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Endogenous Fluorophores (endogenous + fluorophore)
Selected AbstractsMultiphoton microscopy in life sciencesJOURNAL OF MICROSCOPY, Issue 2 2000K. König Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm,2 to GW cm,2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm,2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible. [source] Human liver autofluorescence: An intrinsic tissue parameter discriminating normal and diseased conditions,,LASERS IN SURGERY AND MEDICINE, Issue 5 2010Anna C. Croce PhD Abstract Background and Objective Autofluorescence (AF) emission is an intrinsic parameter that can provide real-time information on morpho-functional properties of biological tissue, being strictly related with their biochemical composition and structural organization. The diagnostic potentials of AF-based techniques have been investigated on normal, fibrotic, and steatotic liver tissues, in reference to histological features as evidenced by specific histochemical stainings. Materials and Methods AF emission under excitation at 366,nm has been examined on cryostatic tissue sections obtained from biopsies collected during surgical operation, by means of fluorescence imaging and microspectrofluorometric techniques. Results NAD(P)H, collagen, and vitamin A were found to be the endogenous fluorophores characterizing normal, fibrotic, and steatotic liver tissue AF, respectively. The differences of their photo-physical properties, in terms of emission amplitude, spectral shape, and response to irradiation, give rise to modifications of overall AF signal collected from tissues that allow the liver conditions to be distinguished. Conclusion The study provides a valid premise for a development of AF-based optical biopsy techniques for a real-time discrimination of liver anatomo-pathological patterns. Lasers Surg. Med. 42:371-378, 2010. © 2010 Wiley-Liss, Inc. [source] Discriminant analysis of autofluorescence spectra for classification of oral lesions in vivoLASERS IN SURGERY AND MEDICINE, Issue 5 2009J.L. Jayanthi MSc, MPhil Abstract Background and Objectives Low survival rate of individuals with oral cancer emphasize the significance of early detection and treatment. Optical spectroscopic techniques are under various stages of development for diagnosis of epithelial neoplasm. This study evaluates the potential of a multivariate statistical algorithm to classify oral mucosa from autofluorescence spectral features recorded in vivo. Study Design/Methods Autofluorescence spectra were recorded in a clinical trial from 15 healthy volunteers and 34 patients with diode laser excitation (404,nm) and pre-processed by normalization, mean-scaling and its combination. Linear discriminant analysis (LDA) based on leave-one-out (LOO) method of cross validation was performed on spectral data for tissue characterization. The sensitivity and specificity were determined for different lesion pairs from the scatter plot of discriminant function scores. Results Autofluorescence spectra of healthy volunteers consists of a broad emission at 500,nm that is characteristic of endogenous fluorophores, whereas in malignant lesions three additional peaks are observed at 635, 685, and 705,nm due to the accumulation of porphyrins in oral lesions. It was observed that classification design based on discriminant function scores obtained by LDA-LOO method was able to differentiate pre-malignant dysplasia from squamous cell carcinoma (SCC), benign hyperplasia from dysplasia and hyperplasia from normal with overall sensitivities of 86%, 78%, and 92%, and specificities of 90%, 100%, and 100%, respectively. Conclusions The application of LDA-LOO method on the autofluorescence spectra recorded during a clinical trial in patients was found suitable to discriminate oral mucosal alterations during tissue transformation towards malignancy with improved diagnostic accuracies. Lasers Surg. Med. 41:345,352, 2009. © 2009 Wiley-Liss, Inc. [source] Molecular Fluorescence Excitation,Emission Matrices Relevant to Tissue Spectroscopy,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2003Ralph S. DaCosta ABSTRACT In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation,emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250,520 nm, with fluorescence emission spectra collected in the range 260,750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal.com). [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] | ||||||||||