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MAF is pleased to announce the publication of a special issue of 21 research articles on the applications and development of Fluorescence Lifetime Imaging (FLIM). The collection highlights and celebrates the advances that were enabled by FLIM, and looks to the future to see where to go next in this highly interdisciplinary field at the physical sciences/life sciences interface.
It is now over 30 years since the first two reports introducing FLIM as a fluorescence microscopy method were published [1, 2].
Rather than performing single point fluorescence spectroscopy through a microscope objective, the work provided, for the first time, proper 2-dimensional images of cell samples on the micrometer scale. The contrast in the images was given by the fluorescence lifetime of the fluorophores, independent of their concentration.
These papers marked the beginning of a whole new field of microscopy, with substantial instrumentation development, data analysis strategies and commercial enterprises to follow.
We are delighted that one of the pioneers of this technique, Karsten König [1], still working with FLIM, has contributed an update of FLIM for clinical applications. Using two-photon excitation, FLIM provides rapid non-invasive and label-free intratissue autofluorescence biopsies of skin with picosecond temporal resolution [3].
FLIM of autofluorescence is a major application of a technique, which is reflected in this article collection. Autofluorescence from cofactors NAD(P)H and FAD, which fluoresce in the blue and green region of the spectrum (460–500 nm (NAD(P)H) and 520–560 nm (FAD)), is an indicator of metabolic activity. Barkauskas et al study mouse livers in vivo following induction of various disease states and show that NAD(P)H amplitude ratio, and the FAD/NAD(P)H fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis, biliary fibrosis and hepatocellular carcinoma [4].
Autofluorescence FLIM can also be used ex vivo to study metabolism in human tumors at the cellular level, as reported by Lukina at al [5].
Indeed, the autofluorescence imaging approach can be extended to archived unstained histopathology tissues samples, as described by Chacko and Eliceiri. They show that the lifetime-based metabolic contrast in a sample is preserved after chemical fixation even after long periods of storage. Autofluorescence FLIM can thus be used to image archived unstained histopathology tissues to draw metabolic interpretations [6].
Rodimova et al report that the metabolic changes in induced pluripotent stem cells undergoing differentiation in two directions, dermal and epidermal, can be visualised by two-photon excited autofluorescence FLIM. This is achieved, again, by measuring the optical redox ratio of FAD/NAD(P)H and mapping and tracing the fluorescence lifetimes of NAD(P)H and FAD [7].
A different type of autofluorescence, namely that of chlorophyll in algae, is affected by pH. Marcek Chorvatova et al show FLIM of algae can be used to understand the responsiveness of algae to acidification, and that chlorophyll fluorescence from algae can potentially serve as a biosensing tool for monitoring pH change in their natural environment [8].
A strategy to optimise an autofluorescence FLIM approach is outlined by Cao et al [9] who share their thoughts on two-photon excitation and emission wavelengths, acquisition time, and fluorescence decay fitting strategies and phasor plot representation of FLIM data.
Regarding the optimisation and development of FLIM, Trinh et al describe the theoretical background that justifies the developments of advanced high-speed single photon counting systems for FLIM [10].
Another advance is described by Ulku et al, who employ a novel wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM. They characterise the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision, and find that the SPAD detector is essentially an ideal shot noise limited sensor, capable of video rate FLIM measurement [11].
A range of FLIM advances are reviewed in Poudel et al, again highlighting SPAD arrays and other detection methodologies, as well as multiparameter FLIM, i.e. its combination with polarization and spectral detection, or other imaging modalities such as total internal reflection fluorescence, lightsheet or stimulated emission depletion microscopy [12].
Rehman et al demonstrate a different advance, stimulated emission-based fluorescence lifetime measurements, that was able to achieve a high temporal resolution of ∼4 ps, for depolarization studies of fluorophore ATTO 647N. It has enabled the authors to measure the time-resolved fluorescence anisotropy as a function of temperature, and utilised high temporal resolution for the measurements of hetero-FRET between ATTO 647N and gold nanorods [13].
A major application of FLIM is to identify Förster Resonance Energy Transfer (FRET), a photophysical effect where the excited state energy of a donor fluorophore is non-radiatively transferred, via a dipole-dipole coupling mechanism, to an acceptor molecule in close proximity. This phenomenon was first described by physical chemist Theodor Förster in the 1940s [14, 15], and has over the last decades gained in importance, because it can be used to infer the proximity of fluorophores on the nanometer scale with optical methods.
Schneckenburger has contributed a brief summary on the topic of FRET [16].
Godet and Mely report on the study of protein-protein interactions with FLIM-FRET in bacteria, based on green fluorescent protein (GFP) as a donor, and its red variant mCherry as an acceptor, and introduce a graphical method to provide information about stoichiometry and binding mode, even in the presence of large differences in protein expression levels [17].
Sizaire et al report on applications of FLIM FRET of genetically-encoded FRET biosensors to detect the kinase AURKA, using a time-gated FLIM approach combined with a spinning disk microscope which allows rapid automatic screen analysis in a 96-well plate format [18].
Tran et al also use FLIM FRET to elucidate 'quinary stoichiometry' of proteins, i.e. a set of macromolecular interactions between proteins that are transient in vivo. They employ FLIM of organic fluorophores linked to proteins in HeLa cells and observe them during cell division. They then monitor fluorescence lifetime dequenching during multiple cell divisions, and analyse the data based on a fluorescence lifetime self-quenching model. They find that at any given point in time, there are five or six weakly interacting partners in the immediate vicinity of any given protein in living HeLa cells [19].
The work of Pliss and Prasad is also related to protein concentration, but they use a different approach to gain information about this, exploiting the fact that the average fluorescence lifetime of GFP is a function of the refractive index. Here, FLIM is used to image the average fluorescence lifetime of GFP, to map the refractive index as a proxy for protein concentration, for example during cell division [20].
Zeng et al also employ GFP, and perform FLIM of oocytes, relatively large samples which are a millimeter in diameter. They find a spatially varying average GFP fluorescence lifetime that decreases from the centre to the edge of the oocyte. They advance several possible reasons for this novel observation, such as GFP self-quenching, pH or the refractive index [21].
Li et al describe a fibre-based intraluminal imaging system for measurements inside a bioreactor for tissue grafts. They take simultaneous and co-registered measurements of endogenous fluorescence lifetimes of FAD and NADH and exogenous marker GFP fluorescence intensity. Using a phantom and millimetre-sized tissue graft samples, they show that this system could be a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in three dimensions [22].
Sapermsap et al describe the use of amplitude weighted lifetime, intensity weighted lifetime and phasor plots to analyse FLIM images of Raw macrophage cells from mice that were infected with bacteria. They find that to distinguish between GFP-labelled proteins and Fluor 546-labelled macrophages, the amplitude weighted lifetime model is superior in visually differentiating bacteria that are in extra- and intracellular and membrane-bounded locations, whereas the intensity weighted lifetime model provides excellent precision [23].
Finally, FLIM does not necessarily have to be performed on living samples, or even life science samples. Its application to forensic sciences [24] and art conservation [25, 26] has been reported in the past, and the present collection includes a report on spectral FLIM of colour centres in diamond by Jones et al where the samples do not move, and do not bleach! [27]
In addition, Xu et al report on the use of light to induce the aggregation from solution of a series of photoluminescent conjugated polyelectrolytes containing tetraphenylethene units. They monitor the photoaggregation process by time-resolved fluorescence imaging techniques, and find that structural differences in the polymer lead to variations in the photo-induced aggregation behaviour [28].
In this special issue we aim to showcase the forefront of FLIM applications and development. Perhaps in the future, the wavelength, position, polarization and arrival time of every photon detected from the sample will be recorded, and thus a maximum of information will be gained about the immediate environment of the fluorophore (ideally with a minimum of acquisition time and maximum of sensitivity). Will it be before or after the 40th anniversary of FLIM? In any case, SPAD arrays are set to play a major role in these advancements [11, 12]. And will the FLIM wavelength range be extended for routine FLIM measurements in the UV and infrared? And what will the 50th anniversary of FLIM bring? For the time being, enjoy reading about the progress, innovative approaches and insight gained by more than 30 years of FLIM.
Klaus Suhling
Simon Ameer-Beg
Marina Kuimova
London 2020