Fluorescence lifetime imaging in biosciences: technologies and applications (original) (raw)
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Fluorescence lifetime imaging microscopy in the medical sciences
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).
Journal of Biomedical Optics
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 intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains 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 Förster 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.
1 Brief history of fluorescence lifetime imaging
Multiphoton Microscopy and Fluorescence Lifetime Imaging, 2018
This review gives an overview of the history of fluorescence lifetime imaging (FLIM) in life sciences. FLIM microscopy based on an ultrafast laser scanning microscope and time-correlated single photon counting (TCSPC) was introduced in Jena/ Germany in 1988/89. FLIM images of porphyrin-labeled live cells and live mice were taken with an unique ZEISS confocal picosecond laser microscope. Five years later, the first in vivo FLIM on human volunteers started with time-gated cameras to detect dental caries based on one-photon wide-field pulsed laser excitation of autofluorescent bacteria. Another five years later, two-photon FLIM of autofluorescent skin was performed on a volunteer with a lab microscope in the frequency domain. The first clinical non-invasive optical, two-photon 3D FLIM biopsies were obtained fifteen years ago in patients with dermatological disorders using a certified clinical multiphoton tomograph based on a tunable femtosecond titanium:sapphire laser and TCSPC. A current major FLIM application in cell biology is the study of protein-protein interactions in transfected cells by FLIM-FRET microscopy. Clinical FLIM applications are still on a research level and include preliminary studies on (i) one-photon FLIM autofluorescence microscopy of patients with ocular diseases using picosecond laser diodes, (ii) time-gated imaging in brain surgery using a nanosecond nitrogen laser, and (iii) two-photon clinical FLIM tomography of patients with skin cancer and brain tumors with near-infrared femtosecond lasers and TCSPC.
Basis: Brief history of fluorescence lifetime imaging
Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine, 2018
This review gives an overview of the history of fluorescence lifetime imaging (FLIM) in life sciences. FLIM microscopy based on an ultrafast laser scanning microscope and time-correlated single photon counting (TCSPC) was introduced in Jena/ Germany in 1988/89. FLIM images of porphyrin-labeled live cells and live mice were taken with an unique ZEISS confocal picosecond laser microscope. Five years later, the first in vivo FLIM on human volunteers started with time-gated cameras to detect dental caries based on one-photon wide-field pulsed laser excitation of autofluorescent bacteria. Another five years later, two-photon FLIM of autofluorescent skin was performed on a volunteer with a lab microscope in the frequency domain. The first clinical non-invasive optical, two-photon 3D FLIM biopsies were obtained fifteen years ago in patients with dermatological disorders using a certified clinical multiphoton tomograph based on a tunable femtosecond titanium:sapphire laser and TCSPC. A current major FLIM application in cell biology is the study of protein-protein interactions in transfected cells by FLIM-FRET microscopy. Clinical FLIM applications are still on a research level and include preliminary studies on (i) one-photon FLIM autofluorescence microscopy of patients with ocular diseases using picosecond laser diodes, (ii) time-gated imaging in brain surgery using a nanosecond nitrogen laser, and (iii) two-photon clinical FLIM tomography of patients with skin cancer and brain tumors with near-infrared femtosecond lasers and TCSPC.
Time-domain fluorescence lifetime imaging applied to biological tissue
Photochemical & Photobiological Sciences, 2004
Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue autofluorescence, it reveals intrinsic excellent contrast between different types and states of tissue. This article aims to review our recent progress in developing time-domain FLIM technology for microscopy and endoscopy and applying it to biological tissue.
2009
Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo. For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we show the application of this method for intravital imaging of direct protein-protein interactions.
Multidimensional multiphoton fluorescence lifetime imaging of cells
Multiphoton Microscopy in the Biomedical Sciences VIII, 2008
We have used an experimental arrangement comprising two photomultipliers and time-correlated single photon counting (TCSPC) detection to measure time and polarization-resolved fluorescence decays and images simultaneously. Polarization-resolved measurements can provide information which may be difficult to extract from lifetime measurements alone. The combination of fluorescence lifetime and time-resolved anisotropy in an imaging modality with two detectors minimizes the errors arising from bleaching of a sample between consecutive measurements. Anisotropy measurements can provide evidence of fluorescence resonance energy transfer between chemically identical fluorophores (homo-FRET). This phenomenon is not detectable in spectral or lifetime changes, yet a lowering of the anisotropy and a faster anisotropy decay can provide evidence for close proximity (≤ 10 nm) of adjacent fluorophores including dimerization and oligomerization of molecules. We have used FLIM and fluorescence anisotropy to measure variations in fluorescence lifetimes and anisotropy of GFP-tagged proteins in cells in immunological synapse samples and also acquire images of BODIPY-stained carcinoma cells.
Journal of Modern Optics, 2002
Wide-®eld¯uorescence lifetime imaging with spectral resolution and optical sectioning has been performed to achieve ®ve-dimensional¯uorescence microscopy. Spectral ®ltering has been shown to have the potential to provide functional information about biological tissue by simultaneously measuring the spectral/lifetime signature of the sample. The potential to use multispectral imaging to separate cellular components spatially by their di erent emission wavelengths has also been demonstrated thus reducing artefacts in the calculated lifetime maps. The instrument is based on diode-pumped solid-state laser technology and an ultrafast gated optical image intensi®er. Also reported is the use of a picosecond blue laser diode as the excitation source to produce ā uorescence lifetime microscope with a footprint of less than 0.25 m 2 .