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Light Imaging Section: Equipment
** Signup for the instruments is done via our scheduling system (NIH use only). **
Zeiss LSM 780
For imaging of fixed samples and live cells; 4 lasers (405nm, 488nm, 561nm, 633nm). Inverted microscope platform. Heating and CO2 in small stage chamber (Pecon). Definite focus for fast autofocusing during live cell image acquisition. Good for FRAP etc. The new, more sensitive 780 essentially replaces the 510.
Zeiss LSM 510 Meta
The 510 Meta is based on an inverted Axiovert 200 microscope. It is equipped with 4 lasers (see Table) and several dichroic filters allowing numerous combinations of fluorophores. Spectral imaging allows the simultaneous visualization of dyes that are difficult to distinguish with filters alone. The microscope is in an environmental box which permits control of temperature, humidity, and CO2.
This is the instrument of choice for time-lapse work with live cells and techniques such as FRAP.
Leica SP5 NLO Confocal Microscope
The SP5 is built around the Leica DMI6000 inverted microscope. It is equipped with 5 lasers (see Table), 5 internal PMT detectors, a transmitted light detector and an external non-descanned detector. The MaiTai infra-red laser, a pulsed Ti:Sapphire laser tunable for excitation from 690 to 1020 nm is used for 2-photon microscopy.
The room is equipped for mouse anesthesia and the SP5 is the instrument to use for intravital microscopy.
The SP5 also includes the photon detection system from Picoquant (shown below) for single molecule techniques including Fluorescence Lifetime Imaging Microscopy (FLIM). This upgrade was added thanks to ARRA funds and became available in 2010.
Motorized upright fluorescence & transmitted light microscope with automated DIC imaging. Filters for blue, green, red and deep red fluorescence. Much brighter than the DMR which it replaces for fluorescence imaging.
The Leica DMR is an upright fluorescence microscope equipped with single bandpass filters for the observation of blue (DAPI, Hoechst), green (FITC, Alexa 488, GFP, YFP), red (Cy3, TRITC, Alexas 543,568, RFP), and deep red (Cy5, Texas Red, Alexa 592, 633, 647) fluorescent conjugates. It also has double bandpass filters for the simultaneous observation of green and red fluorophores or of blue and yellow chromophores (CFP-YFP e.g.). It has 16x, 25x, 40x, 63x, and 100x oil immersion lenses and 4x and 10x air lenses. It can be used for fluorescence and transmitted light imaging in phase, DIC, bright-field, and pseudo-dark-field mode. It is fitted with a black and white digital camera (Hamamatsu ORCA) and with a color camera (Leica) driven respectively by IPLab (from iVision) and by Leica software on a Macintosh computer.
Leica DMI6000 (formerly DMRI)
2012 upgraded inverted fluorescence microscope, for samples on chambered cover glass or plastic dishes. New objectives with correctable optics now provide as good images of samples plated on plastic dishes as from samples on glass coverslips! Filter wheel for time-lapse recordings of cell migration and Eppendorff micro-manipulator for intracellular injections can be mounted if needed.
Zeiss Axio Observer
AvZeiss Axio Observer belonging to the Skin Biology Laboratory can be accessed by prior arrangement. This inverted microscope is equipped with long working distance lenses and uses a contrast method (PlasDIC) which gives much better contrast than classic contrast techniques with cells plated on plastic. Information on the PlasDIC technique can be found here. This microscope therefore allows the examination and photography of fluorescently labeled cells on plastic dishes or plastic coverslips, for example for the evaluation of transfections with fluorescent proteins. However, fluorescence imaging of cells plated on plastic dishes remains inferior to what can be obtained when cells are plated on glass coverslips.
The system is equipped with filters for blue, green, and red fluorophores, can work in fluorescence or transmitted light modes (phase, plasDIC, bright-field), has 5x, 10x, 20x, and 40x air lenses and a Zeiss AxioCam digital camera. You can also use this easy-to-follow Powerpoint manual.
Automated and eyepiece-less microscope, the first of a new generation of instruments that aim at making microscopy easier (think of point & shoot vs. single-lens reflex in photography). Works in fluorescence (blue, green, red) and transmitted light, slides or chambered coverglass.
Specialized Microscopy Instruments
Leica TIRF System
This system is based on the Leica DMI6000 microscope and is equipped with 4 diode lasers (405 nm, 488 nm, 561 nm, 633 nm) for up to 4 color-imaging. It is equipped with an Andor 512x512 EM CCD camera and is in an environmental box to maintain temperature as desired.
Zeiss Laser Microdissection Instrument
Laser microdissection allows the physical capture of a very small area in a tissue sample. The tissue is visualized under the microscope, the area to be captured is highlighted and then cut away from the surrounding tissue by a UV laser. The same laser beam can then be used to catapult the small detached piece of tissue into the cap of a small Eppendorf tube. The captured tissue can then be analyzed, for example after amplification of the DNA.
IncuCyte (Essen Bioscience)
The IncuCyte allows you to visualize your adherent cells while they grow or differentiate over several days. It is an imaging system placed in a regular tissue culture incubator. It contains a tray that accomodates several types of tissue culture vessels (flasks, multi-well plates, dishes, micro-slides). It is used to follow cell growth, motility, morphology, fluorescent protein expression etc. by time-lapse imaging. Our unit has a single 20x objective for high-quality phase-contrast and/or fluorescence microscopy (green fluorophores). We can mix and match multiple vessels and even monitor different experiments running concurrently. Images can be collected at multiple locations as frequently as every few minutes. Vessels can be taken out for feeding or drug additions and returned to continue imaging at the original location.
We also have a module for a scratch wound assay called CellPlayer Assay, including a device to make a reproducible wound and software to automatically image the wounded area and plot recovery of cells migrating into the scratched area.
IncuCyte ZOOM (Essen Bioscience)
The IncuCyte Zoom can work with either 10x or 20x objective and collect 2-color fluorescence images (green- red) instead of the single green channel available on the original IncuCyte. Like the original instrument, it can do time-lapse imaging of preset fields in different tissue culture dishes. The images and some analysis such as cell density can be viewed remotely in real time.
Leica Slide Scanner SCN400F
The Leica slidescanner collects beautiful images of whole slides, either in transmitted light (histology slides) or in fluorescence illumination. Each scan can be zoomed in continuously from thumbnail overview to 40x magnification. Up to 384 slides can be loaded at the same time and ran with one setup. The Digital Image Hub software gives users remote access to their images with full functionality (zoom, rotation, annotation) and is useful for conferencing. Tissue image analysis allows segmentation based on color and quantitation of the images.
Leica DMZ-6 Dissection Microscope
The LIS dissection microscope can be used in transmitted light and in fluorescence; the color camera can be used to take photos of samples such as whole embryose, whole tissues etc.
Leica CM 1850 Cryostat
For sectioning at -20°C of frozen tissues, down to a few microns thickness. Equipped with disposable blades.
Two PC workstations are loaded with the full software versions of Zeiss LSM and Leica LASAF. NIAMS users also have access to the 3-D rendering and image analysis software Imaris (Bitplane AG) and the deconvolution software Huygens for confocal data (from SVI, supported by Bitplane). Information on the specifics of the commercial software packages IPLab, Imaris and Huygens deconvolution can be found on the websites of the parent companies (iVision, Bitplane, SVI). The non-commercial software packages Image J and MIPAV can be accessed freely by everyone at NIH.
A high-resolution scanner is attached to the workstation and is run via Adobe Photoshop.
Updated December 18, 2012