Project description:Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of species involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of species (using Beta-Bernoulli process priors) and absolute maps of these fluorophore species (using Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our framework using a broad range of synthetic and experimental data and demonstrate its robustness across a number of scenarios including cases where we recover lifetime differences between species as small as 0.3 ns with merely 1000 photons.
Project description:Monitoring the morphological and biochemical information of neurons and glial cells at high temporal resolution in three-dimensional (3D) volumes of in vivo is pivotal for understanding their structure and function, and quantifying the brain microenvironment. Conventional two-photon fluorescence lifetime volumetric imaging speed faces the acquisition speed challenges of slow serial focal tomographic scanning, complex post-processing procedures for lifetime images, and inherent trade-offs among contrast, signal-to-noise ratio, and speed. This study presents a two-photon fluorescence lifetime volumetric projection microscopy using an axially elongated Bessel focus and instant frequency-domain fluorescence lifetime technique, and integrating with a convolutional network to enhance the imaging speed for in vivo neurodynamics mapping. The proposed method is validated by monitoring intracellular Ca2+ concentration throughout whole volume, tracking microglia movement and microenvironmental changes following thermal injury in the zebrafish brain, analyzing structural and functional variations of gap junctions in astrocyte networks, and measuring the Ca2+ concentration in neurons in mouse brains. This innovative methodology enables quantitative in vivo visualization of neurodynamics and the cellular processes and interactions in the brain.
Project description:Microfluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm.
Project description:Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.
Project description:Fluorescence lifetime imaging microscopy presents a powerful tool in biology and biophysics because it allows the investigation of the local environment of a fluorochrome in living cells in a quantitative manner. Furthermore, imaging Förster-type resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy enables protein-protein interactions and intermolecular distances to be mapped under physiological conditions. Quantitative and precise data analysis methods are required to access the richness of information that is contained in FRET data on biological samples. Lifetime detection in the frequency-domain yields two lifetime estimations. The lifetime moments analysis (LiMA) provides a quantitative measure of the lifetime distribution broadness by exploiting the analytical relationship between the phase- and demodulation-lifetime estimations and relating them to the weighted average and variance of the lifetime distribution. The LiMA theoretical framework is validated by comparison with global analysis and by applying it to a constrained two-component FRET system using simulations and experiments. Furthermore, a novel LIMA-based error analysis and a more intuitive formalism for global analysis are presented. Finally, a new method to resolve a FRET system is proposed and experimentally applied to the investigation of protein-protein interactions.
Project description:Fluorescence lifetime imaging microscopy (FLIM) provides quantitative readouts of biochemical microenvironments, holding great promise for biomedical imaging. However, conventional FLIM relies on slow photon counting routines to accumulate sufficient photon statistics, restricting acquisition speeds. Here we demonstrate SparseFLIM, an intelligent paradigm for achieving high-fidelity FLIM reconstruction from sparse photon measurements. We develop a coupled bidirectional propagation network that enriches photon counts and recovers hidden spatial-temporal information. Quantitative analysis shows over tenfold photon enrichment, dramatically improving signal-to-noise ratio, lifetime accuracy, and correlation compared to the original sparse data. SparseFLIM enables reconstructing spatially and temporally undersampled FLIM at full resolution and channel count. The model exhibits strong generalization across experimental modalities including multispectral FLIM and in vivo endoscopic FLIM. This work establishes deep learning as a promising approach to enhance fluorescence lifetime imaging and transcend limitations imposed by the inherent codependence between measurement duration and information content.
Project description:NADH intensity and fluorescence lifetime characteristics have proved valuable intrinsic biomarkers for profiling the cellular metabolic status of living biological tissues. To fully leverage the potential of NADH fluorescence lifetime imaging microscopy (FLIM) in (pre)clinical studies and translational applications, a compact and flexible endomicroscopic embodiment is essential. Herein we present our newly developed two-photon fluorescence (2PF) lifetime imaging endomicroscope (2p-FLeM) that features an about 2 mm diameter, subcellular resolution, and excellent emission photon utilization efficiency and can extract NADH lifetime parameters of living tissues and organs reliably using a safe excitation power (~30 mW) and moderate pixel dwelling time (≤10 μs). In vivo experiments showed that the 2p-FLeM system was capable of tracking NADH lifetime dynamics of cultured cancer cells and subcutaneous mouse tumor models subject to induced apoptosis, and of a functioning mouse kidney undergoing acute ischemia-reperfusion perturbation. The complementary structural and metabolic information afforded by the 2p-FLeM system promises functional histological imaging of label-free internal organs in vivo and in situ for practical clinical diagnosis and therapeutics applications.
Project description:Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.
Project description:A fast (up to video rate) two-photon excited fluorescence lifetime imaging system based on interleaved digitization is demonstrated. The system is compatible with existing beam-scanning microscopes with minor electronics and software modification. Proof-of-concept demonstrations were performed using laser dyes and biological tissue.
Project description:Despite recent improvements in microscopy, it is still difficult to apply super-resolution microscopy for deep imaging due to the deterioration of light convergence properties in thick specimens. As a strategy to avoid such optical limitations for deep super-resolution imaging, we focused on super-resolution radial fluctuation (SRRF), a super-resolution technique based on image analysis. In this study, we applied SRRF to two-photon microscopy (2P-SRRF) and characterized its spatial resolution, suitability for deep observation, and morphological reproducibility in real brain tissue. By the comparison with structured illumination microscopy (SIM), it was confirmed that 2P-SRRF exhibited two-point resolution and morphological reproducibility comparable to that of SIM. The improvement in spatial resolution was also demonstrated at depths of more than several hundred micrometers in a brain-mimetic environment. After optimizing SRRF processing parameters, we successfully demonstrated in vivo high-resolution imaging of the fifth layer of the cerebral cortex using 2P-SRRF. This is the first report on the application of SRRF to in vivo two-photon imaging. This method can be easily applied to existing two-photon microscopes and can expand the visualization range of super-resolution imaging studies.