Fluorescence analysis of picoliter samples

The fluorescence intensity of picoliter-volume samples was quantitated by taking samples and standards into a single siliconized capillary, fixing the capillary under the objective of a microscope-fluorometer, and defining an effective “fluorescence chamber” within the capillary by placing an imaging diaphragm in the emission path. Samples were then moved, by pneumatic control with an air syringe, within the capillary to this “fluorescence chamber.” A diaphragm in the excitation path limited the volume of sample excited. Fluorescence from all samples was thus directly determined under identical optical conditions. The co-efficient of variation of replicate measurements was 3%; carry-over from sample to sample within the capillary was less than 2%. A 3-pl sample containing 0.3 amol of sodium fluorescein (about 200,000 molecules) could be discriminated from the background; fluorescence intensity was linear with concentration for three to four orders of magnitude. Fluorescence intensities of NADH and an ammonia-o-phthalaldehyde-thiol adduct were also determined. Using this “fluorescence chamber” allowed straightforward scaling down of a fluorescence assay for urea in 20-pl samples, lowering the limit of detection to 10 fmol, three orders of magnitude below a previously reported microscale assay. This technique is applicable to many fluorescence assays used in studies of cell physiology, and should allow routine measurement of metabolites in individual cells or of enzymes in individual subcellular organelles.