Biology subjects

Bruschini, C.

Publications and source records attributed to Bruschini, C..

8 recordsLinked to original sources

bioRxiv2026

Time-Resolved Laser Speckle Contrast Imaging (TR-LSCI) of Cerebral Blood Flow Response to Intracranial Pressure Elevation

SignificanceCerebral autoregulation (CA) reflects the dynamic coupling among cerebral blood flow (CBF), intracranial pressure (ICP), and arterial blood pressure (ABP); its failure contributes to secondary brain injury. Existing bedside methods rely on indirect or spatially limited CBF surrogates and cannot resolve microvascular flow dynamics across space, depth, and time. AimTo develop, optimize, and apply a scalable, noncontact time-resolved laser speckle contrast imaging (TR-LSCI) platform for depth-sensitive, high-speed, wide-field CBF imaging during controlled ICP perturbations. ApproachTR-LSCI synchronizes a 20-MHz pulsed laser with a time-gated, single-photon avalanche diode (SPAD) camera (512 x 512 pixels) to detect diffuse photons at varying path lengths, enabling depth-resolved microvascular CBF imaging. Benchtop and mobile TR-LSCI systems were applied in adult rats and a neonatal piglet with synchronized invasive ICP and ABP measurements. ResultsTR-LSCI captured spatially heterogeneous, pulsatile CBF dynamics at up to 52 Hz over large cortical fields of view, with heart rate estimates statistically equivalent to those from ICP and ABP. Multivariable analysis identified reproducible, phase-dependent CA transitions encompassing preserved autoregulation, ABP-driven compensation, and ICP-constrained CBF suppression; notably, CBF alone exhibited distinct phase signatures. ConclusionsTR-LSCI enables dynamic, physiology-informed neurovascular monitoring and supports future bedside CA assessment.

neuroscience↗

bioRxiv2026

Fluorescence-lifetime optical electrophysiology in contracting cardiomyocytes

Precise monitoring of cardiac electrophysiology in vitro is crucial to understanding heart function and cardiac disease. However, high-throughput, contact-free methods for directly measuring excitation-contraction coupling remain limited. Here, we introduce a new paradigm for quantitative electrophysiological imaging that combines fluorescence lifetime and intensity information to capture dynamic cardiac signals with high fidelity. We show that lifetime measurements are intrinsically decoupled from motion artifacts and provide calibrated calcium concentration and membrane potential readouts across wide fields of view. Using a gated single-photon avalanche diode camera, we acquire fluorescence lifetime images at up to 200 frames per second with sufficient signal-to-noise ratio such that each frame contains meaningful lifetime information without temporal averaging. This approach yields spatially resolved maps of absolute voltage and calcium values across contracting cardiomyocyte monolayers, revealing heterogeneous cell behaviors within individual assays and uncovering previously unreported dynamics during late-phase repolarization for real-time analysis of excitation-contraction coupling.

bioengineering↗

bioRxiv2025

Real-Time Wide-Field Fluorescence Lifetime Imaging via Single-Snapshot Acquisition for Biomedical Applications

Fluorescence lifetime imaging (FLI) is a powerful tool for investigating molecular processes, microenvironmental parameters, and molecular interactions across tissue to (sub-)cellular levels. Despite its established value in numerous biomedical applications, conventional FLI techniques are hindered by long acquisition times. This limitation restricts their use in real-time scenarios, such as monitoring fast biological processes, studying live organisms, and in environments that require rapid imaging and immediate inference, such as clinical image-guided interventions. Here, we present a novel FLI approach that combines a large-format time-gated SPAD array with dual-gate acquisition capability, alongside a rapid lifetime determination algorithm. This integration allows for real-time fluorescence lifetime estimation through single-snapshot acquisitions, eliminating the need for traditional, time-consuming time-resolved data collection. We demonstrate the scalability and versatility of this method by achieving real-time FLI across challenging biomedical applications, ranging from capturing fast neural dynamics at the microscopic scale, performing multimodal 3D volumetric FLI of tumor organoids at the mesoscopic scale, to macroscale FLI in both direct and highly scattering regimes. Furthermore, we validate its utility in fluorescence lifetime-guided surgical procedures using tissue-mimicking phantoms. Overall, this new methodology significantly enhances the temporal and spatial capabilities of FLI, opening the door to the assessment of fast dynamic biomedical signals. It also enables the seamless integration of FLI into clinical workflows, particularly in applications like fluorescence-guided surgery.

bioengineering↗

bioRxiv2024

Multiplexed lifetime imaging of single molecules with a gated single-photon camera

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512x512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Forster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

biophysics↗

bioRxiv2021

In vitro and in vivo NIR Fluorescence Lifetime Imaging with a time-gated SPAD camera

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche photodiode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Forster Resonant Energy Transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated-ICCD camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

bioengineering↗

bioRxiv2020

Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

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 Megapixel 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 megapixels.

bioengineering↗

bioRxiv2019

Wide-field time-gated SPAD imager for phasor-based FLIM applications

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512x512 pixels), even in the case of small number of gates and limited photon budget.

biophysics↗

bioRxiv2019

Single Photon, Time-Gated, Phasor-based Fluorescence Lifetime Imaging Through Highly Scattering Medium

Fluorescence lifetime imaging (FLI) is a powerful tool for in vitro and non-invasive in vivo biomolecular and cellular investigations. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to some extent, does not depend on excitation intensity and signal level. However, when used in vivo with visible wavelength emitting fluorophores, FLI is complicated by (i) light scattering as well as absorption by tissues, which significantly reduces fluorescence intensity, (ii) tissue autofluorescence (AF), which decreases the signal to noise ratio and (iii) broadening of the decay signal, which can result in incorrect lifetime estimation. Here, we report the use of a large-frame time-gated single-photon avalanche diode (SPAD) imager, SwissSPAD2, with a very short acquisition time (in the milliseconds range) and a wide-field microscopy format. We use the phasor approach to convert each pixels data into its local lifetime. The phasor transformation provides a simple and fast visual method for lifetime imaging and is particularly suitable for in vivo FLI which suffers from deformation of the fluorescence decay, and makes lifetime extraction by standard fitting challenging. We show, for single dyes, that the phasor cloud distribution (of pixels) increases with decay broadening due to scattering and decreasing fluorescence intensity. Yet, as long as the fluorescence signal is higher than the tissue-like phantom AF, a distinct lifetime can still be clearly identified with an appropriate background correction. Lastly, we demonstrate the detection of few hundred thousand A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering phantom layers, despite significant scattering and the presence of the phantom AF.

bioengineering↗