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 d
ata 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.
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 hig
h 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.
Extracellular protein concentrations and gradients queue a wide range of cellular responses, such as cell motility and division. Spatio-temporal quantification of these concentrations as produced by cells has proven challenging. As a result, artifici
al gradients must be introduced to the cell culture to correlate signal and response. Here we demonstrate a label-free nanoplasmonic imaging technique that can directly map protein concentrations as secreted by single cells in real time and which integrates with standard live-cell microscopes. When used to measure the secretion of antibodies from hybridoma cells, a broad range of time-dependent concentrations was observed: from steady-state secretions of 230 pM near the cell surface to large transients which reached as high as 56 nM over several minutes and then dissipated. The label-free nature of the technique is minimally invasive and we anticipate will enable the quantification of deterministic relationships between secreted protein concentrations and their induced cellular responses.
The phenomena of stochasticity in biochemical processes have been intriguing life scientists for the past few decades. We now know that living cells take advantage of stochasticity in some cases and counteract stochastic effects in others. The source
of intrinsic stochasticity in biomolecular systems are random timings of individual reactions, which cumulatively drive the variability in outputs of such systems. Despite the acknowledged relevance of stochasticity in the functioning of living cells no rigorous method have been proposed to precisely identify sources of variability. In this paper we propose a novel methodology that allows us to calculate contributions of individual reactions into the variability of a systems output. We demonstrate that some reactions have dramatically different effects on noise than others. Surprisingly, in the class of open conversion systems that serve as an approximate model of signal transduction, the degradation of an output contributes half of the total noise. We also demonstrate the importance of degradation in other relevant systems and propose a degradation feedback control mechanism that has the capability of an effective noise suppression. Application of our method to some well studied biochemical systems such as: gene expression, Michaelis-Menten enzyme kinetics, and the p53 system indicates that our methodology reveals an unprecedented insight into the origins of variability in biochemical systems. For many systems an analytical decomposition is not available; therefore the method has been implemented as a Matlab package and is available from the authors upon request.
We develop a theoretical approach to the protein folding problem based on out-of-equilibrium stochastic dynamics. Within this framework, the computational difficulties related to the existence of large time scale gaps in the protein folding problem a
re removed and simulating the entire reaction in atomistic details using existing computers becomes feasible. In addition, this formalism provides a natural framework to investigate the relationships between thermodynamical and kinetic aspects of the folding. For example, it is possible to show that, in order to have a large probability to remain unchanged under Langevin diffusion, the native state has to be characterized by a small conformational entropy. We discuss how to determine the most probable folding pathway, to identify configurations representative of the transition state and to compute the most probable transition time. We perform an illustrative application of these ideas, studying the conformational evolution of alanine di-peptide, within an all-atom model based on the empiric GROMOS96 force field.
R.V.Krishnan
,A.Masuda
,V.E.Centoze
.
(2003)
.
"Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera"
.
V. Krishnan Ramanujan
هل ترغب بارسال اشعارات عن اخر التحديثات في شمرا-اكاديميا