Multiphoton confocal microscope. Multiphoton confocal microscope A1R MP

Size: px

Start display at page:

Download "Multiphoton confocal microscope. Multiphoton confocal microscope A1R MP"

Nickolas Horn
6 years ago
Views:

1 Multiphoton confocal microscope Multiphoton confocal microscope A1R MP

Nikon's provides deeper, faster and sharper imaging.

The A1R MP incorporates these newly developed technologies in response to your requirement for the highest level of confocal and multiphoton imaging performance.

scanner, and their movements were simultaneously captured in three successive fluorescence images at 30 fps (30 msec), with three separate color channels.

The long-wavelength ultrafast laser in combination with the ultrahigh-speed resonant scanner effectively reduces photodamage and makes time resolved multiphoton imaging of biomolecules possible.

Satoshi Nishimura, Department of Cardiovascular Medicine, the University of Tokyo, TSBMI, the University of Tokyo, PRESTO, Japan Science and Technology Agency *Point scanning in two-photon microscopy

5-day-old rat embryo after a 24-hour whole embryo culture Neuroepithelial cells and commissural axon of migrating interneurons can be clearly observed. Photographed with the cooperation of: Dr.

Masanori Takahashi, Division of Developmental Neuroscience, Tohoku University Graduate School of Medicine Fixed neuronal cells of mouse brain expressing egfp Excitation with pulsed IR laser (920 nm)

2 Nikon's provides deeper, faster and sharper imaging. The confocal microscope A1R, which has an excellent reputation for its high speed, superb sensitivity and high resolution, now offers multiphoton excitation imaging. The A1R MP incorporates these newly developed technologies in response to your requirement for the highest level of confocal and multiphoton imaging performance. The world's fastest* multiphoton imaging with a high-speed resonant scanner Fast Bright, high-resolution imaging by new high NA objectives for multiphoton microscopy Sharp 40x 20x 40x 20 µm 20 µm 20 µm 20 µm 3.46 sec 3.49 sec 3.52 sec 3.55 sec Visualization of intravital microcirculation Blood cells in blood vessels within a living organism were excited by a femtosecond pulsed IR laser with the A1R MP's ultrahigh-speed resonant scanner, and their movements were simultaneously captured in three successive fluorescence images at 30 fps (30 msec), with three separate color channels. The arrowhead indicates the tracking movement of the white blood cell nucleus. Three fluorescent probes are simultaneously excited and imaged nucleus (blue), endothelium (green), and plasma (red). The long-wavelength ultrafast laser in combination with the ultrahigh-speed resonant scanner effectively reduces photodamage and makes time resolved multiphoton imaging of biomolecules possible. Image resolution: 512 x 512 pixels, Image acquisition speed: 30 fps, Objective: water immersion objective 60x Photographed with the cooperation of: Dr. Satoshi Nishimura, Department of Cardiovascular Medicine, the University of Tokyo, TSBMI, the University of Tokyo, PRESTO, Japan Science and Technology Agency *Point scanning in two-photon microscopy Deep tissue imaging with the new high-sensitivity non-descanned multiphoton detector Deep Fixed spinal cord primordia (neural tube) of a GFP transfected 13.5-day-old rat embryo after a 24-hour whole embryo culture Neuroepithelial cells and commissural axon of migrating interneurons can be clearly observed. Photographed with the cooperation of: Dr. Noriko Osumi, Dr. Masanori Takahashi, Division of Developmental Neuroscience, Tohoku University Graduate School of Medicine Fixed neuronal cells of mouse brain expressing egfp Excitation with pulsed IR laser (920 nm) allowed higher S/N ratio imaging of deep brain tissue areas than with a confocal microscope. Photographed with the cooperation of: Dr. Satoru Kondo, Department of Cellular Neurobiology, Graduate School of Medicine, the University of Tokyo 0µm 0µm Figure 1 Image of deep areas of spinal cord primordia (neural tube) of a 13.5-day-old rat embryo The entire embryo was cultured for approximately 24 hours after transfection with green fluorescent protein (GFP) by electroporation. A fixed cross-sectional slice of spinal cord was embedded in gel and twophoton excitation was conducted using pulsed IR laser. Observation of deep areas of over 600 µm was achieved. Photographed with the cooperation of: Dr. Noriko Osumi, Dr. Masanori Takahashi, Division of Developmental Neuroscience, Tohoku University Graduate School of Medicine High contrast imaging by Nikon's high-speed, high-precision unmixing algorithms Contrast 300 µm 300 µm Figure 2 Image of fixed neuronal cells of mouse brain expressing egfp captured using Non- Descanned s (NDD) Excitation with pulsed IR laser (920 nm) allowed higher S/N ratio imaging of deep brain tissue areas than with a confocal microscope. Optical sections of deep areas of over 600 µm in the Z-axis were achieved. A new CFI Apo LWD 40x WI λs objective was used. Photographed with the cooperation of: Dr. Satoru Kondo, Department of Cellular Neurobiology, Graduate School of Medicine, the University of Tokyo 600 µm 600 µm Triple excitation with 750 nm wavelength laser Alexa 488, MitoTracker Red, DAPI Image of specimen stained with Alexa 488, MitoTracker Red and DAPI was captured with spectral detector during two-photon excitation and unmixed. Excitation with infrared wavelength reduces damage to live cells. Figure 1 Figure 2 2 3

's multiphoton technology High-speed resonant scanner Fast Objectives for multiphoton microscopy Sharp The resonant scanner is capable of imaging a wide area at a much higher speed than a

The NDD* 1 for multiphoton microscopy makes it possible to image fast and deep through the thickest specimens.

Nikon's new objective, CFI Apo LWD 40x WI λs, offers both high NA and long working distance in addition to high transmission made possible by Nikon's exclusive Nano Crystal Coating* 3.

61 mm *1 NDD (Non-Descanned ) Unlike confocal imaging, where emitted light from the specimen passing through a pinhole is descanned before being detected, the A1R MP eliminates the need for a pinhole.

$CFI Plan Fluor 20x A MI Nano Crystal Coat-deposited NA 0.75, W.D. 0.35 mm *3 An ultra-low refractive index film that was initially developed for Nikon's IC steppers.$

3 's multiphoton technology High-speed resonant scanner Fast Objectives for multiphoton microscopy Sharp The resonant scanner is capable of imaging a wide area at a much higher speed than a non-resonant scanner, making it possible for 420- fps imaging, the world's fastest using point scanning technology. The NDD* 1 for multiphoton microscopy makes it possible to image fast and deep through the thickest specimens. Nikon's optical pixel clock system allows more stable and more evenly illuminated imaging even at high speeds. Nikon's new objective, CFI Apo LWD 40x WI λs, offers both high NA and long working distance in addition to high transmission made possible by Nikon's exclusive Nano Crystal Coating* 3. Fine structures and motions inside a specimen are clearly observed with the excellent resolution and brightness. Objectives for multiphoton microscopy CFI Apo LWD 40x WI λs NA 1.15, W.D mm *1 NDD (Non-Descanned ) Unlike confocal imaging, where emitted light from the specimen passing through a pinhole is descanned before being detected, the A1R MP eliminates the need for a pinhole. By locating the NDD close to the back aperture of the objective, more of the scattered fluorescence emissions can be collected, improving the sensitivity of the instrument. CFI Plan Fluor 20x A MI Nano Crystal Coat-deposited NA 0.75, W.D mm *3 An ultra-low refractive index film that was initially developed for Nikon's IC steppers. With its coarse structure nano particles are arranged in a spongy composition thus the Nano Crystal Coating boosts the transmission of light over a wide spectral wavelength range. High-sensitivity 4-channel NDD (Non-Descanned ) for multiphoton microscopy Deep High-speed, high-precision unmixing *4 Contrast Nikon's newly developed 4-channel NDD* 1 for multiphoton microscopy allows imaging deep into a specimen. A detector* 2 with very high sensitivity and a wider sensitive area than conventional models is placed close to the back aperture of the objective where image formation of the specimen takes place. This configuration improves the detection efficiency for scattered fluorescence, and enables the capture of signals from deep within a living organism more clearly and more reliably with higher S/N and less aberrations. In addition to spectral detection and unmixing by the 32-channel detector, unmixing by the 4-channel NDD* 1 for multiphoton microscopy is also realized. Images captured with a resonant scanner can be unmixed, so clear and high-contrast images of areas deep within a thick specimen can be captured in ultrahigh speed. *4 Fluorescence from a specimen stained with multiple fluorescence probes is separately detected by wavelength, and the brightness and the spectral response of the fluorescence probes can be measured. This method allows easy unmixing of fluorescence probe combinations such as GFP and YFP that cannot be unmixed by conventional methods. *2 Detecting as much scattered fluorescence as possible is important for observation of deep parts in a specimen. The detection depth varies depending on the detector sensitivity, size and installation position. Multiphoton laser beam alignment with a single click When the multiphoton laser wavelength or group velocity dispersion precompensation is changed, the multiphoton laser beam positional pointing may drift, resulting in uneven intensity of images, or a slight misalignment of fluorescence images that are produced by a visible light laser and a multiphoton laser. Since the multiphoton laser is invisible to the eye, the laser beam alignment has been difficult and potentially dangerous for users, especially at wavelengths longer than 800 nm. Nikon A1R MP's newly developed auto laser alignment function automatically corrects multiphoton laser misalignments with a single click. Optical path detection unit Incident Optical Unit (with alignment function) Principle of multiphoton excitation The multiphoton confocal microscope enables excitation by simultaneous absorption of two or more near infrared photons by a single fluorescent molecule. When two photons are absorbed simultaneously into a single fluorescent molecule (two-photon excitation), the excitation efficiency is proportional to the square of the excitation light intensity. In order to achieve multiphoton excitation, a pulsed beam with high photon density or flux of photons is used. Because the laser beam is delivered in very short (femtosecond) pulses and is converged on the focal point through an objective lens, the probability of simultaneous absorption of two photons becomes high enough to be Excited level Virtual level Ground level Transition of energy levels of fluorescence molecule useful for imaging. The intensity of the laser beam converged by the objective lens decreases in inverse proportion to the square of the distance from the focal plane. Therefore, the efficiency of two-photon excitation decreases inversely with the fourth power of the distance from the focal plane. As a result, only a fluorescence molecule located within the diffraction-limited focal volume of the objective lens is excited and emits fluorescence. Because of the small amount of absorption and scattering by a specimen, the near infrared light used for excitation penetrates deep into thick tissue without losing much of its strength. Also, the excitation range is only within the diffraction-limited focal volume of the objective lens. Therefore, photodamage to a specimen can be minimized, making it suitable for observation of whole organisms in vivo, live cells and live tissues. The combination of the group velocity dispersion precompensation system incorporated in the multiphoton laser and the multiphoton detector (NDD) allows fluorescence imaging of deeper areas of a specimen than standard confocal technique. Confocal (one-photon) microscopy Multiphoton microscopy Excitation area in confocal microscopy and multiphoton microscopy Focal plane 4 5

4 System diagram Specifications Laser Module for Multiphoton Incident Optics Chameleon or Mai Tai Incident Optical Unit Microscope A1-TI Ti Adapter Set Laser Module for Confocal C-LU3EX 3-laser Module EX A1R MP Scanner Set Scanning Head Diascopic Unit MP Laser for multiphoton microscopy A1-FN1 FN1 Adapter Set LU-LR 4-laser Power Source Rack L4 L3 L2 L1 LU4A 4-laser Module Z-focus Module When pulsed light of very short duration, typically about 100 fs, passes through microscope optics (e.g. objective), the pulse duration increases due to group velocity dispersion, thus causing a reduction of peak power. By retarding longer wavelengths in the pulse relative to the shorter wavelengths with a group velocity dispersion precompensator before the pulsed laser beam enters the microscope, the ideal pulse duration can be maintained in the specimen, preventing the reduction of peak pulse power. This allows bright fluorescence imaging of areas deep within a specimen with minimum laser power. Femtosecond pulsed lasers Unit A1-DUS Spectral Unit Controller Unit for Multiphoton Non-descanned EPI Filter Wheel for VAAS Option A1-DU4 4-detector Unit Remote Controller Compatible microscopes PC Software ECLIPSE Ti-E motorized inverted microscope ECLIPSE FN1 fixed stage microscope (Scheduled to be compatible in 2010.) Input/output port 3 laser input ports 4 signal output ports (for 4-PMT detector, spectral detector, VAAS, optional detector) Laser for confocal microscopy Wavelength and power Laser diode (405 nm, 36 mw), Laser diode (440 nm, 20 mw), Ar laser (457 nm/488 nm/514 nm, 40 mw), Solid-state laser (488 nm, 20 mw), Solid-state laser (561 nm, 10 mw / 561 nm, 25 mw), G-HeNe laser (543 nm, 1 mw), Laser diode (638 nm, 10 mw) Modulation Method: AOTF (Acousto-Optical Tunable Filter) or AOM (Acousto-Optial Modulator) device Control: power control for each wavelength, Return mask, ROI exposure control Laser unit Standard: 4-laser module or 3-laser module EX Optional: 3-laser module EX (when 4-laser module is chosen as standard laser unit) Laser for multiphoton microscopy Wavelength and power Mai Tai HP DeepSee: nm, max. 2.1 W, max. 100 fs at 800 nm Chameleon Vision: nm, max. 3.0 W, 140 fs at 800 nm Modulation Method: AOM (Acousto-Optial Modulator) device Control: power control, Return mask, ROI exposure control Incident optics nm, auto alignment Standard 4-channel detector Wavelength nm ( nm for multiphoton observation) 4 PMT Filter cube 6 filter cubes commonly used for a microscope mountable on each of three filter wheels Recommended wavelengths for multiphoton/confocal observation: 450/50, 482/35, 525/50, 595/50, 700/75, 540/30, 515/30, 585/65 Diascopic detector Wavelength nm PMT NDD for multiphoton microscopy Wavelength nm 4 PMT Filter cube Filter cubes commonly used for a microscope Unmixing Channel unmixing Scanning head Scanning FOV: Square inscribed in a 18 mm circle Standard image acquisition Scanner: non-resonant scanner x2 Pixel size: max x 4096 pixels Scanning speed: standard 1 fps (512 x 512 pixels), max. 4 fps (512 x 512 pixels) Zoom: x continuously variable Scanning mode: X-Y, XY rotation, Free line, Line Z High-speed image acquisition Scanner: resonant scanner (X-axis, resonance frequency 7.8 khz), non-resonant scanner (Y-axis) Pixel size: max. 512 x 512 pixels Scanning speed: 30 fps (512 x 512 pixels) to 420 fps (512 x 32 pixels), 15,600 lines/sec (line speed) Zoom: 7 steps (1x, 1.5x, 2x, 3x, 4x, 6x, 8x) Scanning mode: X-Y, Line Acquisition method: Standard image acquisition, High-speed image acquisition, Simultaneous photo activation and image acquisition*1 Dichroic mirror Low-angle incidence method Position: 8 Standard filter: 405/488, 405/488/561, 405/488/561/638, 457/514, 405/488/543/638, BS20/80, IR, 405/488/561/IR Pinhole µm variable (1st image plane) Spectral detector (option) Number of channels 32 channels Spectral image acquisition speed 4 fps (256 x 256 pixels), 1000 lps Wavelength resolution 2.5 nm, 6 nm, 10 nm Wavelength range variable in 0.25 nm steps Unmixing High-speed unmixing, Precision unmixing Compatible microscopes ECLIPSE Ti-E inverted microscope, ECLIPSE FN1 fixed stage microscope*2 Z step Ti-E: µm, FN1 stepping motor: 0.05 µm Option Motorized XY stage (for Ti-E), High-speed Z stage (for Ti-E), High-speed piezo objective-positioning system (for FN1), VAAS Software Display/image generation 2D analysis, 3D volume rendering/orthogonal, 4D analysis, spectral unmixing Image format Application JP2, JPG, TIFF, BMP, GIF, PNG, ND2, JFF, JTF, AVI, ICS/IDS FRAP, FLIP, FRET, photo activation*1, three-dimensional time-lapse imaging, multipoint time-lapse imaging, colocalization Control computer OS Microsoft Windows Vista Business 64-bit SP1 (Japanese version /English version) CPU Memory Hard disk Data transfer Monitor Intel Xeon X5570 (2.98 GHz/8 MB/1333 MHz) 12 GB SAS (15,000 rpm), 300 GB x2, RAID 0 configuration Dedicated data transfer I/F 1600 x 1200 or higher resolution, dual monitor configuration recommended Vibration isolated table 1800 (W) x 1500 (D) mm recommended *1 Photo activation by a laser for multiphoton microscopy is scheduled to be available in *2 Scheduled to be compatible in Operation conditions Temperature: 20 to 25 C (± 1 C), with 24-hour air conditioning Humidity: 75 % (RH) or less Completely dark room or light shield for microscope Power source A1R MP Multiphoton system (scanner set, laser unit) Ar laser (457 nm, 488 nm, 514 nm) 120 VAC 12.5 A 220 VAC 6.8 A Lazer Except Ar laser (457 nm, 488 nm, 514 nm) 120 VAC 2.5 A 220 VAC 1.4 A 120 VAC 19.2 A Mai Tai HP DeepSea, Newport Corp., Chameleon Vision II, Coherent Inc. Laser for multiphoton microscopy (laser, water chiller, others) Spectra-Physics Lasers Division (Nikon specifications) (Nikon specifications) 220 VAC 10.5 A 120 VAC 4.4 A Microscope Inverted microscope Ti-E with HUB-A and epi-fluorescence illuminator 220 VAC 2.4 A 6 7 Computer unit 120 VAC 6.7 A 220 VAC 3.6 A 120 VAC 12.2 A 220 VAC 6.6 A

5 Layout Approx Unit: mm Incident Optical Unit Laser Chiller for Multiphoton Laser for Multiphoton Laser Controller for Multiphoton 1500 Approx laser Module, 4-laser Power Source Rack 4-detector Unit, Spectral Unit, Controller Scanning Head Non-Descanned Vibration Isolated Table Dimensions and weight PC Monitor Scanning head 276 (W) x 183 (H) x 453 (D) mm Approx. 13 kg Incident optical unit 363 (W) x 186 (H) x 404 (D) mm Approx. 11 kg 1200 Controller 360 (W) x 580 (H) x 600 (D) mm Approx. 45 kg 4-detector unit 360 (W) x 199 (H) x (D) mm Approx. 16 kg (approx. 22 kg with VAAS) Spectral detector unit 360 (W) x 325 (H) x 595 (D) mm Approx. 26 kg Non-descanned detector unit (W) x 54.5 (H) x (D) mm Approx. 5 kg 4-laser module 438 (W) x 301 (H) x 690 (D) mm Approx. 43 kg (without laser) 4-laser power source rack 438 (W) x 400 (H) x 800 (D) mm Approx. 20 kg (without laser power source) 3-laser module EX 365 (W) x 133 (H) x 702 (D) mm Approx. 22 kg (without laser) 700 Dimensions exclude projections. Remote Controller Specifications and equipment are subject to change without any notice or obligation on the part of the manufacturer. August NIKON CORPORATION WARNING TO ENSURE CORRECT USAGE, READ THE CORRESPONDING MANUALS CAREFULLY BEFORE USING YOUR EQUIPMENT. The AOTF incorporated into the 4-laser unit and the AOM optionally incorporated into the 3-laser unit are classified as controlled products (including provisions applicable to controlled technology) under foreign exchange and trade control laws. You must obtain government permission and complete all required procedures before exporting this system. * Monitor images are simulated. Company names and product names appearing in this brochure are their registered trademarks or trademarks. NIKON CORPORATION 6-3, Nishiohi 1-chome, Shinagawa-ku, Tokyo , Japan phone: fax: NIKON INSTRUMENTS INC Walt Whitman Road, Melville, N.Y , U.S.A. phone: ; NIKON (within the U.S.A. only) fax: NIKON INSTRUMENTS EUROPE B.V. Laan van Kronenburg 2, 1183 AS Amstelveen, The Netherlands phone: fax: NIKON INSTRUMENTS (SHANGHAI) CO., LTD. CHINA phone: fax: (Beijing branch) phone: fax: (Guangzhou branch) phone: fax: NIKON SINGAPORE PTE LTD SINGAPORE phone: fax: NIKON MALAYSIA SDN. BHD. MALAYSIA phone: fax: NIKON INSTRUMENTS KOREA CO., LTD. KOREA phone: fax: NIKON CANADA INC. CANADA phone: fax: NIKON FRANCE S.A.S. FRANCE phone: fax: NIKON GMBH GERMANY phone: fax: NIKON INSTRUMENTS S.p.A. ITALY phone: fax: NIKON AG SWITZERLAND phone: fax: NIKON UK LTD. UNITED KINGDOM phone: fax: NIKON GMBH AUSTRIA AUSTRIA phone: fax: NIKON BELUX BELGIUM phone: fax: Printed in Japan ( )T Code No.2CE-SCAH-1 This brochure is printed on recycled paper made from 40% used material. En

Multiphoton confocal microscope. Multiphoton confocal microscope A1R MP

Multiphoton confocal microscope. Multiphoton confocal microscope A1R MP confocal microscope confocal microscope A1R MP A1R MP boosts multiphoton imaging Fast multiphoton imaging, powerful enough for in vivo imaging The A1R MP is capable of high-speed 420-fps imaging, the world's