Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.
Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.
Approach: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Forster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed.
Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

https://www.spiedigitallibrary.org/journals/journal-of-biomedical-optics/volume-25/issue-7/071203/Fluorescence-lifetime-imaging-microscopy--fundamentals-and-advances-in-instrumentation/10.1117/1.JBO.25.7.071203.pdf

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080-human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

/pdf/fluorescence-lifetime-imaging-with-a-megapixel-spad-camera-3fhgc4sqhs.pdf

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation.

Fluorescence lifetime imaging (FLIM) is increasingly used in many scientific disciplines, including biological and medical research, materials science and chemistry. The fluorescence label is not only used to indicate its location, but also to probe its immediate environment, via its fluorescence lifetime. This allows FLIM to monitor and image the cellular microenvironment including the interaction between proteins in their natural environment. It does so with high specificity and sensitivity in a nondestructive and minimally invasive manner, providing both structural and functional information. Time-Correlated Single Photon Counting (TCSPC) is a popular, widely used, robust and mature method to perform FLIM measurements. It is a sensitive, accurate and precise method of measuring photon arrival times after an excitation pulse, with the arrival times not affected by photobleaching or excitation or fluorescence intensity fluctuations. It has a very large dynamic range, and only needs a low illumination intensity. Different methods have been developed to advance fast and accurate timing of photon arrival. In this review a brief history of the development of these methods is given, and their merits are discussed in the context of their applications in FLIM.

/pdf/fast-timing-techniques-in-flim-applications-2yzh2lu52p.pdf

Fast Timing Techniques in FLIM Applications

A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Forster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein-protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

Luminescence lifetime imaging of three-dimensional biological objects.

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.

Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy.

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Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy.