Platinum porphyrins as phosphorescent label for time-resolved microscopy.
We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.
de Haas
R R
RR
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
van Gijlswijk
R P
RP
van der Tol
E B
EB
Zijlmans
H J
HJ
Bakker-Schut
T
T
Bonnet
J
J
Verwoerd
N P
NP
Tanke
H J
HJ
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D015704:CD4 Antigens / Q000032:analysis; D002478:Cells, Cultured; D003306:Coproporphyrins; D015200:Fluoroimmunoassay / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000737:chemistry; D008665:Metalloporphyrins / Q000737:chemistry*; D008958:Models, Molecular; D009944:Organoplatinum Compounds; D010984:Platinum; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D019809:Streptavidin; D013388:Succinimides / Q000737:chemistry
D000345:Affinity Labels; D001426:Bacterial Proteins; D015704:CD4 Antigens; D003306:Coproporphyrins; D007074:Immunoglobulin G; D008665:Metalloporphyrins; D009944:Organoplatinum Compounds; D012339:RNA, Ribosomal, 28S; D013388:Succinimides; C093483:neutravidin; C107920:platinum 3,8,13,18-tetramethyl-21H,23H-porphine-2,7,12,18-tetrapropionic acid; C107919:platinum meso-tetra(4-caroboxyphenyl)porphine; D001360:Avidin; D010984:Platinum; D019809:Streptavidin