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Laser Scanning Cytometry
The laser scanning cytometer (LSC) is manufactured by CompuCyte and can be described as a cross between a flow cytometer and a static image cytometer. It is a method that provides equivalent data to a flow cytometer but which is slide-based. It allows light scatter and fluorescence measurements but also records the position of each measurement. Cells of interest may be re-located, visualised, re-stained, re-measured and photographed. In common with some flow cytometers, the LSC measures multicolour fluorescence (up to four fluorochromes are currently possible) and light scatter on a single cell basis. This technology is of particular use when cell numbers are low eg in FNA samples, or small sub-populations are being examined or morphological information as well as phenotypic information is required.
The LSC is designed around a standard Olympus BX50 fluorescence microscope. One or two laser beams may be directed via an oscillating mirror through an objective lens - through a 20x lens the beam spot size is 5μ. The motorised stage has a computer-controlled stepper motor moving in 0.5μ increments. In this way a pixellated bit map on the slide is built up in 0.5μ squares. For each pixel, the fluorescence value for each measured parameter is recorded. The user defines a threshold parameter which is used to delineate the specific fluorescence from the cells from the background fluorescence of the slide. The computer program then draws a threshold contour within which we can measure a number of parameters, e.g. area (the number of pixels occupied by the cell), the integral value (the sum of the fluorescence values for each pixel in the contour) and the maximum pixel (the position and value of the brightest pixel within the contour). Additional factors can also be measured, e.g. cell perimeter and 'texture'.
The LSC may be seen as a complementary technology to flow cytometry. The table below highlights some of the differences between the two approaches.
| |
Flow Cytometry |
Laser Scanning Cytometry |
| Staining and measurement |
Suspension |
Slide |
| Morphology |
Yes |
Yes |
| Fluorochrome localisation |
No |
Yes |
| Multiparameter |
Yes |
Yes |
| Multiple measurements |
No |
Yes |
| Sequential analysis |
No |
Yes |
| Cell loss |
Significant |
Minimal |
| Tissue sections |
No |
Yes |
| Light scatter |
Yes |
Limited |
| Cell sorting |
Yes |
No |
| Speed |
Up to 25,000 cells/second |
100 cells/second |
Examples of applications to which the LSC has been put include:- Cell cycle studies
- Cell proliferation
- Apoptosis
- Immunophenotyping
- Enzyme kinetics
- Cytogenetics including FISH studies
- Cell-cell interaction
One useful aspect of DNA analysis by Laser Scanning Cytometry is that G2 and M cells can be separately identified - as cells enter mitosis, the DNA contracts and occupies less space - on the LSC this manifests itself as an increase in Maximum Pixel value (as the fluorescence occupies a smaller area). This is illustrated below.
Specific examples where the LSC has been used in this Laboratory are BrdU-staining on adherent cells; Comet analysis for the assessment of apoptosis; assessment of phagocytosis; assessment of cell adhesion and fluorescence localisation within T cells.
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