Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using
two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues.
Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp–mediated
processes in collagen tissues may be used for the selective modification of collagen fibers.
Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.
Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discriminating between
free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ1
and longer τ2), but also with the relative concentrations of both (fractions a1 and a2 correspondingly). Given the role of
NADH in cellular energetics, NADH FLIM has been applied for the noninvasive characterization of metabolic changes
in a range of pathologies. However, for the discrimination of pathological states, a proper characterization of NADH
fluorescence lifetime dynamics at physiological conditions has to be conducted. We have applied FLIM NADH for the
characterization of metabolic changes during cell culture growth. Our results demonstrate that during the exponential
growth stage there's a well expressed trends of gradual decrease of the free/bound ratio, as measured from the center
from the cell colonies. At the same time the cells at the edges of a colony exhibit higher values of the ratio. Several
possible reasons for the phenomena observed are discussed.
Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the
neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity
to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal
pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in
fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues
(97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation
of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon
counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene
product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP
become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP
is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH
and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the
process of polyQ protein aggregation per se.
Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discrimination between
free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ1
and longer τ2), but also with the relative concentrations of both (fractions α1 and α2 correspondingly). Given the role of
NADH in cellular energetics, NADH FLIM has been applied for the noninvasive characterization of metabolic changes
in a range of pathologies. However, for the discrimination of pathological states, a proper characterization of NADH
fluorescence lifetime dynamics at physiological conditions has to be conducted. We have applied FLIM NADH for the
characterization of metabolic changes during cell culture growth. Our results demonstrate that during the exponential
growth stage there's a well expressed trends of gradual decrease of the free/bound ratio, as measured from the center
from the cell colonies. At the same time the cells at the edges of a colony exhibit higher values of the ratio. Several
possible reasons for the phenomena observed are discussed.
The metabolic changes of human mesenchymal stem cells (hMSCs) during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime. An increase in mean fluorescence lifetime and decrease in the ratio between free NADH and protein-bound NADH correlated with our previously reported increase in the adenosine triphosphate (ATP) level of hMSCs during differentiation. These findings suggest that NADH fluorescence lifetime may serve as a new optical biomarker for noninvasive selection of stem cells from differentiated progenies.
Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was ~1.3 ns. The NADH average fluorescence lifetime increased to ~3.5 ns within 15 min after 1 µM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.
One of the major intrinsic fluorophores, reduced nicotinamide dinucleotide (NADH) is as sensitive non-invasive
indicator of the cellular energy metabolism, whereas measurement of its fluorescence lifetime has been demonstrated to
derive more information from the cells, than its spectrum, providing with the information on free and enzyme-bound
states dynamics of the NADH as well as its environment. This attractiveness of NADH as a non-invasive indicator
served as a basis for the rapid increase in it studies, which resulted in a number of diagnostic methods for a range of
pathological conditions, utilizing NADH. Given this growing importance of NADH thorough characterization of its
lifetime dynamics is of high importance. We have conducted a series of NADH lifetime measurements at different cell
density in the early logarithmic growth phase. The results has shown that the decrease in both short and long lifetime
compounds is the earlier event cell culture growth, than the changes in NADH lifetime components preexponential
factors ratio.
KEYWORDS: Fluorescence resonance energy transfer, Fluorescence lifetime imaging, Microscopy, Cell death, Luminescence, Proteins, Molecules, Data modeling, RGB color model, Green fluorescent protein
Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2Apro), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Förster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2Apro released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.
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