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The steady improvement in the imaging of cellular processes in living tissue over the last 10–15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that can measure photosynthesis, linear electron flux, and CO2 assimilation by directly scanning leaves, or by using reconnaissance imaging from a drone, an aircraft or a satellite. Lignin fluorescence can be used in imaging studies of wood for phenotyping of genetic variants in order to evaluate reaction wood formation, assess chemical modification of wood, and study fundamental cell wall properties using Förster Resonant Energy Transfer (FRET) and other methods. Many other fluorescent molecules have been characterized both within the protoplast and as components of cell walls. Such molecules have fluorescence emissions across the visible spectrum and can potentially be differentiated by spectral imaging or by evaluating their response to change in pH (ferulates) or chemicals such as Naturstoff reagent (flavonoids). Induced autofluorescence using glutaraldehyde fixation has been used to enable imaging of proteins/organelles in the cell protoplast and to allow fluorescence imaging of fungal mycelium.

We present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale.

Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER‐integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN‐containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two‐hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited‐state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV‐3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV‐3a to open PD, further indicating interactions between the two proteins. Different vMPs are capable of interacting with RTN3 and RTN6. Coexpression of the vMP CMV‐3a and RTN3 results in proteins changing subcellular localization and reduces the ability of CMV‐3a to open plasmodesmata.

Fluorescence Lifetime Imaging Microscopy (FLIM) quantifies autofluorescence lifetime to assess cellular metabolism, therapeutic efficacy, and disease progression. These dynamic and heterogeneous processes complicate signal analysis. Fit-free analysis methods such as phasor analysis are increasingly used due to limitations of fit-based approaches. However, incorporating photon-counting shot noise often leads to moderate-to-high uncertainty in detecting subtle changes. Common noise-reduction strategies can introduce errors and cause data loss. We developed noise-corrected principal component analysis (NC-PCA), which selectively identifies and removes noise to isolate the signal of interest. We validated NC-PCA by analyzing FLIM images of patient-derived colorectal cancer organoids treated with several therapeutics. First, we show NC-PCA decreases uncertainty by up to 5.5-fold compared to conventional analysis and reduces data loss over 50-fold. Then, using a merged dataset, NC-PCA reveals multiple metabolic states. Overall, NC-PCA offers a powerful, generalizable tool to enhance FLIM analysis and improve detection of biologically relevant metabolic changes.

Comprehensive imaging of both the structural and biochemical characteristics of atherosclerotic plaque is essential for the diagnosis and study of coronary artery disease because both a plaque’s morphology and its biochemical composition affect the level of risk it poses. Optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIm) are promising optical imaging methods for characterizing coronary artery plaques morphologically and biochemically, respectively. In this study, we present a hybrid intravascular imaging device, including a custom-built OCT/FLIm system, a hybrid optical rotary joint, and an imaging catheter, to visualize the structure and biochemical composition of the plaque in an atherosclerotic rabbit artery in vivo . Especially, the autofluorescence lifetime of the endogenous tissue molecules can be used to characterize the biochemical composition; thus no exogenous contrast agent is required. Also, the physical properties of the imaging catheter and the imaging procedures are similar to those already used clinically, facilitating rapid translation into clinical use. This new intravascular imaging catheter can open up new opportunities for clinicians and researchers to investigate and diagnose coronary artery disease by simultaneously providing tissue microstructure and biochemical composition data in vivo without the use of exogenous contrast agent.

INTRODUCTION Early episodic memory impairment in Alzheimer's disease (AD) is linked to synaptic dysfunction from amyloid β‐protein oligomers (oAβ), particularly affecting the dentate gyrus mossy fiber‐CA3 pathway. The APPNL‐G‐F mouse model exhibits early deficits in mossy fiber long‐term potentiation (mf‐LTP). METHODS We administered the β‐adrenergic receptor agonist isoproterenol (ISO) in vivo and phosphodiesterase type 4 inhibitor GSK356278 in vitro to assess their impact on mf‐LTP and contextual fear memory. Fluorescence lifetime imaging (FLIM)‐Förster resonance energy transfer (FRET) microscopy was used to visualize impaired and rescued cyclic adenosine monophosphate (cAMP) signaling in dentate gyrus neurons. RESULTS ISO prevented mf‐LTP impairment at 3–4 mo and improved memory by 7 mo. GSK356278 inhibited mf‐LTP deficits in a dose‐dependent manner. ISO also reduced hyperphosphorylation of synapsin I and microgliosis. DISCUSSION These findings suggest that β‐AR activation and phosphodiesterase 4 (PDE4) inhibition mitigate oAβ‐induced memory deficits, supporting enhanced cAMP signaling as a therapeutic target for early AD. Highlights Early episodic memory deficits in AD linked to oAβ‐induced synaptic dysfunction. Isoproterenol and GSK356278 improve mossy fiber‐LTP and fear memory deficits. FLIM‐FRET shows treatments restore cAMP signaling in dentate gyrus neurons. Isoproterenol reduces synapsin I hyperphosphorylation and microgliosis. Enhancing cAMP signaling may help mitigate early memory deficits in AD.

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)–FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy.

The emergence of nanobodies (Nbs) has kindled an avid interest for their use in genetic engineering and plant biotechnology. In planta expression of Nbs has relied on either stable or transient transformation approaches that are lengthy and cannot support systemic expression, respectively. In addition, there is no precedence for studies on tissue‐specific expression of Nbs. To address these issues, viral vectors could be used as an alternative, but this has not been shown. Here, this proof‐of‐concept study establishes a platform to demonstrate the phloem‐specific targeting of proteins by Nbs expressed from a citrus tristeza virus‐based vector. The vector facilitates anti‐green fluorescent protein (GFP) Nb production within the phloem of transgenic Nicotiana benthamiana plants expressing a GFP‐fused endoplasmic reticulum‐targeting peptide and that of a microtubule marker line expressing GFP‐fused α‐tubulin 6. The interaction between anti‐GFP Nb and the GFP‐tagged peptide/protein is corroborated by both pull‐down assays and fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy (FRET‐FLIM) measurements. This proof‐of‐concept platform—including validation of Nb–antigen interaction in the phloem by FRET‐FLIM analysis, which has not been described in the literature—is novel for exploring Nb‐mediated functions applicable to targeting or identifying phloem proteins and those co‐opted into the virus infection process. This proof‐of‐concept study, with validation by pull‐down assays and FRET‐FLIM measurements, establishes a platform for targeting proteins in the phloem by nanobodies expressed from a viral vector.