Concept for the Traceability of Fluorescence (Beads) in Flow Cytometry: Exploiting Saturation and Microscopic Single Molecule Bleaching

Abstract

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.

Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements

“…The amount of substance can be expressed as cell count for which the coherent SI unit is the number 1. The expression of function can also be expressed as the number of specific antigens that might be determined, e.g., by counting fluorescence markers (Neukammer et al, 2005). Cell viability provides another important dimension for measurements at cellular levels, though the viability of an individual cell is an ordinal quantity that cannot be traceable to the SI.…”

Parallel microscope-based fluorescence, absorbance and time-of-flight mass spectrometry detection for high performance liquid chromatography and determination of glucosamine in urine

“…High sensitivity in LIF was greatly depended on precise optical alignments, for which specific knowledge and professional experiences were vital in LIF detection [24][25][26]. A convenient strategy to achieve on-line LIF detection for HPLC would thus be of great values, particularly for its conjunction with TOF-MS.…”

Keeping particles brilliant – simple methods for the determination of the dye content of fluorophore-loaded polymeric particles

“…dye-labeled beads. 38 Other approaches like controlled dye photobleaching and single molecule spectroscopy are slowly evolving, 39,40 yet necessitate expensive equipment and are thus not suited for process control. Fluorescence techniques enable only the comparison of different batches of fluorescent beads or fluorophore solutions (concept of molecules of equivalent soluble fluorophore, MESF), 31,32 with an accurate quantification of the dye content per bead being hampered by environmentspecific molar absorption coefficients, emission spectra and fluorescence quantum yields, dye-and matrix-specific and concentration-dependent reabsorption effects, and selfquenching.…”