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Single Photon Counting (single + photon_counting)
Selected AbstractsA fluorescence lifetime imaging scanning confocal endomicroscopeJOURNAL OF BIOPHOTONICS, Issue 1-2 2010Gordon T. Kennedy Abstract We describe a fluorescence lifetime imaging endomicroscope employing a fibre bundle probe and time correlated single photon counting. Preliminary images of stained pollen grains, eGFP-labelled cells exhibiting Förster resonant energy transfer and tissue autofluorescence are presented. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Multispectral fluorescence lifetime imaging by TCSPCMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2007Wolfgang Becker Abstract We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [source] Towards metabolic mapping of the human retinaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2007D. Schweitzer Abstract Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40° fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 ± 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510,560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 , 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [source] Measurement of time-resolved autofluorescenceACTA OPHTHALMOLOGICA, Issue 2008D SCHWEITZER Purpose Functional alterations are first signs of reversible pathologic processes. Whereas microcirculation studies metabolism globally, autofluorescence of endogenous fluorophores has the potential for description of cellular basic processes. Therefore, a discrimination of fluorophores is required in the tissue. Methods Besides excitation and emission spectra, the fluorescence lifetime after short-time excitation is a promising substance-specific mark. Using the opto-mechanical system of a HRA II (Heidelberg Engineering), a fluorescence lifetime mapper was developed. Picosecond pulse-lasers (448nm, 468nm, 100ps FWHM, 80MHz) can be used for excitation and the emission will be detected in 2 spectral ranges (490-560nm, 560-700nm). The dynamic fluorescence will be detected in time-correlated single photon counting (SPC 150, Becker/Hickl, Berlin). An on line image registration is realised by simultaneously detected infrared images during measuring time. Approximating the fluorescence decay by 3-exponential model function, images (lifetime and amplitudes), histograms, and cluster diagrams can be calculated for interpretation. Results Examples are given for healthy subjects, AMD patients (non-exudative, exudative, geographic atrophy), diabetic retinopathy, and oedema. Measurements of excitation and emission spectra as well as lifetimes are performed of expected substances and of anatomical ocular structures for comparison. Conclusion Fluorescence lifetime measurement at the eye is a new method for evaluation of functional metabolic state. [source] | ||||||||||