CompuCyte Laser Scanning Cytometer (LSC).
Our laser scanning cytometer (LSC) allows flow cytometric analysis of cells fixed to a microscope slide. This unique instrument platform allows cytometric scatter and fluorescence measurement on adherent cells where removal for traditional flow analysis would disrupt the phenotype (particularly for physiological assays such as apoptosis, plasma membrane or mitochondrial membrane potential, etc.). Since the X-Y position of each cell is saved as a parameter in the generated FCS file, events in cytometric dotplots and histograms can be relocated for brightfield or epifluorescence microscopic observation. The LSC is also useful for flow analysis of extremely low numbers of cells; cells can be wet-mounted under a coverslip and the entire area of the coverslip analyzed. Small cell samples that cannot be practically analyzed by traditional flow (~a few hundred events) can be completely analyzed by LSC. The LSC is also useful cytometric analysis of tissue sections, particularly by whole-section scanning using the "phantom" contouring function.
The laser scanning cytometer (LSC). All samples must be prepared on standard 1 x 3" microscope slides, which are mounted on the Olympus microscope stepper motor stage. Instrument electronics, lasers and PMT detectors are located in the white box on the left, which scan the slide and generate data typical of traditional flow cytometry. The CCD cameras on the microscope can be used to photograph cells relocated from the resulting cytometric data. The instrument uses Compucyte WinCyte software for acquisition, analysis and relocation on a Windows NT platform. Our instrument has been fully upgraded with an improved nanostep stage, near-IR detection optics, a violet laser diode for near-UV excitation and a Photometrix high-resolution CCD camera (the white camera to the rear of the microscope).
Lasers on the LSC. The LSC is equipped with an argon-ion 25 mW 488 nm and a HeNe 17 mW 632 nm lasers (left photo). The LSC has also been upgraded with a Power Technology violet laser diode (VLD) emitting at 405 nm (right photo). The instrument can scan and trigger with any laser individually, or in dual laser mode with argon and HeNe or argon and VLD together (bottom photo).
Filter configuration on the LSC. Like a conventional flow cytometer, the LSC uses reflecting dichroics and narrow bandpass filters to separate fluorescent signals, and photomultiplier tubes (PMTs to detect them. The filters, dichroics and PMTs are in removable modules that can be changed depending on the fluorochromes to be analyzed. Above, filter/dichroic and PMT modules on the LSC. The stepper motor stage and microscope optics are visible to the right. Below, layout of filters/dichroics and PMTs.
LSC conventional configuration for five-color detection. The default configuration uses a series of longpass dichroics to separate FITC, PE, PE-Texas Red and PE-Cy5 signals off the primary argon-ion laser, and APC or Cy5 off the secondary HeNe laser. As with traditional flow cytometry, color compensation is required and is done through the software.
LSC configuration for near-IR probe detection. The LSC can be configured for detection of far red PE-Cy7 and APC-Cy7 tandem conjugates. The conventional PE-Texas Red and PE-Cy5 / APC (650 LP) filter modules are replaced with a PE-Cy5 / APC module (with a 675/50 bandpass filter) and a PE-Cy7 / APC-Cy7 module.
LSC configuration for UV probe detection. In this configuration, the PE-Texas Red optics (normally occupying the third position) are removed, the the FITC and PE filter modules to the FL2 and FL3 positions. The DAPI/Hoechst optics are then placed in the FL1 position.
