Quantification of fluorescence in thick specimens, with an application to cyclin B-GFP expression in starfish oocytes.
BACKGROUND
Fluorescence imaging of living cells is widely used in cell biology. It is now being extended to thick specimens such as large cells or tissues where it is important to establish methods for obtaining quantitative fluorescence data due to the increasing importance of computational and systems biology approaches.


RESULTS
Fluorescent solutions were used as a calibration standard for determining cellular fluorescence concentrations from z series image sequences. The accuracy of the measurements was evaluated using quantitatively injected cells. Different fluorescence attenuation rates of the cytoplasm and nucleoplasm were documented, and autofluorescence levels were determined. This method was used to characterize the effect of cyclin B overexpression on cell-cycle timing in starfish oocytes. The time interval between application of maturation hormone and germinal vesicle breakdown decreased with increasing cyclin B-GFP (green fluorescent protein) concentration to a level of 100-300 nM, beyond which there was no effect.


CONCLUSIONS
Conditions for determining fluorescent probe concentrations in large cells or multicellular tissues were established, which will facilitate the collection of data for quantitative studies. This method was used to characterize the effect of cyclin B-GFP expression levels on cell-cycle timing in starfish oocytes.
Terasaki
Mark
M
Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06032, USA. terasaki@neuron.uchc.edu
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D003911:Dextrans / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008845:Microinjections; D018613:Microscopy, Confocal / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D012235:Rhodamines / Q000494:pharmacology; D013215:Starfish
D019926:Cyclin B; D056744:Cyclin B1; D003911:Dextrans; D005452:Fluoresceins; D012235:Rhodamines; C051311:rhodamine dextran; D049452:Green Fluorescent Proteins; C007740:fluorexon