Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F?rster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Fluorescence lifetime imaging of calcium using Quin-2.

We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.     

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.     

Quantitative imaging of protein interactions in the cell nucleus

Over the past decade, genetically encoded fluorescent proteins have become widely used as noninvasive markers in living cells. The development of fluorescent proteins, coupled with advances in digital imaging, has led to the rapid evolution of live-cell imaging methods. These approaches are being applied to address biological questions of the recruitment, co-localization, and interactions of specific proteins within particular subcellular compartments. In the wake of this rapid progress, however, come important issues associated with the acquisition and analysis of ever larger and more complex digital imaging data sets. Using protein localization in the mammalian cell nucleus as an example, we will review some recent developments in the application of quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. In this report, we review the principles of acquiring fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins. We then discuss how fluorescence lifetime imaging microscopy (FLIM) provides a method that is independent of intensity-based measurements to detect localized protein interactions with spatial resolution. Finally, we consider potential problems associated with the expression of proteins fused to fluorescent proteins for FRET-based measurements from living cells.     

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins.     

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.     

Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. METHODS: A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. RESULTS: In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. CONCLUSIONS: Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.     

A Cell-Permeable Fluorescent Polymeric Thermometer for Intracellular Temperature Mapping in Mammalian Cell Lines

Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.     

Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET–FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET–FLIM to still be quantitative.     

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and space-correlated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10mW/cm²). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.     

Global analysis of fluorescence lifetime imaging microscopy data

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.     

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.     

Application of the stretched exponential function to fluorescence lifetime imaging

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.     

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.