Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.
Fluorescent ion indicators are widely used to measure ion concentrations in living cells. However, despite considerable
efforts in synthesizing new compounds, no ratiometric sodium indicator is available that can be excited at visible
wavelengths. Ratiometric indicators have an advantage in that measured fluorescence intensities can be corrected for
fluctuations of the indicator concentration and the illumination intensity, which is not possible when non-ratiometric
indicators are used. One way to circumvent this problem is to measure fluorescence lifetimes, which are independent of
these factors. Another way to overcome the disadvantages of a
non-ratiometric indicator dye is to embed it, together with
a reference dye, into nanoparticles. By relating the indicator fluorescence to the fluorescence of the reference dye,
inhomogeneities in the nanosensor concentration or the illumination intensity can be cancelled out reliably. In this study
we compare the benefits and drawbacks of these approaches.
Global analysis algorithms perform better than unconstrained data fitting and can improve the accuracy and precision of
the experimental data. Here, we report on different strategies that can be pursued to improve the results derived from
fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally
resolved manner and show how these data sets can be used to evaluate FRET measurements.
Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This
combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane
patches. Global analysis of the data can yield valuable information about the processes underlying channel activation.
We used this approach to study the activation of homotetrameric CNGA2 channels in inside-out membrane patches. By
using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding
and channel activation.
Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Förster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.
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
time-correlated-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.
Measurements of endogeous fluorophores open the possibility for evaluation of metabolic state at the eye. For
interpretation of 2-dimensional measurements of time-resolved auto fluorescence in 2 separate spectral ranges at the
human eye, comparing measurements were performed on porcine eyes. Determining excitation and emission spectra,
attention was drawn of proof of coenzymes NADH and FAD in isolated anatomical structures cornea, aqueous
humor, lens, vitreous, neuronal retina, retinal pigment epithelium (RPE), choroid, and sclera. All these structures
exhibit auto fluorescence, highest in lens. Excitation at 350 nm results in local fluorescence maxima at 460 nm,
corresponding to NADH, in all structures. This short-wave excitation allows metabolic studies only at the anterior
eye, because of the limited transmission of the ocular media. During excitation at 446 nm the existence of FAD is
expressed by local fluorescence maxima at 530 nm. The composition fluorescence spectra allow no discrimination
between single ocular structures. Approximating the dynamic fluorescence by a double exponential function, the
shortest lifetimes were detected in RPE and neuronal retina. The histograms of mean lifetime tM cover each other on
lens with cornea and also on sclera with choroid. Despite the lifetimes are close between RPE and neuronal retina,
the relative contributions Q1 are wide different. The gradient of trend lines in cluster diagrams of amplitudes α2 vs.
α1 allows a discrimination of ocular structures.
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.
We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. The complete setup consists of a laser scanning microscope (LSM-510, Zeiss), a polychromator (250is, Chromex), a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16-channel TCSPC detector head (PML-16, Becker and Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker and Hickl).
With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. The fluorescence emitted by the sample can be recorded in a single measurement. No filters have to be used to separate the contributions of different fluorophores to the overall fluorescence signal. When applied to Forster resonance energy transfer (FRET) measurements, the technique allows to separate the decay components of the donor and acceptor fluorescence. In this way, it is possible to reliably determine FRET efficiencies between acceptor and donor fluorophores in given subcellular structures.
We present a multi-dimensional TCSPC technique that simultaneously records the photon distribution over the time in the fluorescence decay, the wavelength, and the coordinates of a two-dimensional scan. We demonstrate the application of the technique to single-point autofluorescence measurements of skin, to multi-spectral fluorescence lifetime microscopy, and ophthalmic imaging.
Multi-dimensional time-correlated single photon counting (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 in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. When applied to Forster resonance energy transfer (FRET) measurements, these setups are capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.
Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein–protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein–protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.
In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5•106s-1. A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 107 photons per second and records double-exponential FLIM data within less than 10 seconds.
Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging in laser scanning microscopes can be combined with a multi-detector technique that allows to record time-resolved images in several wavelength channels simultaneously. The technique is based on a multi-dimensional histogramming process that records the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number. It avoids any time gating or wavelength switching and therefore yields a near-ideal counting efficiency. We show an instrument that records dual wavelength lifetime images with up to 512 x 512 pixels, and single wavelength lifetime images with up to 1024 x 1024 pixels. It resolves the components of double-exponential decay functions down to 30 ps, and works at the full scanning speed of a two-photon laser scanning microscope. The performance of the instrument is demonstrated for simultaneous lifetime imaging of the donor and acceptor fluorescence in CFP/YFP FRET systems and for tissue samples stained with several fluorophores.
We present a novel time-correlated single photon counting (TCPSC) imaging technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over time, the x-y coordinates of the scanning area and the detector channel number. The histogramming process avoids any time gating or wavelength scanning and therefore yields a near-perfect counting efficiency. Applied to resonance energy transfer (RET) measurements, the setup is capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.
We use a two-photon laser scanning microscope with a new Time-Correlated Single Photon Counting (TCSPC) imaging technique to obtain combined intensity-lifetime images for FRET measurements in living cells. Single photon pulses from a photomultiplier and signals from the scanning head are used to record the three-dimensional photon density over the time- and image coordinates. Double exponential decay analysis delivers the lifetime components of the quenched and the unquenched molecules in all pixels of the image. We use the ratio of the intensity coefficients of the fast and slow decay component to create images that show the size of the FRET effects in different parts of the cell.
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