Knapp Center Microscopes

    Lattice Lightsheet Bessel Beam Illumination Microscope


    Lattice_Lightsheet

    Overview:This is a commerically-produced clone of the Betzig system described in Science October 2014 and currently running at the Janelia Farm Advanced Imaging Center. It is designed for low-light 3D time lapse imaging. This is NOT like the Zeiss Lightsheet.Z1 meant for large preps! Imaging is by means of water-immersion objectives and is limited to less than 100 micrometers depth of penetration. All samples MUST be on 5mm round coverslips. Use of this microscope requires a Staff person to operate the system. Please be sure to arrange your session in advance with Core Staff. Contact Vytas Bindokas or Christine Labno (information at left).

      Features:
    • High speed, low light 3D timelapse imaging.
    • Heated bath chamber for live cell work (no CO2 at this time)
    • Flash4v2+ sCMOS high-sensitivity, low noise camera
    • 100nm pixel size (xy)
    • Nikon 25x NA 1.1 water immersion objective
    • 405nm (DAPI), 488nm (GFP), 561nm (mCherry) and 642 (Cy5) lasers
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    Zeiss Lightsheet Z.1 for Single Plane Illumination Microscopy (SPIM)


    The Zeiss Lightsheet Z.1 was provided by the Institute for Genomics & Systems Biology (IGSB).

    What is SPIM? Single Plane Illumination Microscopy is particularly suited to long-term timelapse imaging of larger living specimens, such as zebrafish, Drosophilia embryos, and small plants. This technology has also been used to image living and fixed 3D cell cultures, including organoids, cysts and 3D cell migration assays.

    Lightsheet Z.1

    Overview: The Zeiss Lightsheet Z.1 is one of the first commercially available Single Plane Illumination microscopes. It allows for temperature controlled, long-term imaging of large samples with up to three colors (cyan, green/yellow and red). Samples can be kept in the chamber and imaged for hours or days. Sensitive cameras and single plane illumination allow for gentle sample illumination, reducing photodamage.

      Features:
    • Water cooled/heated Peltier block for chamber temperature settings from 10C-42C. Temperature feeds back to the software from both the block and the sample chamber.
    • Sample is held in a buffer-filled chamber during imaging. PBS, seawater and nutrient buffers have all been used successfully.
    • Two chilled PCO-Edge scMOS 16-bit cameras with 1920 x 1920 pixels for fast, detailed, simultaneous two color imaging.
    • Green/red or cyan/red simultaneous imaging is possible. Cyan/green or three color serial imaging is also possible.
    • 360 degree rotation plus programming for imaging from multiple angles. Incorporation of beads allows for multi-angle 3D reconstructions.
    • 5x, 20x and 40x objectives plus zoom factors from 0.36x - 2.5x for a range of sample sizes.
    • Off-line version of Zeiss Zen software available on facility workstation>.
      Objectives:
    • 5x / NA 0.16 dry (uses 5x NA 0.1 dry illumination objectives)
    • 20x / 1.0 water dipper (uses 10x NA 0.2 dry illumination objectives)
    • 40x / 1.0 water dipper (uses 10x NA 0.2 dry illumination objectives)
      Excitaiton and Emission:
    • 445nm (CFP), 488nm (GFP/YFP) and 561 (DsRed, mCherry) excitaiton lasers
    • BP 460-500nm (CFP), BP 505-545nm (GFP/YFP), BP550nm (GFP/YFP), LP 585nm (mCherry) emission filters.
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    Leica GSD/TIRFM Ground State Depletion Superresolution Microscope


    The Leica GSD was provided by the Institute for Genomics & Systems Biology (IGSB) and the Institute for Molecular Engineering. It is currently housed in KCBD 1250B.

    Overview:The Leica GSD is a four-color Total Internal Reflection fluorescence (TIRF) and a three-color Ground State Depletion (GSD) microscope. It is not a confocal microscope. There are four excitation lines available, spanning the spectrum from violet to far red. All can be used for TIRF; green to far red can be used for GSD.

    What is GSD? Ground State Depletion is one of the "pointilist" techniques for superresolution microscopy. This group also includes Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated Localization Microscopy (PALM)."Pointilist" superresolution microscopy techniques use various methods to manipulate a fluorescently labeled sample into returning only a handful of diffraction limited (200nm) fluorophore signals at one time. These signals are then mathematically processed to localize them to sub-diffraction (20nm) spots. Thousands of frames worth of these localized spots are collected over a potentially minutes-long imaging time and integrated on the fly to form a complete image. Up to three fluorophores (green, red, deep red) can be imaged serially to create multi-color images. TIRF illuminated, 2D epifluorescent and 3D superresolution imaging are all possible.

    jiping GSD movie
    Depth-coded GSD image courtesy of Jiping Yue, Xiaoyang Wu lab

    TIRF image
    Bovine chromaffin cell exocytosis
    Courtesy of Dr. Li Ma, Philipson Lab
    and Dr. Vytas Bindokas

    What is TIRF? Total Internal Reflection Fluorescence is used to observe the events near the plasma membrane of cells, or the fluorescence of a single molecule, without interference from fluorescence coming from the cell's cytoplasm. An evanescent wave selectively excites fluorophores in a small region (less than 200 nm) above the coverglass. This microscope features TIRF excitation at four wavelengths: 405nm, 488nm, 532nm and 642nm with automated critical angle positioning.

      Features:
    • Inverted platform with small laser safety chamber for imaging on slides or live cell dishes (sorry, NO heated stage).
    • 160x NA 1.43 state of the art, adhesive-free objective.
    • Supressed Motion (SuMo) stage which locks in the 160x objective to minimize sample drift.
    • PiFoc precision focusing control for 160x.
    • iXon Ultra EMCCD camera for high frame rate, low light imaging with low read noise.
    • New for 2016 - Option to use Flash4v2+ sCMOS camera and w-Gemini image splitter operated by a second imaging computer. This allows simultaneous two-color GSD acquisition and/or development of biplane 3D localization.
    • Camera-based, software-driven TIRF laser focusing and alignment.
    • Software-based TIRF angle setting allows for your choice of multiple laser angles, creating varying evanescent wave penetration depths. TIRF imaging direction (north, south, east or west) can also be selected.
    • Two off-line workstations for data processing.

      Objectives:
    • 160x / 1.43 oil WD=0.07mm (TIRF and GSD imaging)
    • 10x, 20x, 40x and 60x DRY objectives are available for viewing and widefield images ONLY
      Lasers:
    • solid state violet -- 405nm (used to "backpump" which increases fluorophore blinking, can also be used for TIRF excitation)
    • solid state blue -- 488nm (Alexa 488, bodipy, GFP)
    • green fiber laser -- 532nm (Alexa 555, rhodmine 6G, Cy3b)
    • red fiber laser -- 642nm (Alexa 647, Cy5)
      Filters:
    • Green: ex 483-493nm with 400-410nm dichroic: 496nm em: 505-605nm with 425-475nm
    • OPTIONAL manual filter for em: 503-547nm
    • Red: ex 527-537nm with 400-410nm dichroic: 541nm em: 550-650nm with 425-475nm
    • OPTIONAL manual filter for em: 582-636nm
    • Far Red: ex 637-647nm with 400-410nm dichroic: 649nm em: 660-760nm with 425-475nm
    • OPTIONAL manual filter for em: 664LP
    • Quad: ex 400-410nm; 483-493nm; 27-537nm; 637-647nm dichroics: 417, 496, 544, 655 em: 421-477nm; 497-519nm; 547-621nm; 666-732nm

    Proper Sample Preparation: Samples should be mounted or grown on #1.5 thickness coverslips (high tolerance Schott glass if possible) and stained with high quantum yield fluorophores such as: AlexaFluor 647, 555, or 488, Atto 647 or 488, Rhodamine 6G, Cy5, Cy3b or bodipy (this is not an exhaustive list). DO NOT use DAPI! Also, DO NOT mount the coverslips to slides. Bring unmounted coverslips in the holding medium of your choice (PBS works well) for imaging. We will provide MEA mounting buffer and apply it just before imaging. For information on GSD sample prep, or to set up a training/demo session, contact Christine Labno or Vytas Bindokas (information at right).

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    Leica SP8 3D, 3-color Stimulated Emission Depletion (STED) Laser Scanning Confocal with Time Gating


    LeicaSP8image
    Overview: The SP8 is Leica's newest high speed, superresolution laser scanning confocal platform. It features a white light laser (WLL) capable of excitation in any wavelength from 470nm to 670nm, and an acousto-optical beam splitter (AOBS) to select/introduce up to eight laser lines (at 8nm intervals) at a time. In addition to the WLL, there are Argon and UV lasers for imaging, photobleacing and photoactivation, so exciation spans the spectrum from UV to deep red. There are also three depletion lines for STimulated Emission Depeletion (STED) superresoluion: 775nm (depletes red and far red dyes), 660nm (depletes orange and yellow dyes) and 592nm (depletes green dyes). The acousto-optical tunable filters (AOTF) make it possible to detect a wide range of emission wavelengths with unlimited range on each of three HyDs (hybrid GaAsP detector) two PMTs (photon multiplier tube). Spectral scanning / unmixing in exciation and/or emission is available. Time gated fluorescence detection is possible on the three HyD detectors. Real-time deconvolution of confocal and STED data is possible with the SVI (Huygens)/Leica collaboration HyVoluiton package.

      Features:
    • White Light Laser allows for selection of any excitation wavelength between 470nm and 670nm. Up to 8 lines can be used simultaneously, must have at least 8nm spacing between lines.
    • Standard Argon (457, 488 and 514nm) and near UV (405nm) lines are also available for excitaiton, photoactivation and photobleaching.
    • Three depletion lines for STimulated Emission Depeletion (STED) superresoluion: pulsed 775nm (depletes red and far red dyes), 660nm (depletes orange and yellow dyes) and 592nm (depletes green dyes).
    • Capable of 2D or 3D superresolution imaging in XY, XZ or XYZ.
    • AOBS (acousto-optical beam splitter) plus sequential scanning capability allows for rapid sequential scanning of fluorophores with minimal bleed-through or cross-talk
    • THREE high sensitivity, low read noise hybrid GaAsP detectors (HyDs) with digital time gating (0.1 nanosecond increments, 3.5 nanosec. (min) to 12 nanosec (max) range) and photon counting mode.
    • Two chilled PMT fluorescence detectors
    • One transmitted light detector with DIC polarizer/analyzer with prisms for 20x and 40x available (63x is coming). 8- or 12-bit output, 16-bit possible in galvo mode.
    • Notch filters for 488, 561, 594 and 633 from the WLL
    • AOTF (acousto-optical tunable filters) for spectral scanning, allowing separation of fluorophores with similar ranges (e.g. GFP and FITC) through spectral unmixing.
    • Time-gating on HyDs allows for differentiation of fluorophores with similar spectra or enhancement of STED resolution from the pulsed 775nm laser.
    • Tandem scanner with dual scanning galvanometer mirrors allows for either high speed scanning (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (5 channels) OR high pixel density scanning (imaging to 4k x 4k [16 megapixels] per channel in galvo mode).
    • Standard galvo scanner includes beam park for FRAP, bleaching and photoactivaiton
    • xy scanning stage for tiling and multi-point sampling
    • NEW in 2017! Beta version of a new, non-rectangular tiling function to increase speed and decrease file size for large tiling projects. Includes a fast "stage overview" mode.
    • z-Galvo stage for precision Z-sectioning
    • Inverted platform for imaging on slides or live cell dishes (Sorry, NO incubator box at this time)
    • Optical zoom for sampling to 5nm pixel size
    • NEW in 2017! Real-time deconvolution with HuygensPro software via the HyVolution module (a collaboration between SVI and Leica)
    • Wizards for FRAP, FRET and Live Data Mode
    • 3D/4D reconstruction software built in to LAS_X image collection software
    • Bridge for passing images between LAS_X image collection software and ImageJ/Fiji
    • LAS_X Leica confocal software on Windows 10
    • Off-line version of LAS_X software available on facility workstation
      Objectives on the Microscope:
    • 10x / NA 0.4 dry
    • 20x / 0.7 multi-immersion (water, oil or glycerol)
    • 40x / 1.25 oil
    • 63x / 1.4 UV oil
    • 100x / 1.45 oil (STED-rated)
      Available Objectives (please ask to have these installed):
    • 93x / NA 1.3 glycerol (STED-rated, motorized correction collar)
      Lasers for excitation:
    • Near IR -- 405 (DAPI)
    • Argon -- 458nm (CFP), 476, 488 (GFP, FITC), 496, 514 (YFP)
    • White light laser -- ANY wavelength from 470nm - 670nm up to 8 lines at once with 8nm spacing
      Lasers for depletion:
    • pulsed 775nm (depletes red and far red dyes)
    • 660nm (depletes orange and yellow dyes)
    • 592nm (depletes green dyes)
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    Leica SP5 II STED-CW Superresolution Laser Scanning Confocal


    What is STED-CW? Stimulated Emission Depletion (STED) is an advanced form of laser scanning confocal microscopy that allows for a 3-4 fold improvement in resolution over traditional laser scanning confocal; from a smallest resolvable feature size of 150-200nm FWHM to approximately 50nm FWHM. This allows us to see objects that were previously below the limit of optical resolution for light microscopy (Abbe diffraction limit) and approaches the resolution of electron microscopy. CW (continuous wave) technology means that the depletion laser is continuous, not pulsed as in previous STED microscopes. This allows for superresolution at higher speeds than previously possible. See the Leica website for more information and example images.

    vcloud STED image
    STED micrograph courtesy of V. Cloud, Doug Bishop lab

    For best results in STED-CW imaging: we recommend placing samples on a #1.5 coverslip (0.170mm thickness) mounted with ProLong Gold (Invitrogen) antifade mounting medium or Thiodiethanol (TDE). Ideally, samples should contain high quantum yield, photostable fluorophores such as: AlexaFluor 450/488/514, Dylight 488, Atto 425/488, eGFP, mCitrine, mVenus, or mCerulean. For information on STED imaging, or to set up a training session, contact Christine Labno or Vytas Bindokas (information at right).

    Overview: In addition to the live cell-capable superresolution features described above, the SP5 II is also an advanced, high speed laser scanning confocal platform. It includes an acousto-optical beam splitter (AOBS) to select/introduce most excitation laser lines. There are eight excitation lines available, spanning the spectrum from green to far red. The acousto-optical tunable filters (AOTF) make it possible to detect a wide range of emission wavelengths with unlimited range on each of three PMTs (photo multiplier tubes), two HyDs (hybrid GaAsP detector) or two APDs (avalanche photodiodes).

      Features:
    • Continuous wave depletion laser at 592nm allows for single or dual color super resolution (STED method) with either the resonant (high speed) or galvo (high pixel density) scanners.
    • STED mode allows for resolution of particles down to 50nm FWHM (cyan, green, yellow fluorophores only)
    • 4 chilled PMT fluorescence detectors (digital spectral definition in 1 nm increments) plus one transmitted light detector with DIC polarizer/analyzer plus prisms for most objectives available. 8- or 12-bit output, 16-bit possible in galvo mode.
    • 1 high sensitivity, low read noise hybrid GaAsP detector HyD with photon counting mode.
    • 2 internal APD detectors for high sensitivity (green/red and CFP/YFP filters available)
    • Tandem scanner with dual scanning galvanometer mirrors allows for either high speed scanning (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (5 channels) OR high pixel density scanning (imaging to 8k x 8k [64 megapixels] per channel).
    • Standard scanner includes beam park for FRAP, bleaching and photoactivaiton
    • New in 2013 -- Full wrap incubator box with warm air heating for live samples
    • xy scanning stage for tiling and multi-point sampling
    • z-Galvo stage for precision Z-sectioning
    • Inverted platform for imaging on slides or live cell dishes
    • Optical zoom for sampling to 5nm pixel size
    • AOBS (acousto-optical beam splitter) plus sequential scanning capability allows for rapid sequential scanning of fluorophores with minimal bleed-through or cross-talk
    • Wizards for FRAP and FRET
    • AOTF (acousto-optical tunable filters) for spectral scanning, allowing separation of fluorophores with similar ranges (e.g. GFP and FITC) through spectral unmixing
    • LAS_AF Leica confocal software on Windows
    • Off-line version of LAS_AF software available on facility workstation
      Objectives on the Microscope:
    • HC PlanApo 10x / NA 0.4 dry WD=2.74mm
    • HC PlanApo 20x / 0.7 multi-immersion (water, oil or glycerol) WD=0.26-0.17mm
    • HCX PlApo 40x / 1.25-0.75 oil WD=0.22
    • HCX PlApo 63x / 1.4-0.6 UV oil WD=0.14
    • HCX PlApo 100x / 1.40 oil STED Orange WD=0.13mm
      Objectives available for use (please ask):
    • HCX PlApo 50x / 0.9 dry WD=0.28mm
    • HCX PlApo 63x / 1.3 glycerol CORR WD=0.30mm
      Lasers for excitation and Depletion:
    • Depletion laser -- 592nm (depletes cyan, green and yellow fluorophores/proteins) for STED imaging
    • Argon -- 458nm (CFP), 476, 488 (GFP, FITC), 496, 514 (YFP)
    • DPSS -- 561nm (Cy3, TexRed, DsRed, mCherry)
    • orange HeNe -- 594nm (TexRed)
    • red HeNe -- 633nm (Cy5)
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    FV1000 Laser Scanning Confocal Microscope


    Click for product information on the Olympus website

    Fluoview_1000

    Overview: The Olympus FV1000 provides laser scanning confocal imaging across the spectrum, from violet to near IR.

      Features:
    • Olympus IX81 inverted platform.
    • Available Bioptics dish heater, Olympus incubation / CO2 control chamber and independent objective heater to minimize thermal drift.
    • DIC prisms available for 60x and 100x objectives. Brightfield and DIC optics in an automated turret.
    • Three fluorescence PMTs and one transmitted light detector for simultaneous or sequential imaging of up to three fluorophorescence channels and one transmitted light channel per experiment.
    • Olympus image capture software with saving as .oib or .oif image format and export to .tif format capable.
    • Capable of z-stack, timelapse, multipoint and photobleaching/photoactivation with software based wizards. Live plot window and image triggering available.
      Objectives:
    • 4x / NA 0.13 dry (UPlanFLN)
    • 10x / NA 0.30 dry (UPlanFl)
    • 40x / NA 0.75 dry (UPlanFl)
    • 60x / NA 1.42 oil (PlanApoN)
    • 100x / NA 1.40 oil (UPlanSApo)
      Excitation Lasers:
    • Four lasers/six lines for excitiaton: 405nm (DAPI), 458 (CFP), 488 (GFP), 515 (YFP), 543 (Cy3, Texas Red), 633 (Cy5)
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    Olympus "live cell" DSU Spinning Disk Confocal


    Click for user's manual or scale bar calibrations

    Overview: The Olympus "live cell" DSU is one of two Olympus spinning disk confocal systems available in the facility. These systems can be used for either widefield or confocal fluorescence capture, and since the entire field of view is illuminated at once, z-stack and time lapse capture can be faster than on the laser scanning confocals. The live cell DSU is on an inverted platform and features water immersion objectives, a motorized stage and filters for a broad range of fluorophores, from blue to far red, plus DIC optics.

      Features:
    • Back thinned EM-CCD Hamamatsu camera for high speed/ high sensitivity imaging
    • Image capture through SlideBook software
    • Fully automated, inverted platform (model IX81) or imaging on slides or live cell dishes
    • Heated stage platform and 5% CO2 micro chamber available for live cell preps
    • Motorized xy-galvo stage for imaging multiple areas per dish and automated tiling of large specimens
    • NEW in 2017 - LED lightsource for even, flicker-free sample illumination
    • 3D and 4D (volume over time) rendering through SlideBook
    • 100% - 1.5% neutral density filters available
    • Interchangable pinhole disks for many magnifications (special request only)
    • Dual cube viewing through the occulars (blue/green, green/red and red/far red)
      Objectives on the Microscope:
    • UPlanFlN 10x / NA 0.3 dry WD=10mm
    • UPlanApo 10x / 0.4 water
    • UApo340 20x / 0.7 water WD=0.35mm
    • UApo340 40x / 1.15 water WD=0.25mm
    • UPlanSApo 60x / 1.2 water WD=0.28mm
    • PlanApo 100x / 1.45 oil TIRFM
      Objectives available for use (please ask):
    • 2x / 0.06 dry
    • 4x / 0.16 dry
    • 40x / 0.6 dry LWD
    • 150x / 1.45 oil
      Excitation and Emission Filters:
    • blue/violet or blue (DAPI) -- ex: 350/50 or ex: 405/12 and em: 457/50
    • CFP -- ex: 436/10 and em: 470/30
    • greens (FITC, Cy2) -- ex: 490/20 and em: 528/38
    • greens (GFP) -- ex: 480/25 and em: 525/40
    • YFP -- ex: 500/20 and em: 535/30
    • red (TRITC, Cy3) -- ex: 555/28 and em: 617/73
    • reds (DsRed) -- ex: 565/25 and em: 620/60
    • far reds (Cy5) -- ex: 635/20 and em: 685/40
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    Olympus DSU "fixed cell" Spinning Disk Confocal


    Click for user's manual or scale bar calibrations

    fixed sample DSU image

    Overview: The Olympus "fixed cell" DSU is one of two Olympus spinning disk confocal systems available in the facility. These systems can be used for either widefield or confocal fluorescence capture, and since the entire field of view is illuminated at once, z-stack and time lapse capture can be faster than on the laser scanning confocals. The fixed cell DSU features high NA oil objectives, high sensitivity camera and filters for a broad range of fluorophores, from blue to far red, plus DIC optics.

      Features:
    • Image capture through SlideBook software
    • Fully-automated, inverted platform (model IX81) for imaging through slides, dishes or plates
    • NEW in 2017 - LED lightsource for even, flicker-free sample illumination
    • Heated stage platform available for live cell preps (35mm round dishes)
    • z-galvo stage for Z-sectioning
    • 3D and 4D (volume over time) rendering through SlideBook
    • Neutral density filters available for long-term light time lapse imaging
    • DIC polarizer/analyzer plus appropriate prisms for most objectives
    • Evolve- backthinned CCD camera (512x512 16um pixels)
      Objectives on the Microscope:
    • UPlanFlN 10x / NA 0.3 dry WD=10mm
    • UPlanFl 20x / 0.5 dry WD=2.1mm
    • Zeiss PlanNeoFl 40x / 1.3 oil WD=0.21mm
    • PlanApo 60x / 1.4 oil
    • UPlanApo 100x / 1.35 oil
    • 60x / 1.2 water
      Available Objectives (please ask):
    • 2x / 0.06 dry
    • 4x / 0.16 dry
    • 40x / 0.6 dry LWD
    • 150x / 1.45 oil
      Excitation and Emission Filters:
    • blue (DAPI) -- ex: 387/11nm and em: 440/40
    • greens (FITC, GFP) -- ex: 485/20 and em: 525/30
    • reds (Cy3, Texas Red) -- ex: 560/25 and em: 607/36
    • far reds (Cy5) -- ex: 650/13 and em: 684/24
    • near IR reds (Cy7) -- ex: 710/40 and em: 775/46, dichroic 741
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    Olympus IX81 Inverted Widefield Microscope


    Click for user's manual or scale bar calibrations

    IX81 image

    Overview: This is an automated fluorescence microscope with a full-wrap incubation chamber that is well suited to multi-well plate imaging, fixed cell fluorescence imaging, live cell imaging with real-time pixel intensity readouts, FRET experiments, ratiometric imaging (such as fura-2 340/380), and checking transfection efficiency.

      Features:
    • Automated, inverted platform (model IX81) for imaging on slides, live cell dishes or multi-well plates
    • Full-wrap incubator box with micro CO2 chamber available (new October 2017)
    • Marzhauzer xy motorized stage for precise control of tiling, multi-point sampling or multi-well plate reading
    • Hamamatsu Orca Flash 4.0 camera with 4 megapixels (6.5um pixel size, 2048 x 2048 pixels). Huge field of view, great for tissue samples and multi-well plates
    • Zero Drift Correction (TM) auto re-focusing system. Especially useful for long term time lapse and / or heated preps
    • Automated filter changer with single color filters and a penta dichoic / filters for fast multi-color fluorescence imaging
    • DIC polarizer/analyzer plus appropriate prisms for most objectives
    • SlideBook imaging software image capture in 2D, 3D, time lapse and/or multipoint / tiling / multi-well
    • Fura-2 340/380 ratiometric imaging available
    • CFP/YFP and Chameleon FRET filter set available
    • Beam splitter for simultaneous capture of two emission wavelenghts with the SAME excitation wavelength. Splitters available are: green/red (520/30 and 630/50) and blue/yellow-orange (460/50 and 570/60). The beam splitter is available, but be aware there is significant vignetting due to the large chip size of the new Flash 4.0 camera
      Objectives:
    • 4x / 0.16 dry
    • UPlanFL 10x / NA 0.3 dry
    • LC PlanFL 20x / 0.4 dry
    • UApo/340 40x / 1.35 oil UV (best for FURA)
    • PlanApo 60x / 1.45 oil (TIRFM rated)
    • 100x 1.45 oil (TIRFM rated)

    • Also available (must be swapped in):
      • 2x / 0.06 dry
      • 40x / 0.6 dry LWD
      • UPlanFl 100x / 1.3 oil WD=0.2mm
      • 150x / 1.45 oil (TIRFM rated)
      Excitation and Emission filters:
    • blue on penta -- ex: 387/11 and em: 440/40 dichroic: 408
    • greens (FITC, GFP) single cube -- ex: 480/40 HQ and em: 510LP
    • greens on penta -- ex: 485/20 HQ and em: 525/32 dichroic: 504
    • reds (Cy3, Texas Red) single cube -- ex: 530-550 and em: 590LP
    • reds on penta -- ex: 560/25 and em: 650/13 dichroic: 581
    • far reds (Cy5) single cube -- ex: 620/60 HQ and em: 700/75 HQ
    • far reds on penta -- ex: 607/36 and em: 684/24 dichroic 667
    • NearIR on penta (Cy7) -- ex: 740/13 and em: 809/81 dichroic 762

    • Also available (must be swapped in):
      • CFP -- ex: 436/20 and em: 480/40 (available, please ask)
      • YFP -- ex: 500/20 and em: 535/30HQ
      • Fura-2 ex: 340; 380 and em: 510/40 (available, please ask)
      • Chameleons 2 ex: 440/20 and em: 535/35; 485/40 (available, please ask)
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    Zeiss Axiovert 200M (inverted widefield fluorescence)


    Click for user's guide

    Overview: The Axiovert 200M is the state of the art in inverted widefield microscopy from Zeiss. It is fully motorized and an absolute pleasure to use. This microscope is used for high resolution immunofluorescence or Nomarksi (DIC) images on fixed or live specimens on slides. The high quality images can be further enhanced by digital deconvolution on the analysis stations. Users may capture their images using the ORCA ER digital camera for fluorescent samples or the Zeiss Axiocam color digital camera for samples which require transmitted light color imaging.

      Features:
    • High resolution, cooled CCD camera system designed for low-light scientific applications. The Hammatsu Orca ER firewire digital monochrome camera provides high resolution at 12 bits. The imaging array is 1344 x 1024 (6.45x 6.45 um pixels).
    • High resolution images with the Zeiss Axiocam digital color camera
      • CCD basic resolution is 1300x1035; maximum file size per image is 3900x3030 @3x14 bit (color);
      • Digitisation is 14 bit/10MHz
      • Pixel size is 6.7 x 6.7 microns
    • 3I SlideBook 5.0 and 5.5 software for image capture (Windows 7)
    • Inverted platform and multiple stages to support slides and dishes for live and fixed samples
    • Automated z-drive for z-stacks
    • Automated xy stage for fine movement
    • Automated objective turret
    • Automated reflector turret with 5 positions and a multitude of fluorescent filters
    • Automated condensor turret
    • Dual filter wheels with individual excitation filters for fluorescence
    • Nomarksi (DIC) imaging
      Objectives on the Microscope:
    • 10x / 0.5 NA
    • 20x / 0.5 NA
    • 40x / 1.3 NA oil
    • 40x Korr
    • 63x / 1.4 NA oil
    • Also available:
      • 1.25x / 0.035 NA
      • 4x / 0.10 NA
      • 25x Korr oil/glycerol
      • 32x / 0.4NA
      • 40x / 0.6 NA
      • 100x/ 1.4 NA oil
      Excitation and emission filters:
    • Single emission filter cubes: Dapi, CFP, Fitc, GFP, eGFP, YFP,Texas red, Cy3, Cy5
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    Zeiss Axiovert 100TV (inverted widefield fluorescence for timelapse) - Dismantled, no longer available for use

    Olympus VivaView in-incubator microscope


    Click for product information on the Olympus website

    vivaview

    Overview: The Olympus LCV110U VivaView is an innovative system for long-term, multi-dimentional, live cell imaging. The system is an inverted widefield fluorescence microscope built into an incubator box. The incubator keeps optimal temperature, humidity and CO2 during long experiments and decreases the effects of thermal drift and evaporation. Allows for imaging of multiple areas in up to eight dishes, kept at 37C, 5% CO2. Brightfield, blue (DAPI), green (GFP) and near red (mCherry, DsRed) imaging available.

      Features:
    • Fully enclosed, humidified incubator set to 37C, 5% CO2.
    • Independent objective and carousel heaters minimize thermal drift.
    • Inverted platform with 8 position motorized carousel for imaging multiple areas across multiple 35mm round live-cell dishes (#1.5 glass bottom dishes recommended, MatTek dishes are available for $2 ea. in the facility).
    • 40x NA 0.95 dry objective for optimized light collection, 0.5x and 2.0x maganification changers available.
    • X-Cite light source can be adjusted to low light intensity to minimize photobleaching.
    • Air-cooled (-35C) CCD camera gives12-bit grayscale images with 1344 x 1024 pixels (6.45 micron pixels).
    • Optional motorized z-stack collection for 3D or focus correction.
    • Transmitted light plus up to three fluorescent colors (blue, green, red).
    • MetaMorph image capture software.
    • Data are saved to hard drive immediately post-capture.
      Objective:
    • 40x / NA 0.95 dry
    • 0.5x and 2x magnification changers create 20x and 80x magnification
      Excitation and Emission Filters:
    • X-Cite eXactate arclamp with liquid light guide
    • blue -- (DAPI, CFP)
    • green -- (GFP, YFP)
    • near red -- (mCherry, DsRed)
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    Zeiss Axioskop (upright histology)


    Click for user's guide

    Axioskop image

    Overview: This microscope is optimized to meet the needs of those who want high resolution histological work. Data can be captured with the digital camera and analyzed on an analysis station.

      Features:
    • Ultra-high resolution with the Zeiss Axiocam digital color camera
      • CCD basic resolution is 1300x1035;
      • Maximum file size per image is 3900x3030 @3x14 bit (color);
      • Digitisation is 14-bit/10MHz
      • Pixel size is 6.7 x 6.7 microns
    • QImaging software on Windows 7
    • Brightfield, phase contrast and Nomarski (DIC)
    • Upright platform

    Fu
    Image obtained from the Zeiss Axioskop

      Features:
    • Achromat 4x / 0.10 NA
    • ECPlanNeoFl 10x / 0.3 NA WD=5.2mm
    • PlanNeoFl 20x / 0.5 NA WD=2.0mm
    • Achrostigmat 40x / 1.3 NA oil WD=0.21mm
    • PHPlanNeoFl 100x / 1.3 NA oil WD=0.2mm

    • Also available:
    • 1.25x /0.035 NA
    • 25x / NA oil/glycerol
    • 40x / 0.6 NA or 40x Korr
    • 63x / 1.4 NA oil
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    Olympus SZX-Zb12 Research Stereomicroscope


    Stereomicroscope image

    Overview: The stereomicroscope is built for imaging large specimens. The scope has fluorescent, transmitted, and oblique illumination, 2 objectives, zoom focusing, and a high resolution color camera.

      Features:
    • Long working distance and wide zoom range (12:86:1)
    • 10x occulars
    • External fiber optic illuminator for oblique lighting
    • Fluorescence light: Stereo fluorescence with 100W mercury lamp, GFP longpass and Cy3 fluorescence filter set.
    • Ultra-high resolution with the Zeiss Axiocam digital color camera
      • CCD basic resolution is 1300x1035;
      • maximum file size per image is 3900x3030 @3x14 bit (color);
      • Digitisation is 14-bit/10MHz
      • Pixel size is 6.7 x 6.7 microns
    • 3I SlideBook software for image capture (Windows 7)
      Objectives:
    • 0.5x Plan fluorite PF (NA 0.055, working distance 70mm) objective plus zoom
    • 1.2x PF2
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    CRi Pannoramic Scan Whole Slide Scanner


    Click for viewer software download or free video tutorials from the 3DHistech website
    Click for our Scanner Slide Preparation Guidelines

    slide scanner photo

    Overview:

    This whole slide scanner from Cambrige Research and Instrumentation (CRi, now owned by Perkin Elmer) creates a virtual library of hisology or multi-channel fluorescent slides. Using the proprietary software available in the facility or as a free download, details on the virual slide can then be zoomed to a virtual magnifications from 0.5x-100x and exported as .tif or .jpeg for analysis or presentation with other software packages.

      Features:
    • Drop off service: drop off your slides and put your name in the queue. We'll do the rest and notify you when your scans are done. Slides are scannned on a first-come, first-served basis.
    • Automated scanning of up to 150 slides per batch.
    • Data is available on our server, so you can download it straight to any campus computer.
    • Virtual magnification range from 0.5x to 100x on high resolution (down to 0.12um/pixel) tiled images.
    • Offline image viewing software available free from 3DHistech (PC only).
    • Two cameras -- a Zeiss AxioCam MRm high sensitivity, 12-bit greyscale, 6.45um per pixel camera for fluorescence imaging and an Allied Vision Technologies Stingray F146C color, 4.6 um per pixel camera for histology.
    • 40x NA 0.95 LWD Zeiss objective.

    CRi fluorescence animation
    Sample Images from the whole slide scanner

      Fliter Cubes Available:
    • Full color histology imaging with white light
    • Up to four colors with fluorescent light: blue (DAPI), green (FITC, GFP), near red (Cy3, TexasRed, AlexaFluor 500 series) and deep red (Cy5, AlexaFluor 600 series).
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    Leica TCS SP2 AOBS Laser Scanning Confocal - Dismantled, no longer available for use

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    Abbott Hall (AB-129) Microscopes

    Leica SP5 Tandem Scanner Spectral 2-Photon Confocal


    2 photon image

    Overview: This laser scanning confocal system features software selectable conventional or high-speed resonance scanner galvanometer system with three (chilled, high sensitivity) internal PMT detectors (spectral) and two external (NDD) PMT detectors. Six visible laser lines and a tunable NIR pulsed laser (Spectra Physics Mai Tai broadband 710-990nm) provide excitation. The system features turnkey operation and full software control in addition to the features listed below.

      Features:
    • Custom fit full incubation jacket (clear) for increased thermal stability during live cell experiments
    • Automated notch filters
    • 3 chilled PMT fluorescence detectors (digital spectral definition in 1 nm increments) plus 2 HyD Hybrid GaAsP detectors, 2 NDD non-descanned detectors and one transmitted light detector. Potential for 8-, 12-, or 16-bit output.
    • Spectra Physics Mai Tai broadband Ti:sapphire laser with 10W pump (tunable from 710 to 990 nm) (up to 2W output at 810 nm)
    • Dual scanning galvanometer mirrors (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (4 channels). Standard scanner with beam park, bleaching, imaging to 8k x 8k (64 megapixels) per channel.
    • "SuperZ" galvo focusing stage, 1.5 mm range, "instantaneous" XZY scanning. Demonstrated capability of three cell volumes per second (12 x 1-mm optical slices each)
    • AOTF control of visible laser lines, EOM and ND filter attenuation of NIR
    • Excitation and emission scanning capability with linear unmixing
    • Automatically optimized confocal pinhole apertures
    • Digital zoom to 40X
    • Transmitted detector with LWD and oil lenses and filters suited for SHG imaging
    • FRET, deconvolution, and 3D software options
    • Inverted platform for imaging of slides or dishes
    • LSM objective inverter device (allows system to be used as if it were an upright microscope platform)
      Objectives on the microscope:
    • 20x / NA 0.8 dry
    • 40x / 0.8 water
    • 63x / 0.9 water
    • 63x / 1.4 oil
    • 100x / 1.46 oil
      Objectives available for use (please ask):
    • 25x / NA 0.95 water
    • Objectives listed under the SP5 STED description
      Excitation Laser Lines:
    • Violet/near UV -- 405nm (DAPI, CFP)
    • Argon -- 458nm, 476nm, 488nm (GFP, FITC), 496nm, 514nm (YFP)
    • Orange HeNe -- 561nm (Cy3) and 594nm (Texas Red)
    • Red HeNe -- 633nm (Cy5)
    • MaiTai -- tunable from 710 - 990nm
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    Marianas Yokogawa type spinning disk (inverted confocal fluorescence)


    Overview: Fully automated Yokogawa type spinning disk confocal ideal for imaging live samples. Features an automated XY stage and piezo-controlled fine Z stage (focus) positioning for mutlipoint scanning plus incubator box for temperature, CO2 and humidity control. Slidebook software controls filter cube turret, objective turret, condenser, DIC and brightfield optics and high-speed brightfield shutter.

      Features:
    • OKO full environmental control chamber (constant temperature, humidity and CO2)
    • Zeiss inverted microscope base
    • Sperical Aberration correction (SAC) optics for sharp images through thicker specimens
    • Motorized XY stage for multipoint timelapse and tiling
    • Piezo controlled fine Z-stage positioning for 3D imaging
    • Fast shutter speeds and channel switiching for high speed imaging
    • Vector high-speed point scanner for photoactivation/photoablation and FRAP experiments
    • Full microscope automation through Slidebook software
    • Two Evolve- backthinned, air chilled (-85C) CCD cameras (512x512 16um pixels)
    • DualCam DC2 beam splitter for simultaneous imaging of two channels (fliters for CFP/YFP and green/red)
      Objectives:
    • 20x Plan-Neofluar / NA 0.5 dry
    • 40x Plan-Apochromat / NA 1.3 oil
    • 63x W Plan-Apochromat / NA 1.0 water dipping
    • 100x Alpha Plan-Fluar / NA 1.45 oil
      Fluorescence Filters:
    • blue (DAPI, Alexa 350)
    • cyan (CFP)
    • green (GFP/FITC)
    • red (mCherry, Texas Red)
    • far red (Cy5)
      Lasers (all solid state):
    • 405nm (excites DAPI, Alexa 350)
    • 445 nm (excites CFP)
    • 488 nm (excites GFP, Alexa 488, FITC, Cy2)
    • 561 nm (excites Cy3, mCherry, Texas Red, Alexa 543)
    • 640 nm (excites Cy5, Alexa 633, Alexa 647)
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    Zeiss Axiovert135 (inverted widefield fluorescence)


    Click for user's manual

    Axiovert135 image

    Overview: A manual microscope with simple controls for checking fluorescent protein expression. Can do simple image capture (multi color stills, single color time lapse).

      Features:
    • Manual inverted microscope platform
    • Image capture with MicroMax camera and MicroManager software
      Objectives:
    • 20x / NA 0.5 dry
    • 40x / 1.3 oil
    • 63x / 1.4 oil
      Fluorescence Excitation and Emission Filters:
    • blue (DAPI) -- ex: 365/50; em: 450/60
    • green (GFP/FITC) -- ex: 450-490; em: 515-565
    • red (rhodamine) -- ex: 546; em: 590LP
    • far red (Cy5) -- ex: 628/40; em: blank
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    Leitz Diaplan (upright histology and fluorescence microscope)


    Leitz image

    Overview: A small, upright microscope with high-quality color camera for histology. Also features limited manual fluorescence, brightfield, DIC and darkfield imaging.

      Features:
    • QImaging MicroPublisher 3.3 RTV firewire camera
    • Darkfield and DIC filters (optimal on 25x and 40x) plus brightfield illumination
    • 50/50 ocular and camera split view
    • White balance correction through the QImaging capture software suite
      Objectvies:
    • 2.5x / NA 0.08
    • 6.3 / 0.20
    • 25 / 0.6
    • 40 / 0.7
    • 100 / 1.25 oil
      Fluorescence Excitation / Emission Filters:
    • blue -- (DAPI)
    • green -- (GFP/FITC)
    • red -- (rhodamine)
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