Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

Abstract

Significance: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine.Aim: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year.Approach: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as singlemolecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements.Conclusions: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.

Alteration of Mitochondrial Integrity as Upstream Event in the Pathophysiology of SOD1-ALS

“…Changes in shape, motility and hypopolarization of mitochondria in SOD1 mutant MNs should disrupt their metabolic function significantly and lead to a reduction in cellular ATP. We have used the Förster resonance energy transfer (FRET)-based ATP biosensor A-team as recently described by [ 47 , 60 ] to establish a robust and efficient way to measure relative changes in ATP concentration in individual neurons and their subcompartments. In this approach, binding of ATP by the sensor leads to its conformational change and an increase in FRET between donor (CFP) and acceptor (YFP) subunits.…”

“…In this approach, binding of ATP by the sensor leads to its conformational change and an increase in FRET between donor (CFP) and acceptor (YFP) subunits. To avoid artifacts associated with measurements of fluorescent intensity-based FRET, we utilized Fluorescence Lifetime Imaging (FLIM) approaches [ 47 , 60 ]. FLIM microscopy measures multiple single-photon fluorescence events and allows the building of a histogram, and therefore an estimation of fluorescence lifetime and amplitude (i.e., number of detected photons) for each fluorophore.…”

Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system

“…We also investigated the influence of IFN-γ on neutrophil metabolic activity by means of optical metabolic imaging, which quantifies relative amounts of reduced NADH and FAD [72,73], and by extension the redox ratio. The optical redox ratio is used to obtain information on the dynamic changes in oxidation-reduction rates in cells and is sensitive to alterations in cellular metabolic rates [72].…”

NAD(P)H Fluorescence Lifetime Imaging of Live Intestinal Nematodes Reveals Metabolic Crosstalk Between Parasite and Host

“…The time-resolved NAD(P)H uorescence signal provided further information on the molecular environment in cells and tissue. The coenzymes NADH and NADPH are able to bind to diverse enzyme and participate in this way to different metabolic pathways and to reductive biosynthesis within cells 29,[37][38][39][40] . The uorescence lifetimes of both unbound NADH and unbound NADPH lay at ≈450 ps 41,42 , both being the average of the uorescence lifetime over two folding states of each type of coenzyme molecule (≈200 ps and ≈700 ps) 43 .…”

“…As such, uorescence lifetime imaging has been used to quantify different vital parameters in cells and tissues, e.g. ionic strength, calcium levels, pH values, protein folding and cleavage using Foerster Resonant Energy Transfer (FRET), temperature or viscosity [16][17][18][19][20][21][22][23][24][25][26][27][28][29] . The uorescence of the ubiquitous co-enzymes NADH and NADPH has been extensively used for FLIM to monitor metabolic activity 27 .…”