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 ophthalmoscopy.

The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.

Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity an...

https://www.worldscientific.com/doi/pdf/10.1142/S1793545819300039

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Photoluminescence lifetime imaging of upconverting nanoparticles is increasingly featured in recent progress in optical thermometry. Despite remarkable advances in photoluminescent temperature indicators, existing optical instruments lack the ability of wide-field photoluminescence lifetime imaging in real time, thus falling short in dynamic temperature mapping. Here, we report video-rate upconversion temperature sensing in wide field using single-shot photoluminescence lifetime imaging thermometry (SPLIT). Developed from a compressed-sensing ultrahigh-speed imaging paradigm, SPLIT first records wide-field luminescence intensity decay compressively in two views in a single exposure. Then, an algorithm, built upon the plug-and-play alternating direction method of multipliers, is used to reconstruct the video, from which the extracted lifetime distribution is converted to a temperature map. Using the core/shell NaGdF4:Er3+,Yb3+/NaGdF4 upconverting nanoparticles as the lifetime-based temperature indicators, we apply SPLIT in longitudinal wide-field temperature monitoring beneath a thin scattering medium. SPLIT also enables video-rate temperature mapping of a moving biological sample at single-cell resolution. Photoluminescence lifetime imaging of upconverting nanoparticles is useful for optical thermometry, but is limited for dynamic samples. Here, the authors present a wide-field and single shot approach based on compressive sensing, for video-rate upconversion temperature sensing of moving samples.

Fast wide-field upconversion luminescence lifetime thermometry enabled by single-shot compressed ultrahigh-speed imaging

We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the “start” input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the “stop” input. The time between start and stop is directly converted into position, with a resolution of 200–250 μm. The data acquisition software builds up the distribution of the photons over the...

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode.

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.

Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.

/pdf/wide-field-tcspc-based-fluorescence-lifetime-imaging-flim-3es5ydf55m.pdf

Wide-field TCSPC-based fluorescence lifetime imaging (FLIM) microscopy

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A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode.

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

Wide-field TCSPC-based fluorescence lifetime imaging (FLIM) microscopy