mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy (original) (raw)
Related papers
Multi-dimensional fluorescence lifetime measurements
Progress in Biomedical Optics and Imaging, 2007
In this study, we present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated singlephoton- counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope (LSM 510, Zeiss) and a Titanium:Sapphire-laser (Mira 900D, Coherent) that is used for pulsed one- and two-photon excitation. Fluorescence light emitted by the sample is dispersed by a polychromator (250is, Chromex) and recorded by a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16 channel TCSPC detector head (PML-16, Becker & Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker & Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages over fluorescence techniques that are already well established in the field of confocal microscopy, such as spectrally resolved intensity measurements or single-wavelength fluorescence lifetime measurements.
Fast fluorescence lifetime imaging techniques: A review on challenge and development
Journal of Innovative Optical Health Sciences, 2019
Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research¯elds, because of its advantages of high speci¯city and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the e±ciency at which it determines°u orescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.
A novel fluorescence lifetime imaging system that optimizes photon efficiency
Microscopy Research and Technique, 2008
Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost Digital Frequency Domain FLIM (DFD-FLIM) system which can provide lifetime resolution with quality comparable to timecorrelated single photon counting methods. Our implementation provides data natively in the form of phasors. Based on the mathematical model, we present an error analysis which shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion.
Analytical Biochemistry, 1992
We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-topixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created lifetime images of Y t-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples. The phenomenon of fluorescence is widely utilized for research in the biosciences (1-8). These applications have been focused on two divergent disciplines, time-resolved fluorescence and fluorescence microscopy. In the former one takes advantage of the high information content of the time-dependent fluorescence decays to uncover details about the structure and dynamics of macromolecules (4). Such measurements are performed almost exclusively using picosecond laser sources coupled with high-speed photodetectors. Due to
Journal of Modern Optics, 2002
Wide-®eld¯uorescence lifetime imaging with spectral resolution and optical sectioning has been performed to achieve ®ve-dimensional¯uorescence microscopy. Spectral ®ltering has been shown to have the potential to provide functional information about biological tissue by simultaneously measuring the spectral/lifetime signature of the sample. The potential to use multispectral imaging to separate cellular components spatially by their di erent emission wavelengths has also been demonstrated thus reducing artefacts in the calculated lifetime maps. The instrument is based on diode-pumped solid-state laser technology and an ultrafast gated optical image intensi®er. Also reported is the use of a picosecond blue laser diode as the excitation source to produce ā uorescence lifetime microscope with a footprint of less than 0.25 m 2 .
Journal of Biomedical Optics
Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.
Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting
Medical Photonics, 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.
Multi-dimensional fluorescence lifetime measurements
Multiphoton Microscopy in the Biomedical Sciences VIII, 2008
In this study, we present two different approaches that can be used for multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak-camera system, the other technique is based on the timecorrelated-single-photon-counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope and a Titanium:Sapphire-laser that is used for pulsed one-and two-photon excitation. Fluorescence light emitted by the sample is fed back through the scan head and guided to one of the confocal channels, where it is coupled into an optical fiber and directed to a polychromator. The polychromator disperses the emitted light according to its wavelength and focuses the resulting spectrum on the entrance slit of a streak camera or a 16 channel PMT array, which is connected to a TCSPC imaging module. With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages and pitfalls of fluorescence lifetime measurements.
Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy
2012
Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue, in vivo [1-2]. Phosphorescence lifetime imaging microscopy (PLIM) [3] is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching [4-5]. Point-measurement PLIM [6] of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM [7, 8]. However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here we experimentally demonstrate a parallel-excitation/parallel collection MPM-PLIM system that increases pixel rate by a factor of 100 compared with conventional configurations while simultaneously acquiring lifetime and intensity images at depth in vivo. Full-frame three-dimensional in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging. Current technologies for overcoming the fundamental pixel rate limitation of serialacquisition MPM require parallel excitation and imaging a sample onto multi-element detectors (typically CCD) [9-10]. While satisfactory for thin tissue slices or non-scattering samples, thick scattering samples typically encountered in in vivo applications cause crosstalk between excited pixels when imaged onto a detector array, resulting in smeared