Single-sample image-fusion upsampling of fluorescence lifetime images.

Fluorescence lifetime imaging microscopy (FLIM) provides detailed information about molecular interactions and biological processes. A major bottleneck for FLIM is image resolution at high acquisition speeds due to the engineering and signal-processing limitations of time-resolved imaging technology. Here, we present single-sample image-fusion upsampling, a data-fusion approach to computational FLIM super-resolution that combines measurements from a low-resolution time-resolved detector (that measures photon arrival time) and a high-resolution camera (that measures intensity only). To solve this otherwise ill-posed inverse retrieval problem, we introduce statistically informed priors that encode local and global correlations between the two "single-sample" measurements. This bypasses the risk of out-of-distribution hallucination as in traditional data-driven approaches and delivers enhanced images compared, for example, to standard bilinear interpolation. The general approach laid out by single-sample image-fusion upsampling can be applied to other image super-resolution problems where two different datasets are available.

Fluorescence lifetime Hong-Ou-Mandel sensing.

Fluorescence Lifetime Imaging Microscopy in the time domain is typically performed by recording the arrival time of photons either by using electronic time tagging or a gated detector. As such the temporal resolution is limited by the performance of the electronics to 100's of picoseconds. Here, we demonstrate a fluorescence lifetime measurement technique based on photon-bunching statistics with a resolution that is only dependent on the duration of the reference photon or laser pulse, which can readily reach the 1-0.1 picosecond timescale. A range of fluorescent dyes having lifetimes spanning from 1.6 to 7 picoseconds have been here measured with only ~1 s measurement duration. We corroborate the effectiveness of the technique by measuring the Newtonian viscosity of glycerol/water mixtures by means of a molecular rotor having over an order of magnitude variability in lifetime, thus introducing a new method for contact-free nanorheology. Accessing fluorescence lifetime information at such high temporal resolution opens a doorway for a wide range of fluorescent markers to be adopted for studying yet unexplored fast biological processes, as well as fundamental interactions such as lifetime shortening in resonant plasmonic devices.

Single-shot time-folded fluorescence lifetime imaging.

Fluorescence lifetime imaging is an important tool in bioimaging that allows one to detect subtle changes in cell dynamics and their environment. Most time-domain approaches currently involve scanning a single illumination point across the sample, which can make imaging dynamic scenes challenging, while single-shot "rapid lifetime determination" can suffer from large uncertainties when the lifetime is not appropriately sampled. Here, we propose a time-folded fluorescence lifetime imaging microscopy (TFFLIM) approach, whereby a time-folding cavity provides multiple spatially sheared replicas of the lifetime, each shifted temporally with respect to a fixed time gate. This provides a robust, single-shot FLIM approach that we experimentally validate across a broad lifetime range on fluorescent beads and Convallaria samples.

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation.

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080-human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

Twin-Airy Point-Spread Function for Extended-Volume Particle Localization.

The localization of point sources in optical microscopy enables nm-precision imaging of single-molecules and biological dynamics. We report a new method of localization microscopy using twin Airy beams that yields precise 3D localization with the key advantages of extended depth range, higher optical throughput, and potential for imaging higher emitter densities than are possible using other techniques. A precision of better than 30 nm was achieved over a depth range in excess of 7 µm using a 60×, 1.4 NA objective. An illustrative application to extended-depth-range blood-flow imaging in a live zebrafish is also demonstrated.

3D + time blood flow mapping using SPIM-microPIV in the developing zebrafish heart.

We present SPIM-µPIV as a flow imaging system, capable of measuring in vivo flow information with 3D micron-scale resolution. Our system was validated using a phantom experiment consisting of a flow of beads in a 50 µm diameter FEP tube. Then, with the help of optical gating techniques, we obtained 3D + time flow fields throughout the full heartbeat in a ∼3 day old zebrafish larva using fluorescent red blood cells as tracer particles. From this we were able to recover 3D flow fields at 31 separate phases in the heartbeat. From our measurements of this specimen, we found the net pumped blood volume through the atrium to be 0.239 nL per beat. SPIM-µPIV enables high quality in vivo measurements of flow fields that will be valuable for studies of heart function and fluid-structure interaction in a range of small-animal models.

High-speed extended-volume blood flow measurement using engineered point-spread function.

Experimental characterization of blood flow in living organisms is crucial for understanding the development and function of cardiovascular systems, but there has been no technique reported for snapshot imaging of thick samples in large volumes with high precision. We have combined computational microscopy and the diffraction-free, self-bending property of Airy-beams to track fluorescent beads with sub-micron precision through an extended axial range (up to 600 µm) within the flowing blood of 3 days post-fertilization (dpf) zebrafish embryos. The spatial trajectories of the tracer beads within flowing blood were recorded during transit through both cardinal and intersegmental vessels, and the trajectories were found to be consistent with the segmentation of the vasculature recorded using selective-plane illumination microscopy (SPIM). This method provides sufficiently precise spatial and temporal measurement of 3D blood flow that has the potential for directly probing key biomechanical quantities such as wall shear stress, as well as exploring the fluidic repercussions of cardiovascular diseases. Although we demonstrate the technique for blood flow, the ten-fold better enhancement in the depth range offers improvements in a wide range of applications of high-speed precision measurement of fluid flow, from microfluidics through measurement of cell dynamics to macroscopic aerosol characterizations.