Strong fluorescence signals indicate the presence of a large number of fluorochrome molecules on a cell/particle. The peak emission wavelengths of FITC and PE (530 nm and 570 nm, respectively) are distant enough so that their signals can be picked up by different detectors. The absorption spectra of FITC and PE peak at approximately 495 nm and are easily excited by the argon laser's 488 nm wavelength (Note: PE has a double absorption maximum and peaks at 545 nm as well). Two of the most widely used fluorochromes that meet these criteria are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). Various fluorochromes can be used in combination if each is excited at the same wavelength (in this case, 488 nm) and their peak emission wavelengths are not too close to each other. The argon ion laser, which emits a 488 nm light, is most commonly used in cytometry because it can excite multiple fluorochromes. 2-D scatter plot of a peripheral blood sample. Lower flow rates result in greater resolution, since most of the cells pass through the center of the laser, and are typically used in applications where higher clarity is critical (e.g., DNA analysis). Generally, higher flow rates mean lower resolution and best used for qualitative measurements (e.g., immunophenotyping). A wider sample core can result in some cells passing through the laser slightly off-center and therefore intercept the beam at a less than optimal angle. Conversely, lowering the sample pressure decreases the flow rate by compressing the sample core, thereby allowing fewer cells to pass through the laser beam at one time (Figure 3). Flow rate is increased by increasing the sample pressure, which widens the sample core so that more cells pass through the interrogation point at one time. The sample flow rate is controlled by adjusting the sample pressure relative to the sheath pressure using a pressure regulator. The sample pressure is always greater than the sheath fluid pressure. Operating on principles relating to laminar flow, the sample is injected into the center of a stream of sheath fluid.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |