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Damm, Germany

Becker W.,Becker and Hickl GmbH
Journal of Microscopy | Year: 2012

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gain-modulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady-state fluorescence techniques. © 2012 Royal Microscopical Society. Source

Becker1 W.,Becker and Hickl GmbH
Springer Series in Chemical Physics | Year: 2015

Classic time-correlated single photon counting (TCSPC) detects single photons of a periodic optical signal, determines the times of the photons relative to a reference pulse, and builds up the waveform of the signal from the detection times. The technique achieves extremely high time resolution and near-ideal detection efficiency. The modern implementation of TCSPC is multi-dimensional. For each photon not only the time in the signal period is determined but also other parameters, such as the wavelength of the photons, the time from the start of the experiment, the time after a stimulation of the sample, the time within the period of an additional modulation of the excitation light source, spatial coordinates within an image area, or other parameters which can either vary randomly or are actively be modulated in the external experiment setup. The recording process builds up a photon distribution over these parameters. The result can be interpreted as a (usually large) number of optical waveforms for different combination of the parameters. The advantage of multidimensional TCSPC is that the recording process does not suppress any photons, and that it works even when the parameters vary faster than the photon detection rate. Typical multi-dimensional TCSPC implementations are multi-wavelength recording, recording at different excitation wavelengths, time-series recording, combined fluorescence and phosphorescence decay recording, fluorescence lifetime imaging, and combinations of these techniques. Modern TCSPC also delivers parameter-tagged data of the individual photons. These data can be used to build up fluorescence correlation and cross-correlation spectra (FCS and FCCS), to record fluorescence data from single molecules, or to record time-traces of photon bursts originating from single molecules diffusing through a small detection volume. These data are used to derive multi-dimensional histograms of the changes in the fluorescence signature of a single molecules over time or over a large number of different molecules passing the detection volume. The chapter describes the technical principles of the various multi-dimensional TCSPC configurations and gives examples of typical applications. ©Springer International Publishing Switzerland 2015 Source

Yaseen M.A.,Massachusetts General Hospital | Sakadzic S.,Massachusetts General Hospital | Wu W.,Massachusetts General Hospital | Becker W.,Becker and Hickl GmbH | And 2 more authors.
Biomedical Optics Express | Year: 2013

Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism. © 2013 Optical Society of America. Source

Becker W.,Becker and Hickl GmbH
Medical Photonics | Year: 2015

Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye. © 2015 Source

Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2007-1.2-2 | Award Amount: 7.53M | Year: 2008

The proposal aims at the development and clinical validation of advanced non-invasive optical methodologies for in-vivo diagnosis, monitoring, and prognosis of major neurological diseases (stroke, epilepsy, ischemia), based on diffuse optical imaging by pulsed near infrared light. Established diagnostic imaging modalities (e.g. X-ray Computed Tomography, Magnetic Resonance Imaging, Positron Emission Tomography) provide 3D anatomical, functional or pathological information with spatial resolution in the millimetre range. However, these methods cannot be applied continuously or at the bedside. Diffuse optical imaging is expected to provide a valuable complementing tool to assess perfusion and blood oxygenation in brain tissue and their time evolution in a continuous or quasi-continuous manner. The devices will be portable and comparably inexpensive and can be applied in adults and in children. Time-domain techniques are acknowledged as offering superior information content and sensitivity compared to other optical methods, allowing for separation between contributions of surface tissues (skin and skull) and brain tissue. Time-domain imaging can also differentiate between the effects of scatter and those of absorption.The consortium plans major developments in technology and data analysis that will enhance time-domain diffuse optical imaging with respect to spatial resolution, sensitivity, robustness of quantification as well as performance of related instruments in clinical diagnosis and monitoring. The diagnostic value of time-domain diffuse optical imaging will be assessed by clinical pilot studies addressing specific neurological disorders, in comparison with established neurophysiological and neuroimaging techniques. Perspectives regarding clinical application of time-domain diffuse optical brain imaging will be estimated and a reliable basis for a potential commercialisation of this novel technique by European system manufacturers will be created.

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