Two-dimensional fluorescence intensity distribution analysis: theory and applications.
A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.
Kask
P
P
EVOTEC BioSystems AG, D-22525 Hamburg, Germany.
Palo
K
K
Fay
N
N
Brand
L
L
Mets
U
U
Ullmann
D
D
Jungmann
J
J
Pschorr
J
J
Gall
K
K
D000465:Algorithms; D000937:Antigen-Antibody Reactions; D055592:Biophysical Phenomena; D001703:Biophysics; D005069:Evaluation Studies as Topic; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D008962:Models, Theoretical; D017785:Photons; D017481:Receptors, Somatostatin / Q000378:metabolism; D012235:Rhodamines; D013004:Somatostatin / Q000378:metabolism; D013806:Theophylline / Q000032:analysis / Q000276:immunology
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005456:Fluorescent Dyes; D017481:Receptors, Somatostatin; D012235:Rhodamines; D013004:Somatostatin; D013806:Theophylline