Volume 2, Issue 1 - Special Issue: FLIM in material and life sciences; Guest Editors: Werner Zuschratter and Uwe Ortmann

Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These are critical for cellular processes and implicated in diseases like cancer and neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM), FLIM-FRET, and fluorescence correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, and their responses to environmental stimuli in live cells. This perspective highlights the utility of time-resolved fluorescence and single-molecule spectroscopy techniques for shedding light on condensate functions, properties, and interactions with membranes, offering insights into cellular physiology and pathology.

In this review paper, we summarize the significant advancements in the field of fluorescence lifetime imaging microscopy (FLIM), particularly wide-field FLIM with single-molecule sensitivity, achieved using the time-correlated single-photon counting-based position-sensitive LINCam system. Fluorescence lifetime adds valuable information beyond conventional intensity-based imaging, enabling diverse applications across research fields. Here, we focus on three primary bioimaging applications: (I) single-molecule FLIM in the far-red spectral region, (II) fast and multiplexed super-resolution imaging of cells, and (III) three-dimensional super-resolution imaging with high axial localization precision. Recent advances in position-sensitive detector technologies offer exciting opportunities for high-throughput super-resolution imaging with enhanced localization precision.

Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) is revolutionizing plant biology, by enabling the study of protein–protein interactions (PPIs) within live cells. This manuscript describes the principles of FRET and the practical application of FRET acceptor photobleaching (FRET-APB) in exploring PPIs in vivo . It mainly focuses on the superior characteristics of FRET-FLIM and details the materials and methods for implementing this technique in plants. It provides a profound overview about the required instruments, protocols for sample preparation, methods for calibration and acquisition, and pipelines for data analyses including novel analyses for binding and FRET efficiencies. Furthermore, it discusses the potential pitfalls and challenges related to the sample autofluorescence, protein expression heterogeneity, and acquisition photodamage or bleaching. This works aims to highlight the great prospects of FRET-FLIM in advancing our understanding of PPIs in living plant cells.

In fluorescence microscopy, discrimination of fluorochromes in multi-color labeling was originally based on the emission spectrum only, then on emission and distinct excitation wavelengths. With the advent of faster and easier to use fluorescence lifetime imaging microscope (FLIM) systems, an additional, third level of discriminating fluorochromes becomes feasible. In this tutorial, we describe how to separate two fluorochromes, one with shorter and one with longer fluorescence lifetime, in a single spectral channel. The separation is done with the help of a phasor diagram of the lifetime information. We applied the method on images made by confocal or stimulated emission depletion (FLIM-STED) microscopy but it is transferable to other FLIM methods. This approach works with considerable less photons than separation by curve fitting. Images can be recorded at speeds comparable to normal confocal or STED microscopy. One shown example has two spectral channels with two fluorochromes each, plus another neighboring color channel in which spectral bleed-through and reflection is corrected by lifetime properties. All fluorochromes as well as the hard- and software used are commercially available. Lifetime separation generally may double the number of fluorochromes that can be used in fluorescence microscopy.

This tutorial focuses on presenting experimental protocols for acquiring instrument response functions (IRF) and for calibrating the instruments using reference dyes with validated lifetime in time-domain fluorescence lifetime measurements. Step-by-step preparation of different samples used for the calibrations (scatter solutions, crystals generating second harmonic signal and reference dyes) and the corresponding instrument settings in one- and two-photon excitation are explained. The expected shape of the IRF curves and reference decays are shown using experimentally acquired examples, followed by troubleshooting of the instruments when the expected results are distorted. The discussions focus on the importance of using IRF and reference dyes for adjusting the acquisition parameters of the time-resolved instrument, for data analysis and for comparison and extrapolation of lifetime values between different biological systems.

Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) is a powerful tool for studying conformational changes of biosensors, protein-protein interactions and the dynamics of molecular processes in living cells. This approach enables measurements of molecular distances at the nanometer scale, with high spatial and temporal resolution. FLIM-FRET relies only on donor and acceptor lifetime changes induced by energy transfer thus avoiding possible artifacts of pure intensity-based FRET measurements. This tutorial provides a step-by-step workflow of widefield FLIM measurements of immune competent cells, including instrument setup, sample preparation, data acquisition and analysis. Practical guidelines are offered to optimize experimental conditions and troubleshoot some common issues. A case study highlights the application of FLIM-FRET to investigate protein conformational changes during T-cell receptor activation, demonstrating the technique’s sensitivity and versatility in the context of cellular signaling.

We present a protocol for micro-endoscopic fluorescence lifetime imaging in the femoral marrow of mice allowing the analysis of NAD(P)H-dependent metabolism at single cell level, in vivo . Therefore, we employed a gradient refractive index (GRIN) lens system fixed to the mouse femur by a specialized implant. We provide step-by-step instructions for the practical usage of the microendoscopic femoral implant and discuss experimental parameters required for reliable NAD(P)H FLIM analysis, particularly referring to photon statistics and signal-to-noise ratio. Representative results indicate metabolic heterogeneity both in marrow tissue environment and among marrow LysM + myeloid cells in vivo . We expect the here presented microendoscopic FLIM approach to enable the analysis of cellular functions and dysfunctions, beyond cellular metabolism, providing a better understanding of bone biology, in health and disease.

We describe the use of a single-photon counting imaging device to scan the surface of a flat fossil specimen. We studied an Early Triassic shrimp identified as Anisaeger longirostrus , from the Paris Biota (Idaho, USA; ca 249.1 Ma). Chemically, the specimen consists of a polycrystalline material composed of relatively monodisperse transparent crystals about 10 µm in size. We collected time-resolved fluorescence spectra at each pixel along 300 µm wide ribbons at a rate of 10 µm per second with a spatial resolution of 1 µm × 10 µm. Fluorescence is excited along a line. We demonstrate the presence of two types of crystals whose luminescence significantly differs. The first type emits at 450 nm with a decay time of 840 ps. The second type contains two independent emission centres emitting at 437 nm and 490 nm. They exhibit multiexponential decays with lifetimes of 1 ns and more than 5 ns, respectively. Although a variable proportion of long emitters was observed, the fluorescence of the fossil appeared to be relatively uniform. Thus, over time, fossilization processes have therefore resulted in a homogeneous distribution of fairly pure crystals.

During a precipitation experiment in microfluidics, crystals appear in the flow. Using a molecule that turns fluorescent in the solid phase, these crystals can be counted, measured and their polymorph identified. From this collection of data, the nucleation rate, the growth rate and the polymorph branching ratio are measured. Hundreds of crystals are analysed at a rate of one per second with a transit time in the field of view of 80 ms. Principal component analysis of the ensemble of the fluorescence photons is less efficient than the analysis of individual flowing particles identified by their burst of long-lived photons in the flow. Even if burst as small as 10 photons are detected, the smallest stable crystal , the nucleus, is not detected. The storage capacity of single photons allows deep post-analysis of transient phenomena.

The investigation of fluorescence lifetime became an important tool in biology and medical science. So far, established methods of fluorescence lifetime measurements require the illumination of the investigated probes with pulsed or amplitude-modulated light. In this paper, we examine the limitations of an innovative method of fluorescence lifetime using the strong time-frequency correlation of entangled photons generated by a continuous-wave source. For this purpose, we investigate the lifetime of IR-140 to demonstrate the functional principle and its dependencies on different experimental parameters. We also compare this technique with state-of-the-art FLIM and observed an improved figure-of-merit. Finally, we discuss the potential of a quantum advantage.