A new technique for multiparameter imaging microscopy: Use of long decay time photoluminescent labels enables multiple color immunocytochemistry with low channel‐to‐channel crosstalk

Abstract

In this report, we describe luminescence imaging microscopy using five different photoluminescent dyes in a single specimen. We combined the long decay time luminophores, europium(III) chelate, terbium(III) chelate, palladium(II) coproporphyrin, and platinum(II) coproporphyrin, with a green nuclear stain, Syto 25 trade mark, that emits conventional fast decaying fluorescence. The luminescence emissions from the five different luminophores were separated from each other by the differences in spectra and decay times using time-resolved detection. Applicability of this dye-combination for multiparameter analysis of a biological object was verified in a mixed population of peripheral blood leukocytes. Leukocyte cytocentrifugates were incubated in one step with a cocktail of luminophore-conjugated antibodies recognizing neutrophil- and lymphocyte-specific markers, followed by rapid staining with a mixture of nuclear stain and Pt-porphyrin as an eosinophil stain. The results show that multiple luminescent dyes with long decay time can be used together, and in combination with a conventional fluorophore. The separation of the signals of the long decay time labels was distinctive and enabled reliable identification of different leukocyte types, as well as an automated cell count. The long decay time luminophores together with time-resolved luminescence imaging microscopy (TR-LIM) provide a unique tool for studies of simultaneous expression of multiple antigens at the level of a single cell. In comparison with other multiparameter imaging techniques, the described technique offers increased accuracy of results, simplification of preparation procedure, and dramatic shortening of the total processing time. To our knowledge, this is the first time that simultaneous fivefold labeling/staining and analysis in a single specimen has been performed in the field of immunocytochemistry.

A Phosphorescent Iridium(III) Complex‐Modified Nanoprobe for Hypoxia Bioimaging Via Time‐Resolved Luminescence Microscopy

“…Time‐resolved luminescence imaging technology (TRLI) can effectively eliminate the interferences of short‐lived autofluorescence from long‐lived phosphorescence 13, 14, 15, 16, 17, 18, 19, 20. It includes phosphorescence lifetime imaging microscopy (PLIM) and time‐gated luminescence imaging technology (TGLI).…”

Multiparametric Cell Cycle Analysis by Automated Microscopy

“…For example, a luminescence imaging microscopy approach was described to detect 5 photoluminescent dyes in a single specimen, based on the separation of photoluminescent signals by their differences in spectra and decay times using timeresolved detection. 8 Another approach took advantage of the serial removal of the fluorescence stain using a combination of denaturation and elution techniques to successfully detect the concentration of at least 6 different antigens in each cell.…”

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

“…The long-decay emission of these probes allows time-resolved fluorescent imaging in the microsecond time domain, which can increase the sensitivity and contrast of the sensing system (11) and facilitate multiplexing (40). Phosphorescence lifetime-based oxygen imaging can be applied, allowing more straightforward determination of the absolute oxygen concentrations and eliminating the need of frequent calibrations (46).…”