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Fluorescence instrument response standards in two-photon time-resolved spectroscopy. 2010

Rafal Luchowski, and Mariusz Szabelski, and Pabak Sarkar, and Elisa Apicella, and Krishna Midde, and Sangram Raut, and Julian Borejdo, and Zygmunt Gryczynski, and Ignacy Gryczynski
Center for Commercialization of Fluorescence Technologies, Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107, USA. rafal.luchowski@unthsc.edu

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti:sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

UI MeSH Term Description Entries
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D013997 Time Factors Elements of limited time intervals, contributing to particular results or situations. Time Series,Factor, Time,Time Factor
D019265 Spectroscopy, Near-Infrared A noninvasive technique that uses the differential absorption properties of hemoglobin and myoglobin to evaluate tissue oxygenation and indirectly can measure regional hemodynamics and blood flow. Near-infrared light (NIR) can propagate through tissues and at particular wavelengths is differentially absorbed by oxygenated vs. deoxygenated forms of hemoglobin and myoglobin. Illumination of intact tissue with NIR allows qualitative assessment of changes in the tissue concentration of these molecules. The analysis is also used to determine body composition. NIR Spectroscopy,Spectrometry, Near-Infrared,NIR Spectroscopies,Near-Infrared Spectrometries,Near-Infrared Spectrometry,Near-Infrared Spectroscopies,Near-Infrared Spectroscopy,Spectrometries, Near-Infrared,Spectrometry, Near Infrared,Spectroscopies, NIR,Spectroscopies, Near-Infrared,Spectroscopy, NIR,Spectroscopy, Near Infrared
D036641 Microscopy, Fluorescence, Multiphoton Fluorescence microscopy utilizing multiple low-energy photons to produce the excitation event of the fluorophore (endogenous fluorescent molecules in living tissues or FLUORESCENT DYES). Multiphoton microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal confocal microscopes. Ultimately this allows spatial isolation of the excitation event, enabling deeper imaging into optically thick tissue, while restricting photobleaching and phototoxicity to the area being imaged. Fluorescence Microscopy, Multiphoton,Multiphoton Fluorescence Microscopy,Multiphoton Excitation Microscopy,Excitation Microscopies, Multiphoton,Excitation Microscopy, Multiphoton,Microscopies, Multiphoton Excitation,Microscopy, Multiphoton Excitation,Microscopy, Multiphoton Fluorescence,Multiphoton Excitation Microscopies

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