The "pentamethyl BODIPY stain" http://www.probes.com/servlets/product?region=USA&item=3922 is maximally excited by the 488-nm argon-ion laser and its fluorescence is nowhere near as solvatochromic as nile red. At high concentrations, however, it begins to show excimers with a shift of its green fluorescence to red (with a >515 nm long-pass filter this may appear to be yellow). Cytometry 1994 Oct 1;17(2):151-8 Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Gocze PM, Freeman DA. Department of Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City. Neutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid-stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4-fold more variability than cell size and 2.6-fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet-specific fluorescent dye, bodipy, reduced the variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S-phase cells, which in turn contained more lipid than G0 + G1 cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate-treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescent variability could be reduced by an average of 44% when a more lipid droplet-specific stain was used instead of nile red. A BODIPY cholesterol analog http://www.probes.com/servlets/product?region=USA&item=3927 should work similarly and be even more likely to stain only lipids. I am surprised by the quenching of the chlorophyll when stained with nile red. I would expect the red fluorescence to have increased due to FRET from the nile red to the chlorophyll. Perhaps you are seeing quenching of a second excited state singlet because the chlorophyll is excited at 488 nm. The broad emission of nile red has a gradient of maxima with the lipid environment not specifically yellow and red. Its quantum yield also tends to decrease considerably if in the polar lipids. Its emission actually overlaps that of chlorophyll too to some extent. Absorption and fluorescence emission spectra of nile red bound to phospholipid bilayer membranes: [Spectra: 12KB] Adelina de la Jara Valido wrote: > Hello dear all, > > I am trying to measure neutral and polar lipids in microalgae on a > FACSCalibur flow cytometer equipped with an argon laser using the dye Nile > red. With phytoplankton cells I get yellow fluorescence (FL2) for neutral > lipids and red fluorescence (FL3) for polar lipids and chlorophyll. Looking > at my results I think I am probably getting some kind of antagonistic effect > between both, the polar fraction and the chlorophyll fluorescences, since > the red fluorescence produced by non stained cells (chlorophyll in control > samples) is higher than that one produced by stained cells (polar lipids + > chlorophyll). > > My question is: is it possible to properly compensate FL2 and FL3 using > stained cells with the solvatochromic dye Nile red (since this single dye is > supposed to fluoresce in different channels depending on the chemical > environment)?, do you know about any other possibility?. > > And a last question, does anyone know about a more specific dye for long > chain fatty acids that can be excited at 488 nm? > > Thank to all of you for your invaluable help. > > Adelina de la Jara > Centro de Algología Aplicada > Gran Canaria > Spain > > _________________________________________________________________ > Charle con sus amigos online usando MSN Messenger: http://messenger.msn.com
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