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4B-O-3
BIOMEDICAL PHOTONICS
Resolving Ultrafast Fluorescence Decays with Hybrid Detectors
V. Shcheslavskiy1'2. V. Elagin2, E. Shirshin3, M. Shirmanova2, and W. Becker1
1- Becker&Hickl GmbH, Nunsdorder Ring 7-9, 12277 Berlin, Germany 2- Privolzhsky Research Medical University, Minin and Pozharsky Sq. 10/1, 603005 Nizhny Novgorod, Russia 3- Moscow State University, Vorob'evy Gory, 119435 Moscow, Russia Main author email address: vis@becker-hickl.de
It is commonly believed that autofluorescence lifetimes of biological material are in the range from a few 100 ps to about 5 ns. This is supported by fluorescence-decay data of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), an endogenous fluorophore that plays a crucial role in redox reactions in living cells and exhibits a lifetime of about 400 ps for the free form and from 2 to 5.7 ns for the protein-bound form, depending on the protein to which the fluorophore is bound. [1] While protein binding to NAD(P)H causes an increase in fluorescence lifetime and quantum yield, protein binding of flavine adenine dinucleotide (FAD) to many protein complexes (stack conformation) will typically cause significant quenching and a decrease in lifetime from 2.3 to 2.9 ns for its free state (open conformation) to 0.1 ns in its stacked conformation. [1] Lifetimes of other endogenous fluorophores are in the same range, with fast decay components down to about 200 ps. The fact that there are no faster decay time known may in part be due to the instruments. Commonly used FLIM systems have instrument response functions with a full width at half-maximum (FWHM) of about 250 ps (when using PMT detectors) and around 120 ps (when using hybrid detectors with GaAsP cathodes) [2]. This is not much faster than the dominating decay time of the unbound NADH and comparable to the fluorescence lifetime of FAD in the stacked conformation, and to melanin [1]. Therefore, one can expect that faster detectors may improve the accuracy of the fluorescence decay analysis of these endogenous fluorophores. However, there is a problem. The GaAsP cathode of the typical high- efficiency FLIM detectors limits the speed to about 120 ps. Faster detectors either have extremely small active areas (SPADS) and are thus not applicable to NDD detection in two-photon microscopes, or they have conventional photocathodes with low quantum efficiency (PMTs). A possible compromise are the new Hamamatsu R10467-06 and -07 hybrid detectors with high-efficiency bi-alkali and multi-alkali cathodes. Although the photocathodes do not reach the quantum efficiency of a GaAsP cathode the hybrid detector principle makes up for a part of the loss: unlike a conventional PMT a hybrid detector has no loss of photoelectrons at the first dynode. Virtually all photoelectrons that leave the photocathode also cause a pulse at the output of the detector. This talk presents the investigation of use of ultrafast hybrid detector based on Hamamatsu R10467-06 and its application to record the decay functions of such endogenous fluorophores as NAD(P)H, FAD, both in solutions and cells, and melanin in melanoma.
Acknowledgements: The authors acknowledge the support of the studies related to singlet oxygen detection from the Russian Science Foundation (Grant # 20-65-46018).
[1] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer New York, 3 ed. (2006).
[2] W. Becker, B. Su and A. Bermann, Better FLIM and FCS data with by GaAsP hybrid detectors, Proc. SPIE 7569, Multiphoton
Microscopy in the Biomedical Sciences X, 75690S (26 February 2010)
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