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Light Microscopy Facility
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Reimann Building, Room R371 215-728-5675 215-265-2296 (Pager) Michal.Jarnik@fccc.edu |
Function
Three unique imaging systems are available in the Light Microscopy Facility. One is a Zeiss Radiance 2000 laser scanning confocal microscope (LSCM), another consists of two inverted epifluorescence microscopes equipped with digital camera acquisition systems and image processing and analysis software, and another is a fluorescent dissecting microscope equipped with a z-motor driven by another acquisition and analysis program.
The Radiance 2000 LSCM has its own acquisition and analysis software, LaserSharp. The software runs on a Dell server PC, with an additional copy available on a facility workstation computer. The LSCM system provides three independent lines of excitation from three separate laser sources. The Radiance 2000 also provides three photomultiplier tubes for simultaneous imaging of three confocal channels. There is a fourth channel for non-confocal transmitted light images. The LSCM provides acquisition, display, and analysis of fluorescence and reflectance images in three dimensions. The system is housed on a Nikon E800 upright microscope. The main advantage of the LSCM over conventional microscopy is that the photodetectors only register in-focus light. The improvement in results is two-fold. First, because it blocks the out-of-focus flare, the LSCM produces sharp images. The value of this becomes apparent when the investigator visualizes cellular structures and their organization. Second, the LSCM can handle thicker samples (over 200 microns) than an epifluorescence microscope. Thus, tissue specimens such as bone, epithelia, and muscle can be imaged. The investigator can image one or many optical sections through a sample with an axial resolution of 700 nm. These sections can be displayed both as a sequence and as a three-dimensional composite. Some projects the facility has supported include localization of proteins in embryos and extensive protein localization and co-localization.
Description
The inverted epifluorescence microscopes represent a significant upgrade. The two microscopes are a Nikon TE2000 and a Nikon TE300, equipped for epifluorescence, phase contrast, and differential interference contrast optics. The TE2000 is equipped with a motorized stage, z-axis motor and is housed in a chamber with a forced-air heater, allowing for temperature control for time-lapse image acquisition. There is also a five-position excitation filter wheel, and this system is useful for multiple focal plane (z-direction) images, timelapse and multi-spectral timelapse with the use of a multiple band-pass filter cube. It is equipped with a Cascade 650 monochrome CCD, and the entire system is integrated and run by MetaVue software. The TE300, also housed in a chamber allowing for environmental control, can be used to acquire single focal plane images, and short-term timelapse. It is equipped with a SpotRT monochrome CCD, and is also run by MetaVue software. These image acquisition systems are supported by two workstation computers for image processing and analysis. The workstation software includes MetaVue and MetaMorph software for image processing and analysis, as well as AutoDeBlur software for deconvolution purposes, and Photoshop CS software used to assemble figures for publication. Investigators have used these systems extensively for timelapse imaging of green fluorescent protein (GFP), cell motility, cell migration, and cell fate determination. Others have used the analytical capabilities to quantitate relative fluorescence, quantitate motility rates, determine protein co-localization, and examine DNA damage and repair.
A Nikon SMZ1500 Zoom Stereomicroscope is equipped with three objectives, 0.5X, 1.0X, and 1.6X with an adjustable internal lens that ranges from 0.75X to 11.25X magnifcation. Together, these lenses give a range of magnification from 0.35X to 180X for the microscope. There is a mercury arc lamp attached to the microscope as well as a Prior z-drive and a color Cool-Snap CCD camera. The z-drive and camera are controlled via a computer interface running ImagePro Plus software. This system can produce dramatic images from the cellular to the small-animal scale, with the ability to image both brightfield and fluorescent specimens. Investigators have used the system to automatically quantify colony-forming assays. Others have examined GFP tagged cells in transgenic animals and whole animals injected with GFP tagged cells, as well as traditional histology specimens.
The three systems have complementary strengths. The LSCM provides for excellent three-dimensional analysis of preparations, and the advantage of the conventional epifluorescent systems lies in their sensitivity, versatility, and ease of use. The strength of the dissecting microscope lies in the variety of samples that may be imaged with it: for both brightfield and fluorescent samples, and with the color camera on this system, it is the preferred system for use with histology samples.