Slide Guide Used in Fluorescence Microscope
- Thursday, 01 February 2007
The CellTracks Analyzer II is a type of epi-fluorescence microscope, where observation of fluorescence are from above (“epi”) the sample. Like a regular microscope, a fluorescent microscope produces a magnified image of the sample, only the image emanates from fluorescence of the subject, rather than from the light originally used to illuminate the sample. As a unit, CellTracks Analyzer II consists of a dedicated computer loaded with CellTracks software, mouse, and keyboard. Immunicon uses a “systems” approach to cancer detection. The CellTracks included a tube developed to preserve blood, presumably with CTC cells, en route to a lab. The device contains a system that standardizes and automates sample preparation. Images of all filters are compiled and presented in a gallery format for final cell classification by a lab technician on the computer. In a typical epi-fluorescence microscope, the illumination light (with regard to the CellTrack Analyzer II, from a mercury arc-discharge lamp) first filtered of unnecessary wavelengths and bounced off a dichroic mirror (a type of mirror that reflects some wavelengths but allows others to pass through it) to the sample. Light coming from the sample passes through the dichroic mirror again, and is then separated further from the much weaker emitted fluorescence via an emission filter. The dichroic mirror is mounted to the filter cube. The excitation and emission filters are usually also affixed to the filter cube.
In a typical protocol is as follows: a blood sample is collected in the tube and placed in a cartridge containing a reaction chamber. The cartridge, with the sample, is inserted into a cell presentation device, a fixture of two magnets held together by steel. Cells are then subjected to magnetic nanoparticles, called ferrofluids, that are conjugated to antibodies that target rare cells including CTCs. As part of the cell isolation process, a separate device that is part of the CellTracks system, called a MagNest, magnetizes the sample. This causes the targeted cells to become immuno-magnetically labeled — targeted cells that have the ferrofluids bound to them via the antibodies collect in the magnetic field. The magnetized cells are also brought closer to the surface of the reaction chamber, ready to be scanned. The magnets are then retracted, the cells are re-suspended, and staining reagents are added. Staining (also called labeling), involves target-specific, fluorescent dyes called fluorophores. In the case of the CellTracks Analyzer, three fluorophores are used, one to color the nucleus of the cell, another stains the cell membrane and other non-nucleic structures, and another colors specifically white blood cells. Cancerous cells are identified when they register with the first two dyes, but not the third. The specimen is then illuminated with light at a specific wavelength that is absorbed by the fluorophores, causing them to emit longer wavelengths of light; a common fluorophore, GFP, is harvested from jellyfish, and fluoresces a characteristic bright green when exposed to blue light. The process is very specific, allowing doctors to find a relatively small amount of targeted cells, or even one, among several billions of others.