Laser Scanning Cytometry (LSC)
The Compucyte Laser Scanning Cytometer (LSC) provides data similar to that obtained from a flow cytometer, but has the advantage of being microscope-slide based. This system is a hybrid instrument between a flow cytometer and a microscope-based imaging system. It consists of an Olympus BX50 upright light microscope attached to a laser scanning system. The system is equipped with two lasers: an argon ion (approximately 488 nm) and a helium/neon (approximately 633 nm), and three filter block/photomultiplier tube combinations allowing for simultaneous imaging of up to three fluorophores, or two fluorophores and forward scatter. Software for analysis and data processing are part of the system, as well as a color ccd camera for image capture.
The LSC has the capability to analyze all the cells of a very small specimen. There is no need for centrifuging with potential cell loss, no need for hazardous waste disposal, specimens can be restained and observed morphologically, and the slide can be archived for later examination.
The LSC in our lab is capable of measuring 3-color fluorescence and forward light scatter and records the position (X and Y coordinates) and time of measurement of each cell. Cells of interest can be relocated by Compusort, visually confirmed using epifluorescence or rescanning with the laser, restained, remeasured and photographed using brightfield illumination.
The LSC is equipped with 2 lasers (Argon-488 and Helium/Neon-633) which are directed to a computer-controlled scanning mirror, through a scanning lens and an objective lens (10X, 20X, or 40X) of an Olympus BX-50 microscope onto a specimen on a microscope slide. The laser scan and the slide position automatically move and the fluorescent energy emitted by the excited fluorochrome is collected by the microscope's objective lens. It is partially reflected to a CCD camera to image the cells, and directed back through the scan lens and scanning mirror to a series of dichroic mirrors/interference filters, to 3 photomultipliers detecting wavelengths in the following ranges: 530-555nm (green), 600-640nm (orange-red), and 650nm (far red).
The following measurements can be made for each cell:
- Integrated and peak fluorescence.
- Area and perimeter.
- Absolute position on the slide (x,y coordinates).
- Time when the cell is measured.
- Number of probe spots (for in situ hybridization).
- Distance between probe spots.
Types of specimens that can be analyzed on the LSC:
- Cell cultures grown on microscope slides or coverslips.
- Touch preps.
- Cell suspensions.
- Paraffin-embedded tissue.