PlanApo 60X oil immersion objective with an NA of 1.40. An adapter is needed to connect the confocal head directly to the camera port of standard microscopes (e.g., Olympus, Nikon, Zeiss, and Leica). Please contact us to inquire about the adapter options we currently have available as well as the possibility of having our machine shop provide an adapter for your microscope if there is not one currently available. VCM Series of Confocal Camera Systems ITEM# VCM405 VCM635 VCM405/635 $ £ $ 33,000.00 $ 33,000.00 $ 33,000.00 £ 20,790.00 £ 20,790.00 £ 20,790.00 € € 30.690,00 € 30.690,00 € 30.690,00 RMB DESCRIPTION ¥ 315,150.00 ¥ 315,150.00 ¥ 315,150.00 Confocal System with 405 nm Source Confocal System with 635 nm Source Confocal System with 405 nm and 635 nm Sources www.thorlabs.com 29 Real-Time Video-Rate Laser Imaging Confocal Camera System In addition to Confocal Camera Systems in which the confocal head mounts directly to the camera port via an adapter, leading to a compact ready-to-use module, Thorlabs also offers a VCM-TS Confocal Translation Stand for those customers preferring open access to the microscope. The VCM-TS Stand includes a mechanism for coarse/fine vertical camera translation, an objective turret, and a sample platform on which a user-supplied sample translation stage, such as the MAX312 Flexure Stage (see photo below) can be mounted. Thorlabs’ VCM405 Confocal Camera System shown with the VCM-TS Confocal Translation Stand Confocal Translation Stand ITEM# VCM-TS $ $ € £ 2,000.00 £ 1,260.00 € RMB 1.860,00 ¥ DESCRIPTION 19,100.00 Confocal Translation Stand Confocal Imaging: Backscattering Mode The VCM Series of Confocal Camera Systems offers highly versatile imaging systems. The standard reflection mode allows surface imaging of the highly reflective, opaque materials found in microelectronics, material science, and surface studies (see the images below). In addition, this confocal imaging mode can also provide optical slicing of semitransparent scattering samples, as evidenced by the image to below. Leaf Psuedo-colored 3D projection and crosssectional confocal scattering image of a green leaf. The data was obtained using a 60X objective lens and a VCM635 confocal camera that was modified to provide fiber-coupled excitation at 660 nm. 80 μm 250 μm Figure 1 185 μm Microelectronics Figure 1. Confocal scattering image of a memory chip with XZ and YZ cross-sectional images taken with a 60X objective. The total image size is 75 x 55 µm. 1. +20 0 -20 55 μm 30 www.thorlabs.com 75 μm Figure 2. Reconstructed 3D projection model of the confocal backscattering signal from a portion of the circuit of a microchip, obtained using a 100X objective. 2 110 μm 150 μm Real-Time Video-Rate Laser Imaging Confocal Imaging: Fluorescence Mode Peach Worm Below are pseudo-colored confocal fluorescence images of a peach worm obtained with a confocal camera system using a 60X water immersion objective. A total of 256 Z-slices (0.3 µm step size) were used to create the pseudo-3D projection shown at the bottom of the page. The total 80 µm Z-scan is represented below by a selection of 8 images that were taken in 10 µm increment (see images 1-8); each Z-slice measures 440 µm x 430 µm. 1 Confocal laser scanning microscopy is most frequently used with fluorescent samples since the technique spatially separates the desired fluorescence signal from the out-of-focus background fluorescence, thereby allowing optical sectioning along the Z-plane. Combining this method with imaging processing software enables cross-sectional imaging, 2D projections, and pseudo3D rendering of the optically sectioned sample as demonstrated by the data presented here, which was obtained with a VCM405 confocal system that has been reconfigured as shown in Fig. 3 for fluorescence collection. 2 (optional) 3 4 Figure 3 shows a VCM405 confocal head reconfigured for fluorescence imaging. By swapping out the beamsplitter in the standard VCM405 setup, replacing it with a dichroic filter, and adding the appropriate excitation and emission filters, the system becomes a powerful fluorescence imaging tool. In most situations, a PMT should also replace the fast photodetector used for confocal imaging in the reflection mode. The Confocal Camera is Easily Reconfigured for Fluorescence Imaging Figure 3 Pollen 5 6 7 8 Below are pseudo-colored 2D projections and 3D confocal fluorescent images of pollen taken with a VCM405 confocal camera using a 60X objective. Pollen grains were mounted on a standard microscope slide and excited with 405 nm light from the laser diode (Thorlabs Item# DL5146-152) incorporated into the head of the confocal camera. The emission signal was selected using a dichroic mirror (DMLP505) with a cutoff wavelength of 505 ± 15 nm, collected 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) www.thorlabs.com 31 Real-Time Video-Rate Laser Imaging Confocal Fluorescence Imaging Rabbit Artery Human Skin and Sweat Gland A 250 µm x 210 µm image showing the top view projection of a rabbit artery slice mounted on a standard microscope slide is shown below. Here, the pseudo-colored confocal fluorescent image was taken using a confocal camera in fluorescence mode and a 60X infinity-corrected objective. Below is a view of a sample slice of human skin and sweat gland that was mounted on a standard microscope slide and observed with a confocal camera using an infinitycorrected 60X water immersion objective. Here, a series of individual Z-slices were used to create the pseudo-colored projection. Multichannel Fluorescence Imaging For most fluorescence applications, multiple dyes are used to distinguish visually and chemically specific cell structures or metabolic processes. Whether the protocol uses a combination of external fluorophores like DAPI, Alexa Fluor 488, and Rhodamine or multiple fluorscent proteins, it is useful to have multichannel detection. As shown in Fig. 4, the VCM series of confocal camera systems is easily reconfigured to meet these experimental requirements. The system shown to the left was used to obtain the two-color images below. Although this example utilizes two-channel detection, simply adding a third PMT allows the confocal camera to produce three-color images, making this system comparable to a large commercial confocal fluorescent microscopy system but with a smaller footprint and at a fraction of the cost. Figure 4 Confocal Camera in the Multichannel Configuration Plant Sectioning The pseudo-colored 3D images below show an optically sliced fluorescent plant sample, excited at 405 nm using a modified VCM405 Confocal Camera. Channel 1 (shown in red) collected fluorescence in the 505 – 555 nm range, while channel 2 (shown in green) was used to collect light from 569 – 600 nm. 32 www.thorlabs.com Real-Time Video-Rate Laser Imaging Nonlinear Imaging Using The VCM Series of Confocal Cameras Many new techniques have been developed to enhance imaging contrast and biological and chemical specificity. With the advent of turnkey ultrafast lasers, nonlinear imaging methods such as multiphoton fluorescence imaging (e.g., 2-photon or 3-photon microscopy), second harmonic imaging microscopy (SHIM), and resonant Raman techniques like CARS are increasingly being adopted by microscopists to expand their biochemical understanding of natural processes. Often, more than one of these techniques is used simultaneously, which necessitates a custom system or a modification to a standard confocal laser scanning microscope. As is the case with other Thorlabs’ products, we have designed the VCM series of confocal camera systems to provide you with the tools needed to create your own photonics solutions. The reconfigurable confocal camera head is ideally suited for nonlinear microscopy setups. In many cases, it is cheaper and more convenient to convert an existing confocal laser scanning microscope system into a multiphoton system rather than purchasing a commercial version; in particular, for SHIM and CARS applications, a customized system is the only solution due to the lack of industry options. In the confocal camera head, internal tap holes are provided for changing optical components, and the compact footprint allows it to be adapted to any available microscope port with minimal mounting hardware. Emission Filter, Laser Blocking 750-1000 nm Dichroic Filter, 750 nm Long Pass Focus Lens Confocal Camera Reconfigured for Multiphoton Imaging Figure 5 Please contact us to inquire about the adapter options currently available to connect the confocal head directly to standard camera ports as well as the possibility of having our machine shop provide an adapter for your microscope if there is not one currently available. Multiphoton Fluorescence Microscopy Multiphoton microscopy combines fluorescent dyes with an ultrafast laser to allow fluorescence imaging using less damaging IR radiation. With the increased penetration depth and the inherently localized nature of the ultrafast pulse, multiphoton microscopy provides noninvasive sub-cellular imaging of living systems. The confocal head can be reconfigured for multiphoton microscopy or second harmonic imaging microscopy (SHIM) simply by replacing the single mode fiber before the detector with the appropriate bandpass and emissions filters, as shown in Fig. 5. For SHIM, the appropriate filters select light at half the excitation wavelength. The setup shown in Fig. 5 was used to image a slice of rat kidney using a confocal head that was reconfigured for two-photon fluorescence imaging using a TC780-150 femtosecond laser (Menlo GmbH) for excitation. For more information on this femtosecond laser, please contact Menlo Systems Inc. directly at 973-300-4490 or by emailing [email protected]. Like mulitiphoton microscopy, SHIM uses an ultrafast laser for signal generation, but in this case, no fluorophore is used; instead, the signal is generated from noncentrosymmetric features within the sample, making this technique advantageous in many material science applications. In addition, SHIM is increasingly being used in biological applications, including mammalian studies. For example, the large signal created by myelin and collagen yields excellent cellular structure determination without the need for either an intrinsic or externally applied fluorophore. 1 2 3 4 5 6 7 8 www.thorlabs.com 33 Real-Time Video-Rate Laser Imaging Multiphoton Imaging With and Without a Dispersion-Compensating Mirror Pair Thorlabs has teamed up with its strategic partner, Menlo Systems Inc., to provide a pair of Dispersion-Compensating Mirrors that correct for the phase distortions that occur when ultrashort pulses travel through an optical system. Since femtosecond pulses are comprised from many different wavelengths of light, pulse broadening, as a result of dispersion, will occur when the laser light passes through a dielectric medium (e.g., glass in the optical system). This pulse broadening is attributed to the wavelength dependence of the refractive index of the optical components through which the light travels. Shorter wavelengths are associated with higher indices of refraction than longer wavelengths; thus, when a femtosecond pulse travels through an optical system, the shorter wavelengths will travel slower than the longer ones. The pulse dispersion caused by the wavelength-dependent nature of the refractive index can be corrected using Menlo Systems’ dispersioncompensating mirror pair. These mirrors are specifically designed so that longer wavelengths experience larger group velocity delay than shorter wavelengths, thereby negating the pulse broadening caused by the optical elements within the imaging system. Features ■ ■ ■ ■ ■ ■ Advanced Coating Layer Composition Corrects for Dispersive Elements in the Beam Path Reflectivity: >99.5% from 700-1000 nm Extremely Flat Polished Substrates to Maintain Beam Quality Dispersion per Reflection: -175 fs2 at 800 nm Coated Surface Dimensions: 10 mm x 50 mm Thickness: 12 mm For more information on Menlo Systems’ OCTAVIUS-1G Ti:Sapphire Oscillator used to obtain the mouse images shown below, please contact Menlo Systems Inc. via telephone (973-300-4490) or email ([email protected]). Mouse Kidney The two-photon images of a mouse kidney shown here demonstrate the benefits of using the DispersionCompensating Mirror Pairs manufactured by Thorlabs’ strategic partner, Menlo Systems Inc., for increasing image quality. Figure 1 shows an image of a mouse kidney specimen that was taken without the use of the DispersionCompensating Mirror Pair, whereas Fig. 2 shows the same image acquired after adding the mirror pair to the experimental setup. In the mouse kidney specimen (Molecular Probes®, Invitrogen Corp.), the glomeruli and convoluted tubules are labeled with Alexa Fluor 488 (green) and cell nuclei are labeled with DAPI (blue). These pseudocolored images were obtained using Thorlabs’ inhouse developmental multiphoton microscope equipped with Figure 1. Uncompressed Pulse Figure 2. Compressed Pulse a 40X Olympus objective (NA = 0.75). Two-photon excitation was provided by Menlo Systems’ Octavius-1G, a Ti:Sapphire oscillator that provides a repetition rate of 1 GHz and ultra short (<6 fs) pulses. The group delay dispersion (GDD) attributed to the optical elements in the microscope is ~4200 fs2. GDD was compensated by adding the Dispersion-Compensating Mirror Pair into the beam path prior to the imaging system entrance. An intensity analysis of the images shown in Figures 1 and 2 indicates that the pulse compression provided by the mirror pair increases the signal to noise by a factor of ~38 (~16 dB), thereby providing a higher quality image of the mouse kidney. $ £ € RMB DESCRIPTION $ 5,000.00 £ 3,150.00 € 4.650,00 ¥ 47,750.00 Dispersion-Compensating Mirror Pair ITEM # DCMP175 34 www.thorlabs.com