Frequency-domain Fluorescence Lifetime Imaging Research Articles

Phase-Sensitive Fluorescence Image Correlation Spectroscopy.

Fluorescence lifetime imaging microscopy is sensitive to molecular interactions and environments. In homo-dyne frequency-domain fluorescence lifetime imaging microscopy, images of fluorescence objects are acquired at different phase settings of the detector. The detected intensity as a function of detector phase is a sinusoidal function that is sensitive to the lifetime of the fluorescent species. In this paper, the theory of phase-sensitive fluorescence image correlation spectroscopy is described. In this version of lifetime imaging, image correlation spectroscopy analysis (i.e., spatial autocorrelation) is applied to successive fluorescence images acquired at different phase settings of the detector. Simulations of different types of lifetime distributions reveal that the phase-dependent density of fluorescent objects is dependent on the heterogeneity of lifetimes present in the objects. We provide an example of this analysis workflow to a cervical cancer cell stained with a fluorescent membrane probe.

Identification of different plastic types and natural materials from terrestrial environments using fluorescence lifetime imaging microscopy

Environmental pollution by plastics is a global issue of increasing concern. However, microplastic analysis in complex environmental matrices, such as soil samples, remains an analytical challenge. Destructive mass-based methods for microplastic analysis do not determine plastics’ shape and size, which are essential parameters for reliable ecological risk assessment. By contrast, nondestructive particle-based methods produce such data but require elaborate, time-consuming sample preparation. Thus, time-efficient and reliable methods for microplastic analysis are needed. The present study explored the potential of frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) for rapidly and reliably identifying as well as differentiating plastics and natural materials from terrestrial environments. We investigated the fluorescence spectra of ten natural materials from terrestrial environments, tire wear particles, and eleven different transparent plastic granulates <5 mm to determine the optimal excitation wavelength for identification and differentiation via FD-FLIM under laboratory conditions. Our comparison of different excitation wavelengths showed that 445 nm excitation exhibited the highest fluorescence intensities. 445 nm excitation was also superior for identifying plastic types and distinguishing them from natural materials from terrestrial environments with a high probability using FD-FLIM. We could demonstrate that FD-FLIM analysis has the potential to contribute to a streamlined and time-efficient direct analysis of microplastic contamination. However, further investigations on size-, shape-, color-, and material-type detection limitations are necessary to evaluate if the direct identification of terrestrial environmental samples of relatively low complexity, such as a surface inspection soil, is possible.Graphical

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

Multilayer Perceptron Development to Identify Plastics Using Fluorescence Lifetime Imaging Microscopy

Abstract Existing plastic analysis techniques such as Fourier transform infrared spectroscopy and Raman spectroscopy are problematic because samples must be anhydrous and identification can be hindered by additives. This article describes a new approach that has been successfully demonstrated in which plastics can be classified by neural networks that are trained, validated, and tested by frequency domain fluorescence lifetime imaging microscopy measurements.

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy.

Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, imaging is still rarely investigated in general and in carbon dots (CDs) in particular. To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and , together with the steady-state images of fluorescence intensity (FI) and FA (). The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. The FLT of fluorescein samples was found to decrease from to , whereas both and were significantly increased from to and to , respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of from to and from to for the first CDs and from to and to for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, , and ). Nevertheless, was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

Abstract 644: Angiogenic Blood Vessels Reconfigure/ Attenuate Hypoxic Tumor Microenvironments In A Quantitative In Vitro Model

Introduction: Angiogenesis is crucial in the spread of cancer cells, and hypoxic tumor microenvironment (TME) plays a vital role in its initiation. Hypoxia stimulates the release of pro-angiogenic factors, promoting the growth of new blood vessels towards the tumor, which further alters the TME. The variation of in vivo hypoxic TME have been studied recently. However, due to technical limitations such as highly invasive procedure, high cost, tedious process, and poor signal-to-noise ratio, it is challenging to perform quantitative oxygen sensing. Here we develop an advanced in vitro model directly investigating the interaction between a solid tumor and blood vessels for an extended period. Hypothesis: The angiogenic blood vessels can reconfigure and attenuate the hypoxic TME, which can be directly measured Methods: The model is built using a microfluidic device with a sealed microwell for 3D tumor spheroid culture (human colon cancer cell line, HCT116), and a microfluidic channel beneath the microwell for endothelium formation (human umbilical vein endothelial cell, HUVEC), connected by a small hole for interaction between the cell types. Oxygen sensitive fluorescence particles are distributed in the hydrogel matrix, and the widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) is exploited to reliably characterize the oxygen tension around the tumor spheroid with minimal sensitivity to ambient optical noise. Results: The oxygen tension of TME around the tumor spheroid within the distance of 0 to 0.7 mm rapidly decreases during tumor growth, from 15% at day 1 to less than 6% at day 14. The presence of endothelial cells slows down this decreasing rate and maintains it in a stable range, from 16% to 14%. The average oxygen tension difference between the two experiments, with and without co-culture of endothelial cells, is statistically significant after 5 days of observation (p&lt;0.01). Conclusions: We develop a quantitative in vitro method to study the hypoxic TME for its dynamic pattern and as a target for cancer treatment. By observing the effect of angiogenesis on it, this method can aid in exploring optimal cancer management strategies, such as chemotherapy, immunotherapy, tailored anti-angiogenic agents and other targeted therapies.

Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation.

Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns. In vivo endogenous, dual-excitation (375nm/445nm), multispectral (four bands) FD-FLIM imaging of human skin and oral mucosa at 12.5 kHz pixel rate and room-light conditions was also successfully demonstrated. This versatile, simple, compact, and cost-effective FD-FLIM implementation will facilitate the clinical translation of FLIM imaging and microscopy.

Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

Cases of the spontaneous regression of multiple pulmonary metastases, after radiofrequency ablation (RFA), of a single lung metastasis, have been documented to be mediated by the immune system. The interaction of immune checkpoints, e.g., PD-1/PD-L1 and CTLA-4/CD80, may explain this phenomenon. The purpose of this study is to identify and quantify immune mechanisms triggered by RFA of pulmonary metastases originating from colorectal cancer. We used two-site time-resolved Förster resonance energy transfer as determined by frequency-domain FLIM (iFRET) for the quantification of receptor-ligand interactions. iFRET provides a method by which immune checkpoint interaction states can be quantified in a spatiotemporal manner. The same patient sections were used for assessment of ligand-receptor interaction and intratumoral T-cell labeling. The checkpoint interaction states quantified by iFRET did not correlate with ligand expression. We show that immune checkpoint ligand expression as a predictive biomarker may be unsuitable as it does not confirm checkpoint interactions. In pre-RFA-treated metastases, there was a significant and negative correlation between PD-1/PD-L1 interaction state and intratumoral CD3+ and CD8+ density. The negative correlation of CD8+ and interactive states of PD-1/PD-L1 can be used to assess the state of immune suppression in RFA-treated patients.

An algorithmic method for the identification of wood species and the classification of post-consumer wood using fluorescence lifetime imaging microscopy

Abstract. In this contribution the frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) technique is evaluated for post-consumer wood sorting. The fluorescence characteristics of several wood samples were determined, whereby two excitation wavelengths (405 and 488 nm) were used. The measured data were processed using algorithmic methods to identify the wood species and post-consumer wood category. With the excitation wavelength of 405 nm, 16 out of 19 samples could be correctly assigned to the corresponding post-consumer wood category by means of the fluorescence lifetimes. Thus, the experimental results revealed the high potential of the FD-FLIM technique for automated post-consumer wood sorting.

Improved Protoporphyrin IX-Guided Neurosurgical Tumor Detection with Frequency-Domain Fluorescence Lifetime Imaging

Precise intraoperative brain tumor visualization supports surgeons in achieving maximal safe resection. In this sense, improved prognosis in patients with high-grade gliomas undergoing protoporphyrin IX fluorescence-guided surgery has been demonstrated. Phase fluorescence lifetime imaging in the frequency-domain has shown promise to distinguish weak protoporphyrin IX fluorescence from competing endogenous tissue fluorophores, thus allowing for brain tumor detection with high sensitivity. In this work, we show that this technique can be further improved by minimizing the crosstalk of autofluorescence signal contributions when only detecting the fluorescence emission above 615 nm. Combining fluorescence lifetime and spectroscopic measurements on a set of 130 ex vivo brain tumor specimens (14 low- and 56 high-grade gliomas, 39 meningiomas and 21 metastases) coherently substantiated the resulting increase of the fluorescence lifetime with respect to the detection band employed in previous work. This is of major interest for obtaining a clear-cut distinction from the autofluorescence background of the physiological brain. In particular, the median fluorescence lifetime of low- and high-grade glioma specimens lacking visual fluorescence during surgical resection was increased from 4.7 ns to 5.4 ns and 2.9 ns to 3.3 ns, respectively. While more data are needed to create statistical evidence, the coherence of what was observed throughout all tumor groups emphasized that this optimization should be taken into account for future studies.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery

Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τD = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τD) &amp;lt; 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τϕDA, was directly related to the amplitude-weighted lifetime τaveDA inferred from the known FRET efficiency (EFRETtrue) of the constructs. A linear fit to a plot of (ω·τϕDA) vs. (ω·τaveDA) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent EFRETapp based on the measured phase lifetime and EFRETtrue for frequencies such that (ω · τD) &amp;lt; 1.1. We had previously reported a similar relationship between EFRETapp and EFRETtrue at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between τϕDAand τaveDA has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.

Dual-modality optical coherence tomography and frequency-domain fluorescence lifetime imaging microscope system for intravascular imaging.

.Significance: Detailed biochemical and morphological imaging of the plaque burdened coronary arteries holds the promise of improved understanding of atherosclerosis plaque development, ultimately leading to better diagnostics and therapies.Aim: Development of a dual-modality intravascular catheter supporting swept-source optical coherence tomography (OCT) and frequency-domain fluorescence lifetime imaging (FD-FLIM) of endogenous fluorophores with UV excitation.Approach: We instituted a refined approach to endoscope development that combines simulation in a commercial ray tracing program, fabrication, and a measurement method for optimizing ball-lens performance. With this approach, we designed and developed a dual-modality catheter endoscope based on a double-clad fiber supporting OCT through the core and fluorescence collection through the first cladding. We varied the relative percent of UV excitation launched into the core and first cladding to explore the potential resolution improvement for FD-FLIM. The developed catheter endoscope was optically characterized, including measurement of spatial resolution and fluorescent lifetimes of standard fluorophores. Finally, the system was demonstrated on fresh ex vivo human coronary arteries.Results: The developed endoscope was shown to have optical performance similar to predictions derived from the simulation approach. The FLIM resolution can be improved by over a factor of 4 by primarily illuminating through the core rather than the first cladding. However, time-dependent solarization losses need to be considered when choosing the relative percentage. We ultimately chose to illuminate with 7% of the power transmitting through the core. The resulting catheter endoscope had lateral resolution for OCT and lateral resolution for FD-FLIM. Images of ex vivo coronary arteries are consistent with expectations based on histopathology.Conclusions: The results demonstrate that our approach for endoscope simulation produces reliable predictions of endoscope performance. Simulation results guided our development of a multimodal OCT/FD-FLIM catheter imaging system for investigating atherosclerosis in coronary arteries.

Direct frequency domain fluorescence lifetime imaging using field programmable gate arrays for real time processing.

Frequency domain (FD) fluorescence lifetime imaging (FLIM) involves the excitation of the sample of interest with a modulated light source and digitization of the fluorescence emission for further analysis. Traditional FD-FLIM systems use heterodyne or homodyne detection, where the excitation light source and detector are modulated at specific frequency(s). More recently, FD-FLIM systems that use reflection of the light source as a trigger or phase reference for lifetime calculations have been developed. These detection schemes, however, require extra components that increase the cost and complexity of the FD-FLIM system. Here, we report a novel FD-FLIM detection scheme whereby the light source modulation and emission digitization are implemented using Field Programmable Gate Arrays (FPGAs), and fixed gain avalanche photodiodes are used for fluorescence detection. The reported FD-FLIM system was designed for probing nanosecond lifetime fluorophores (2-10 ns) at three emission bands simultaneously. The system utilizes a 375 nm diode laser for excitation at multiple simultaneous modulation frequencies (between 1 MHz and 83 MHz, bandwidth limited intentionally by using a lowpass filter) and three fixed gain avalanche photodiodes for simultaneous detection of three emission bands: 405/20 nm, 440/40 nm, and 525/50 nm (center/FWHM). Real-time computation of the modulation and phase lifetimes is simply performed by direct application of the discrete Fourier transform (max. of 10 frequencies) to the digitized fluorescence emission signals. The accuracy and sensitivity of this novel FD-FLIM detection scheme was demonstrated by imaging standard fluorophores and ex vivo unfixed human coronary artery tissue samples.

Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery.

.Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.Results: Our imaging system enabled macroscopic FLIM of a field of view at spatial resolutions . A frame of with a lifetime accuracy was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology.Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

Spatiotemporal Subcellular Characterization of Absolute NADH Concentration Over the Dynamic Course of Metaphase, Anaphase, Telophase and Cytokinesis using the Phasor Approach to FLIM

Absolute concentration measurements of free and bound nicotinamide adenine dinucleotide hydride (NADH) using the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) is a powerful method for discerning metabolic robustness in living cells with subcellular resolution. Cell cycle and metabolism are at the forefront of ongoing cancer research to explore new targets for cancer therapeutics. A key feature of cancer cells is continual mitotic division. Completion of mitosis ends with the progression of cells through metaphase, anaphase, telophase and cytokinesis. Each of these phases last approximately 3 minutes, presenting a large challenge for researchers to assess metabolism at these stages using available biochemical approaches. Using the phasor FLIM paired with Digital Frequency Domain (DFD) FLIM hardware, we spatiotemporally measure the absolute concentration of NADH (a measure of metabolic robustness) and free/bound NADH ratio (a measure of metabolic mode) in individual mitochondria of cells progressing from metaphase through cytokinesis. Our timelapse FLIM measurements of HeLa cells characterize the metabolic heterogeneity of mitochondria metabolism based on spatial location at the cell equator or spindle poles during the temporal progression of cells from metaphase through cytokinesis. We observe mitochondria NADH concentration and free/bound NADH at the spindle equator and pole correlate in early metaphase, but diverge starting late metaphase to early anaphase coinciding with onset of anaphase cell elongation. Our subcellular NADH FLIM analysis results support the existence of a spatiotemporal specific mechanism for mitochondria function during the course of cell division. Supported by NIGMS P41GM103540 to EG and MD.

FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.

.We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system’s sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards’ high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions () is significantly () longer than the lifetime of the unloaded TSMod probe (). The pixel dwell time was for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency () of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.

Coding Scheme Optimization for Fast Fluorescence Lifetime Imaging

Fluorescence lifetime imaging (FLIM) is used for measuring material properties in a wide range of applications, including biology, medical imaging, chemistry, and material science. In frequency-domain FLIM (FD-FLIM), the object of interest is illuminated with a temporally modulated light source. The fluorescence lifetime is measured by computing the correlations of the emitted light with a demodulation function at the sensor. The signal-to-noise ratio (SNR) and the acquisition time of a FD-FLIM system is determined by the coding scheme (modulation and demodulation functions). In this article, we develop theory and algorithms for designing high-performance FD-FLIM coding schemes that can achieve high SNR and short acquisition time, given a fixed source power budget. Based on a geometric analysis of the image formation and noise model, we propose a novel surrogate objective for the performance of a given coding scheme. The surrogate objective is extremely fast to compute, and can be used to efficiently explore the entire space of coding schemes. Based on this objective, we design novel, high-performance coding schemes that achieve up to an order of magnitude shorter acquisition time as compared to existing approaches. We demonstrate the performance advantage of the proposed schemes in a variety of imaging conditions, using a modular hardware prototype that can implement various coding schemes.

Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera with lateral electric field charge modulators.

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD-FLIM system is a valuable tool in the biomedical field.