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Register a new userProbing subtle fluorescence dynamics in cellular proteins by streak camera based Fluorescence Lifetime Imaging Microscopy
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Added by
V. Krishnan Ramanujan
Publication date
2003
fields
Biology
and research's language is
English
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We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility. We demonstrate the applicability of this instrument in living cells for measuring the effects of protein targeting and point mutations in the protein sequence which are not obtainable in conventional intensity based fluorescence microscopy methods. We discuss the relevance of such time resolved information in quantitative energy transfer microscopy and in measurement of the parameters characterizing intracellular physiology.
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Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.
We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging.
Fluorescence lifetime imaging microscopy (FLIM) systems are limited by their slow processing speed, low signal-to-noise ratio (SNR), and expensive and challenging hardware setups. In this work, we demonstrate applying a denoising convolutional network to improve FLIM SNR. The network will be integrated with an instant FLIM system with fast data acquisition based on analog signal processing, high SNR using high-efficiency pulse-modulation, and cost-effective implementation utilizing off-the-shelf radio-frequency components. Our instant FLIM system simultaneously provides the intensity, lifetime, and phasor plots textit{in vivo} and textit{ex vivo}. By integrating image denoising using the trained deep learning model on the FLIM data, provide accurate FLIM phasor measurements are obtained. The enhanced phasor is then passed through the K-means clustering segmentation method, an unbiased and unsupervised machine learning technique to separate different fluorophores accurately. Our experimental textit{in vivo} mouse kidney results indicate that introducing the deep learning image denoising model before the segmentation effectively removes the noise in the phasor compared to existing methods and provides clearer segments. Hence, the proposed deep learning-based workflow provides fast and accurate automatic segmentation of fluorescence images using instant FLIM. The denoising operation is effective for the segmentation if the FLIM measurements are noisy. The clustering can effectively enhance the detection of biological structures of interest in biomedical imaging applications.
Using a streak camera, we directly measure time- and space-resolved dynamics of N2+ emission from a self-seeded filament. We observe characteristic signatures of superfluorescence even under ambient conditions and show that the timing of the emitted light varies along the length of the filament. These effects must be taken into consideration for accurate modelling of light filaments in air, and can be exploited to engineer the temporal profile of light emission in air lasing.
Fluorescence-lifetime imaging microscopy (FLIM) was applied to investigate the donor distribution in SrTiO3 single crystals. On the surfaces of Nb- and La-doped SrTiO3, structures with different fluorescence intensities and lifetimes were found that could be related to different concentrations of Ti3+. Furthermore, the inhomogeneous distribution of donors caused a non-uniform conductivity of the surface, which complicates the production of potential electronic devices by the deposition of oxide thin films on top of doped single crystals. Hence, we propose FLIM as a convenient technique (length scale: 1 $mu$m) for characterizing the quality of doped oxide surfaces, which could help to identify appropriate substrate materials.
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