Light Microscopy Equipment

Location: Thomas Building, DE-512
Contact phone: (206) 667-4205
Contact e-mail: imaging@fredhutch.org

Cellomics Arrayscan Vti

Instrument type: High content reader for automated imaging and analysis of microplates.

General info: The Cellomics ArrayScan is a dedicated microscope for the automated imaging and analysis of samples in multi-well plate format, and is specifically designed for high content analysis and for image-based screening applications in multi-well plate format. The resource provides two Chamlide microcope incubator systems. The systems are portable and can be used on several microscopes. The small chamber can accept a variety of vessels, including mutli-well coverglass chambers, standard and coverglass 35 mm dishes and TC plates. The system includes a temperature controller that maintains the sample chamber at constant temperature, an objective heater to prevent heat loss from immersion objectives, a CO2 mixer and CO2 humidifier. A multigas mixer (CO2, O2 and N2) is also available, e.g. for experiments that require low oxygen conditions.

Analysis using Cellomics Arrayscan systemThe system also includes a server with 2 TB of data storage and the Cellomics High Content Informatics suite. This analysis suite includes a dozen different bio-applications allowing a variety of analysis routines, including nuclear translocation, compartmental analysis, measurements of fluorescence intensity over time, and cellular morphology.

The instrument is primarily designed for the imaging of fluorescent samples (including DAPI, Green, red and far red channels), but is also capable of collecting transmitted light images. The system is also equipped with an environmental module to provide controlled temperature and CO2 for time lapse experiments of live samples. Please contact the Resource for a list of compatible plates.

Specifications:

  • Zeiss Observer Z1 inverted microscope with transmitted light and fluorescence
  • EXFO Excite metal halide arc lamp for fluorescence excitation
  • Filter sets for DAPI, FITC, TRITC and Cy5 (Blue, Green, Red and Far Red dyes)
  • 5x/0.25 Fluar, 10x/0.3 Plan Neofluar and 20x/0.4 Epi Plan Zeiss long working distance objectives
  • High resolution Zeiss 10x/0.45 and 20x/0.8 PlanApo objectives
  • Hamamatsu Orca ER CCD camera
  • Thermo Catalyst 5 robotic arm
  • Cellomics High Content analysis software suite
  • 2 TB IBM data storage server.

Recommended Uses: high-throughput microscopy; high content analysis; high content screening; image-based screens (fixed and live samples); plate imaging; automated image analysis.

Deltavision Elite

Instrument type: 3-D deconvolution (image restoration) microscope system.

General Info: The Deltavision Elite image restoration system uses knowledge of the 3-D imaging properties of the microscope (point spread function or PSF) to deblur three-dimensional microscopy data by remapping the out-of-focus signal, thereby enhancing image resolution and contrast and providing more quantitatively accurate data. (Instructions available.)

Before and After Deconvolution

The proprietary illumination light path of the new DeltaVision Elite has been redesigned to produce intense and even illumination over the captured field of view. This results in greater sensitivity (i.e. very short exposure times, ideal for fast image acquisition), and reduces background for high contrast images. The system uses a novel light engine that provides seven user-selectable excitation bands suitable for a wide array of fluorescent labels and fluorescent proteins. The elimination of excitation filters allows very fast switching for rapid image acquisition, specially in combination with double and triple band pass emission filters. This feature is particularly useful for FRET and colocalization studies in living specimens.

DV Elite systemFinally, since the light engine produces no UV light (as opposed for instance to mercury and metal halide lamps), the risk of phototoxicity due to UV leakage is greatly reduced in live cell experiments. The instrument is ideal for high resolution imaging of thin fixed specimens (yeasts, cells, thin sections), and for time lapse imaging of live cells.

Capabilities: Multidimensional acquisition (x,y,z, wavelength, time). Up to 5 channels (fluorescence and brightfield/DIC). Multi-point acquisition. Large area acquisition (tiling). Autofocus. Fast acquisition mode.

Recommended uses: High resolution multicolor fluorescence imaging of thin fixed specimens (yeast and mammalian cells, thin tissue sections, worm embryos). Time lapse imaging of live cells and other suitable specimens. High-precision colocalization studies. FRET.

Instrument Specifications:

Microscope body: Olympus IX71 inverted microscope with proprietary high precision Applied Precision x,y, z stage, and light delivery optics.

Excitation light source: Solid state light engine with seven excitation bands at 390, 440, 475, 510, 545, 580, and 640 nm.

Emission filter sets:

  • Filter Set 1 (Standard): DAPI, FITC, TRITC, Cy5, and similar dyes.
  • Filter Set 2: DAPI, FITC, Texas Red, Cy5, and similar dyes (similar to set 1 but with red shifted red channel). Suitable for GFP/ mCherry.
  • Filter Set 3: CFP, YFP, mCherry
  • Filter Set 4: CFP, YFP, mCherry (Fast): triple band pass for very fast switching between channels (live imaging, FRET)
  • Filter Set 5: GFP, mCherry ((Fast): double band pass for very fast switching between channels (live imaging, FRET)

The filter sets are all installed and can be selected in the software.

Camera: Scientific grade high resolution cooled Photometrics HQ2 CCD camera with 6.45 x6.45 micron pixels (chip size 1024 x 1024 pixels; 12-bit).

Olympus Objectives:

  • 10x/0.4 UPlanApo (air)
  • 20x/0.75 UApo (air)
  • 40x/0.75 UPlanApo (air)
  • 40x/1.3 UPlanFl N (oil)
  • 60x/1.42 PlanApo N (oil)
  • 100x/1.4 UPlanSApo (oil)
  • 60x/1.2 PlanApo (water).

Accessories: Environmental chamber with temperature and CO2 control. "Ultimate Focus" hardware autofocus system to keep specimen in focus during extended time lapse experiments.

ImageXpress Confocal High Content System

Instrument type: High content reader for automated imaging and analysis of microplates.

General info: The ImageXpress is a high content analysis system for image-based screening applications in microplate format. The instrument is capable of imaging in fluorescence (Blue, Green, Red and Far Red channels), and transmitted light mode. The system can image in widefield (conventional fluorescence) or confocal mode. This is specially useful for 3D cell cultures and large specimens such as spheroids. The system includes an automated incubator and robotics for automated plate imaging. It is highly recommended to contact the Resource for experiment planning and for a list of compatible plates.

Incucyte

The Essen IncuCyte is an incubator microscope system for live cell imaging and cellular assays. The system allows time lapse live cell imaging of cells in a variety of vessels, including dishes, tissue culture plates, and multi-well chambers. Images can be reviewed and analyzed from a networked remote computer without opening the incubator or disturbing the cells. The system is specially suited for monitoring cell lines (cell growth, transgene expression, cell migration), and for high content assay development. Analysis software suite includes validated assays for cell migration, apoptosis, cell proliferation, angiogenesis, reporter gene expression, and more. Cellular Imaging operates two similar systems, an older Zoom, and a newer S3.

The system includes:

  • 4x, 10x, and 20x Nikon objectives
  • Enhanced phase contrast and two color (green/red) fluorescence
  • 30 TB data server
  • Incucyte image viewing and analysis software. Includes many validated assays (see web site below).

Accepts micro-plates, 35 mm dishes, and slide chambers. For a list of accepted vessels, please contact the resource.

More information on Incucyte Zoom can be found at: http://www.essenbioscience.com/essen-products/incucyte

Nikon Eclipse E800

Instrument type: Fixed stage upright transmitted light and fluorescence microscope.

General info: The Nikon Eclipse E800 is an upright fixed stage microscope, suitable for collecting 2-D images (no stacks) in transmitted light and/or fluorescence mode.

Fluorescence image by Nikon E800The microscope is all manual and easy to use. Fluorescence channels include the standard Blue, Green, Red and Far Red channels (for DAPI, FITC, Rhodamine, Texas red, Cy5, and similar dyes). Transmitted light imaging includes Phase Contrast and Differential Interference Contrast (DIC). See the Nikon E800 tutorial for instruction on use. The microscope is fitted with a sensitive high resolution monochrome camera for fluorescence image capture (Photometrics Coolsnap HQ), and a 5 Megapixel Nikon DigitalSight DS 5Mc color camera for true color transmitted light imaging. Both cameras are controlled by Nikon NIS Elements software.

Transmitted light image of lung tissue stainedObjectives: A wide selection of high quality Nikon objectives with magnifications from 1x to 100x are available, including objectives for fluorescence, DIC, and phase contrast imaging. For details, please see our Objectives database.

Cameras: Photometrics Coolsnap HQ 12-bit monochrome CCD camera for fluorescence imaging; Nikon Digital Sight DS 5Mc color CCD camera.

Recommended Uses: Transmitted light, fluorescence, immunohistochemistry, differential interference contrast (DIC), phase contrast. Image capture of fixed specimens (slides).

Nikon Ti Live

Instrument Type: Transmitted light and fluorescence widefield microscope system; swept field confocal microscope system with FRAP.

General info: The Nikon Live is a dual microscope capable of conventional (widefield) and confocal (swept field) fluorescence imaging. The microscope is primarily dedicated to time lapse imaging of living samples.

Microtubules fluorescently labeledConfocal imaging uses the Prairie Technologies swept field system to record images on a Photometrics QuantEM 512x512 pixel EMCCD camera. The confocal module is equipped with three lasers and allows imaging of CFP, GFP, YFP , RedFP, and other cyan, green and red fluorescent dyes.

A Photoactivation/FRAP system from Prairie Technologies is available in conjunction with swept field confocal imaging to allow photobleaching/photoactivation of the specimen with 405 nm, 488 nm, and 355 nm (UV) light. Cell ablation experiments are also possible with the high power UV laser.

The Nikon Live can also be used as a conventional fluorescence andn transmitted light microscope. Excitation and emission filters are available for a wide variety of fluorescent dyes and proteins, from UV dyes such as DAPI to far red dyes such as Cy5. Transmitted light imaging is also possible, including phase contrast and DIC. Images are captured on a high resolution Photometrics Coolsnap HQ2 scientific CCD camera.

The system is highly automated, allowing the imaging of multiple locations over time. The system is also equipped with a fast focus tracking mechanism (Nikon's "Perfect Focus" system) to keep samples within the focal plane during extended imaging sessions. This is one of our most versatile systems for the imaging of a variety of samples, including plates, and for time lapse microscopy.

The Prairie Technologies Photoactivation device was custom built to incorporate a UV laser, and was generously funded by the M.J. Murdock Charitable Trust.

Microscope: Nikon Ti fully automated inverted microscope with Perfect Focus and motorized x,y stage.

Objectives: Extensive range of Nikon objectives is available, with magnifications from 1x to 100x. Dry (air), oil, and water immersion objectives are available, including long working distance and Phase Contrast.

Widefield microscope specifications: High power metal halide lamp with filter sets for DAPI, CFP, GFP, FITC, YFP, Rhodamine, RFP, Texas Red, mCherry, Cy5 and similar dyes. DIC and Phase optics. Photometrics Coolsnap HQ2 high resolution CCD camera. Controlled via Nikon NIS Elements software.

Swept Field confocal specifications: 454, 488, 514 and 561 nm lasers for excitation of CFP, GFP, YFP, RFP, mCherry, FITC, Rhodamine, Texas Red and similar dyes. Photometrics QuantEM 512 x 512 pixel EMCCD camera (16 micron pixel size electron multiplication camera). Controlled via PrairieView software.

Photoactivation/Frap device specifications: 405 nm, 488 nm, and UV (355 nm) high power lasers for photoactivation, photoconversion, photobleaching and cell ablation. Controlled via PrairieView software.

Recommended Uses: Fluorescence, Phase Contrast, Differential Interference Contrast (DIC), Time lapse, Live cell imaging, Photobleaching/Photoactivation/Cell ablation.

Nikon TIRF/STORM

Instrument type: Widefield fluorescence and total internal reflection fluorescence microscope (TIRF) with super resolution (N-STORM).

General info: Total Internal Reflection Fluorescence microsopy (TIRF) uses evanescent wave technology to restrict fluorescence excitation to a very thin layer of approximately 100-200 nanometers near the coverslip surface, greatly reducing background fluorescence and improving image contrast. Therefore, TIRF is ideally suited for the detection of faint signals at or near the cell surface, and for the detection of molecules in in vitro reconstituted systems.

STORM (Stochastic Optical Reconstruction Microscopy) is a super-resolution technique that relies on the precise localization of individual molecules to provide image resolution down to a few tens of nanometers.

Vinculin stained and illuminated with TIRF on rightOur Nikon system combines TIRF and STORM on a single microscope. The system is equipped with the latest EMCCD technology (Andor iXon X3 EMCCD camera), capable of providing single molecule detection sensitivity. The system includes a four-laser launch with 405, 488, 561 and 640 nm laser lines for excitation of blue, green, red and far red dyes and fluorescent proteins. The system is capable of TIRF (as well as conventional fluorescence) imaging in four channels (Blue, green, red, far red). The system can also provide super-resolution images (tested with one channel; additional channels potentially possible).

Microscope: Nikon Ti fully automated inverted microscope with Perfect Focus, motorized x,y stage, and piezo z stage. Nikon NIS Elements software.

Objectives: Nikon 10x/0.3 objective for sample set-up, and dedicated Nikon 60x/1.49 CFI Apo TIRF oil immersion and Nikon 100x/1.49 CFI Apo TIRF oil immersion objectives for TIRF imaging. Other Nikon objectives available.

System specifications: 405 nm, 488 nm, 561 nm and 640 nm for TIRF imaging of Blue, green, red and far red dyes and fluorescent proteins. High power 640 nm laser for STORM imaging. Andor iXon X3 EMCCD camera with 512 x 512 pixel chip (16 micron pixels).

Recommended Uses: High contrast fluorescence imaging of molecules at the cell membrane (vesicular transport, adhesion, membrane receptors), including time lapse; imaging of in vitro reconstituted systems (such as molecular motors, microtubule assembly, etc); single molecule detection; super resolution imaging (localization microscopy) of fixed specimens.

The Nikon TIRF/STORM Microscope was funded in part by a generous grant by the M.J. Murdock Charitable Trust.

Tissuefax

Instrument type: digital pathology system / slide scanner.

General info: The Tissuefax is an automated microscope for the imaging of histology slides, including large tissue sections and tissue micro-array (TMA) slides. The microscope includes a motorized stage that can accept up to eight slides. Slides can be rapidly pre-viewed (imaged) at low resolution. The preview images are used to define regions of interest (ROIs) for acquisition at high magnification/resolution.

TMA galleryThe microscope is capable of either transmitted light (IHC samples) or fluorescence (IF samples) imaging. After regions have been defined, most of the imaging is automated, including slide positioning and focussing, for higher throughput and image consistency. The Tissuefax system includes two software packages, TissueQuest and HistoQuest, for the analysis and scoring of IHC and IF slides.

Specifications:

  • Zeiss AxioImager Z2 upright microscope
  • EXFO Excite metal halide arc lamp for fluorescence excitation LED illumination for transmitted light
  • DAPI, FITC, Rhodamine and Cy5 fluorescence filter cubes for fluorescence imaging of Blue, Green, Red and Far Red fluorescent dyes. 10x to 100x objectives
  • PCO Pixelfly QE high performance 1.4 MP (1392 x 1024 pixels) monochrome camera for fluorescence imaging
  • Pixelink 2 MP (1600 x 1200 pixels) color camera for transmitted light true color IHC imaging

Analysis screens for Tissuefax

Recommended Uses: Imaging of whole histology slides and tissue micro-array slides; transmitted light and fluorescence microscopy; imaging or large tissue sections. Slide digitization and archival; digital pathology; quantitation and scoring of IHC and IF staining.

Ultraview Spinning Disk

Instrument type: Spinning disk confocal microscope with FRAP/Photoactivation.

General info: The PerkinElmer Ultraview Vox is a high performance spinning disk confocal microscope system for fast imaging of living specimens.

The system is equipped with four lasers (405, 488, 561 and 640 nm) for excitation of Blue, Green, Red and Far Red fluorescent dyes and fluorescent proteins, and is fitted with two sensitive Hamamatsu ImagEM EMCCD cameras for simultaneous acquisition of green and red channels.

The system includes a Photokinesis module for photoactivation and photobleaching experiments. A high power laser for cell ablation and wounding (Photonic Instruments Micro-Point) is also available.

The instrument is reserved exclusively for live cell imaging. A stage top incubator with a temperature controller and gas mixer is available to provide a controlled environment for live cell experiments.

Specifications:

  • Nikon Ti fully motorized inverted microscope with Perfect Focus; motorized x,y stage; piezo z stage
  • 405/488/561/640 nm lasers for fluorescence excitation and photoactivation/photobleaching
  • Nikon 10x and 20x dry, 40x, 60x, 100x high NA oil immersion objectives; other objectives, including high NA water immersion objectives available upon request
  • Two Hamamatsu ImagEM EMCCD cameras (512 x 512 pixels, 16 micron pixel size, 90% Quantum Efficiency)
  • Photokinesis device for FRAP/Photoactivation
  • High power Andor/Photonic Instruments Micro-Point nitrogen laser for wounding and cell ablation (365, 405 and 440 nm wavelengths available)
  • Multi-point marking and imaging capability
  • Volocity acquisition and visualization software

Recommended Uses: Time-lapse fluorescence microscopy, live cell imaging, photobleaching/photoactivation, laser wounding and cell ablation.

The Perkin Elmer Ultraview spinning disk system was funded in part by a generous grant by the M.J. Murdock Charitable Trust.

Zeiss LSM 780 Confocal and Multi-Photon with Airyscan

Instrument type: Confocal and multi-photon microscope

General Information: This Zeiss LSM 780 NLO, installed in early 2013, is a state of the art confocal and multi photon microscope that offers outstanding versatility and sensitivity, and is the Resource's workhorse for imaging the broadest range of specimens, including cells, tissue sections, biopsies, fly embryos, embryonic and adult worms, and more. The system is equipped with a filter-less spectral detector (Zeiss QUASAR detector), allowing users complete freedom in setting up detection bands. The system is also ideal for the acquisition of spectral scans and the separation of spectrally overlapping signals.

Confocal microscopy uses optical methods to remove out-of-focus signal, thereby providing high contrast images of thick fluorescent specimens.

The benefits of multi photon imaging include deeper sample penetration, excitation of molecules that are not readily excited with conventional visible-range lasers, and increased viability of certain types of specimens during time lapse imaging.

Instrument Specifications: The system includes five lasers in the visible range providing up to seven excitation lines (405, 456, 488, 514, 561, 594, and 640 nm). The spectral detection system can be freely tuned, allowing the imaging of virtually any dye in the visible spectrum, and includes Gallium Arsenide Phosphide (GaAsP) photomultipliers with extremely high sensitivity.

The instrument is also equipped with a Coherent Chameleon Vision II ultrafast pulsed IR laser. This laser has a broad, continuous tuning range (680-1080 nm), and allows two photon and multi photon excitation of suitable dyes and fluorescent proteins.

A wide range of top-of-the-line Carl Zeiss objectives are available for most imaging needs. The system includes advanced ZEN acquisition software with a number of automated features such as large area acquisition (tiling), and multi-point visiting. Photoactivation and photobleaching (FRAP) experiments are also possible. Stage-top incubators providing controlled temperature and atmosphere for live cell experiments are available for the system. A user manual for the LSM 780 is available from the Resource upon request.