Wide-field Fluorescence Imaging Research Articles

Slicing Network for Wide-Field Fluorescence Image Based on the Improved U-Net Model.

Fluorescence imaging stands as a pivotal component in biomedical research, requiring the elimination of out-of-focus background noise resulting from wide-field volumetric illumination of the whole field-of-view and scattering within thick biological tissues. Traditional methods struggle to effectively address varying degrees of defocusing in fluorescence images. This study introduces the utilization of upU-Net, 3D U-Net, and 3D upU-Net as defocusing networks tailored for 2D and 3D wide-field fluorescence images, yielding notable enhancements. These advancements facilitate more economically viable confocal microscopy, delivering significant advantages to biologists presently utilizing wide-field fluorescence microscopy.

Exploring the Interplay of Wavelength, Quantum Yield, and Penetration Depth in In Vivo Fluorescence Imaging.

The intricate interplay between the irradiation wavelength, the fluorophore quantum yield (QY) and penetration depth profoundly influences the efficacy of in vivo fluorescence imaging in various applications. Understanding the complex behavior of fluorescence in vivo, specifically how variations in wavelength affect the QY of commonly used dyes and the depth of imaging is crucial for optimizing fluorescence imaging techniques, as it directly impacts the accuracy and efficiency of imaging in biological tissues. In our study, we explore these dynamics through Monte Carlo simulations conducted under conditions reflective of wide-field fluorescence imaging, examining how variations in wavelength impact the dye's QY and depth of imaging, and consequently, the fluorescence behavior. A transition in the exponential decay of the emission depth exponent is observed around the 500-600nm range, indicating varying degrees of influence of depth on the fluorescence emission. The analysis of the fluorophore's QY reveals wavelength-dependent variations, with the most significant impact observed in the 600-700nm range. Moreover, we continued our investigation to explore multiplexing, unveiling insights into the spacing between identical spots in multiplexing images across various depths and wavelengths.

High-speed in vivo calcium recording using structured illumination with self-supervised denoising

High-speed widefield fluorescence imaging of neural activity in vivo is fundamentally limited by fluctuations in recorded signal due to background contamination and stochastic noise. In this study, we show background and shot noise-reduced imaging of the ultrafast genetically encoded Ca2+ indicator GCaMP8f in CA1 pyramidal neurons using periodic structured illumination (SI) with computational image reconstruction. We implement what we believe to be a novel reconstruction method for data acquired using periodic structured illumination, termed pseudo-HiLo (pHiLo), that combines a pseudo-widefield (pWF) reconstruction with individual SI frames to perform a HiLo reconstruction. We compare this new technique to interleaved optical sectioning structured illumination microscopy (OS-SIM) and pWF reconstruction. We quantify the performance of each reconstruction by evaluating contrast, transient peak-to-noise ratio (PNR), pairwise correlation coefficients between ΔF/F time courses extracted from individual in-focus cells, and correlation coefficients between each cell with surrounding cell-free background pixels. We additionally incorporate a self-supervised deep learning method for real-time noise suppression (DeepCAD-RT) into our data preprocessing pipeline. At 500 Hz frame rates, we demonstrate a 75% increase in PNR using the denoised pHiLo reconstruction compared to pWF. Utilizing DeepCAD-RT, we show significant PNR improvements using both structured illumination (SI) reconstruction methods with OS-SIM showing a 59% increase in PNR after denoising. Both pHiLo and OS-SIM reconstructions result in a ≈65% decrease in the mean correlation coefficient of the ΔF/F time courses between ROIs in comparison with pWF, indicating the potential to remove background fluorescent transients from out-of-focus cells.

A cross-reactive imaging matrix of membrane protein profiling for single-cell analysis

Membrane proteins play a critical role in cellular processes such as chemical transport, signal transduction, and enzymatic catalysis. A comprehensive understanding of membrane protein types and accurately measuring their expression levels are essential for advancing cell biology. However, current methods often fail to characterize the types and amounts of multiple membrane proteins in specific cell types due to inherent measurement limitations. Here, we have developed a single-cell image analysis technique based on a cross-reactive imaging matrix using membrane protein tags. This technique allows quantitative and qualitative analysis of multiple membrane protein types and their expression levels within single cells. As a proof of concept, we used four specific DNA aptamers to tag four different membrane proteins in seven different cell types. The four proteins are MUC1, EGFR, HeR2, and TLR4. The seven cell lines are 3t3, 4T1, A549, CT26, HeLa, Hepa1–6, Raw, respectively. We captured a comprehensive image matrix using wide-field fluorescence imaging, allowing precise quantification of protein expression at the single-cell level. Our approach introduces a multi-labeling analysis method for membrane proteins and reveals the influence of membrane protein diversity on cell heterogeneity analysis. We found that the accuracy of cell heterogeneity recognition can be affected by adjusting the number of membrane proteins. As the diversity of membrane protein species increases, the accuracy of cell type recognition increases. Our approach facilitates the evaluation of heterogeneity in diverse cells, providing new insights into cellular diversity and a powerful tool for studying single-cell biology.

Intraoperative confocal laser endomicroscopy during 5-aminolevulinic acid-guided glioma surgery: significant considerations for resection at the tumor margin.

Because gliomas have poorly defined tumor margins, the ability to achieve maximal resection is limited. To better discern these margins, fluorescence-guided surgery has been used to aid maximal safe resection. The authors describe their experience with the simultaneous use of intraoperative fluorescein sodium (FNa) confocal laser endomicroscopy (CLE) and operating microscope 5-aminolevulinic acid (5-ALA) fluorescence imaging for glioma resection to improve CLE use for better margin discrimination. FNa CLE and 5-ALA wide-field imaging were used in 33 patients with gliomas. CLE imaging was enhanced with the use of a telesurgical pathology software platform that enables real-time conversation between the operating neurosurgeons and the pathologists located remotely. CLE was used for imaging tumor regions that were subjectively regarded as tumor margins under normal visualization with the operative microscope. After FNa CLE imaging, 5-ALA wide-field imaging was performed in the same regions. Tissue was biopsied at imaging locations, and interpretations of FNa CLE and 5-ALA wide-field imaging were compared to those of permanent histological sections. Eighty-eight deep- and superficial-margin regions of interest (ROIs) were imaged with FNa CLE and 5-ALA imaging. Most of the ROIs interpreted by the neuropathologist as infiltrative glioma based on FNa CLE imaging lacked 5-ALA-induced fluorescence. Permanent histological sections from the corresponding regions were concordant with the interpretation of FNa CLE images in 57 of 88 (65%) ROIs and with the interpretation of 5-ALA imaging in 43 of 88 (49%) ROIs. The sensitivity and specificity of FNa CLE for the interpretation of tumor margins were 73% and 41%, respectively, and those of 5-ALA were 38% and 82%, respectively. Positive and negative predictive values for CLE were 79% and 33%, respectively, and those for 5-ALA were 86% and 31%, respectively. Conventional intraoperative evaluation of tumor margins, based on MRI and wide-field fluorescence imaging, can underestimate the invasiveness of gliomas. FNa CLE showed higher accuracy in detecting regions with infiltrating tumors than intraoperative 5-ALA imaging. Future considerations should include more rigorous comparisons of FNa CLE imaging and 5-ALA-guided resections on a larger cohort of patients.

Refining hemodynamic correction in in vivo wide-field fluorescent imaging through linear regression analysis

Accurate interpretation of in vivo wide-field fluorescent imaging (WFFI) data requires precise separation of raw fluorescence signals into neural and hemodynamic components. The classical Beer-Lambert law-based approach, which uses concurrent 530-nm illumination to estimate relative changes in cerebral blood volume (CBV), fails to account for the scattering and reflection of 530-nm photons from non-neuronal components leading to biased estimates of CBV changes and subsequent misrepresentation of neural activity. This study introduces a novel linear regression approach designed to overcome this limitation. This correction provides a more reliable representation of CBV changes and neural activity in fluorescence data. Our method is validated across multiple datasets, demonstrating its superiority over the classical approach.

Automatic dendritic spine segmentation in widefield fluorescence images reveal synaptic nanostructures distribution with super-resolution imaging.

Dendritic spines are the main sites for synaptic communication in neurons, and alterations in their density, size, and shapes occur in many brain disorders. Current spine segmentation methods perform poorly in conditions with low signal-to-noise and resolution, particularly in the widefield images of thick (10 μm) brain slices. Here, we combined two open-source machine-learning models to achieve automatic 3D spine segmentation in widefield diffraction-limited fluorescence images of neurons in thick brain slices. We validated the performance by comparison with manually segmented super-resolution images of spines reconstructed from direct stochastic optical reconstruction microscopy (dSTORM). Lastly, we show an application of our approach by combining spine segmentation from diffraction-limited images with dSTORM of synaptic protein PSD-95 in the same field-of-view. This allowed us to automatically analyze and quantify the nanoscale distribution of PSD-95 inside the spine. Importantly, we found the numbers, but not the average sizes, of synaptic nanomodules and nanodomains increase with spine size.

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo.

Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

Fluorescence lifetime imaging with distance and ranging using a miniaturised SPAD system

In this work we demonstrate a miniaturised imaging system based around a time-gated SPAD array operating in a “chip-on-tip” manner. Two versions of the system are demonstrated, each measuring 23 mm × 23 mm × 28 mm with differing fields of view and working distances. Initial tests demonstrate contrast between materials in widefield fluorescence imaging (WFLIm) mode, with frame rates of > 2 Hz achievable. Following this, WFLIm images of autofluorescence in ovine lung tissue are obtained at frame rates of ~ 1 Hz. Finally, the ability of the second system to perform simultaneous WFLIm and time of flight (aka Flourescence Lifetime Imaging Distance and Ranging, FLImDAR) is also tested. This shows that the system is capable of 4 mm resolution of object separation when tested on 3D printed samples. It is further demonstrated as being able to perform scene reconstruction on autofluorescent lung tissue. This system is, to date, the smallest chip on tip WFLIm system published, and is the first demonstration of the FLImDAR technique in a compact, portable system.

Improved intraoperative identification of close margins in oral squamous cell carcinoma resections using a dual aperture fluorescence ratio approach: first in-human results.

Surgical excision is the main treatment for solid tumors in oral squamous cell carcinomas, where wide local excision (achieving a healthy tissue margin of around the excised tumor) is the goal as it results in reduced local recurrence rates and improved overall survival. No clinical methods are available to assess the complete surgical margin intraoperatively while the patient is still on the operating table; and while recent intraoperative back-bench fluorescence-guided surgery approaches have shown promise for detecting "positive" inadequate margins (), they have had limited success in the detection of "close" inadequate margins (1 to 5mm). Here, a dual aperture fluorescence ratio (dAFR) approach was evaluated as a means of improving detection of close margins. The approach was evaluated on surgical specimens from patients who were administered a tumor-specific fluorescent imaging agent (cetuximab-800CW) prior to surgery. The dAFR approach was compared directly against standard wide-field fluorescence imaging and pathology measurements of margin thickness in specimens from three patients and a total of 12 margin locations (1 positive, 5 close, and 6 clear margins). The area under the receiver operating characteristic curve, representing the ability to detect close compared to clear margins () was found to be 1.0 and 0.57 for dAFR and sAF, respectively. Improvements in dAFR were found to be statistically significant (). These results provide evidence that the dAFR approach potentially improves detection of close surgical margins.

Low-frequency background estimation and noise separation from high-frequency for background and noise subtraction.

In fluorescence microscopy, background blur and noise are two main factors preventing the achievement of high-signal-to-noise ratio (SNR) imaging. Background blur primarily emanates from inherent factors including the spontaneous fluorescence of biological samples and out-of-focus backgrounds, while noise encompasses Gaussian and Poisson noise components. To achieve background blur subtraction and denoising simultaneously, a pioneering algorithm based on low-frequency background estimation and noise separation from high-frequency (LBNH-BNS) is presented, which effectively disentangles noise from the desired signal. Furthermore, it seamlessly integrates low-frequency features derived from background blur estimation, leading to the effective elimination of noise and background blur in wide-field fluorescence images. In comparisons with other state-of-the-art background removal algorithms, LBNH-BNS demonstrates significant advantages in key quantitative metrics such as peak signal-to-noise ratio (PSNR) and manifests substantial visual enhancements. LBNH-BNS holds immense potential for advancing the overall performance and quality of wide-field fluorescence imaging techniques.

Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution

Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces.

Deep learning based depth map estimation of protoporphyrin IX in turbid media using dual wavelength excitation fluorescence.

This study presents a depth map estimation of fluorescent objects in turbid media, such as biological tissue based on fluorescence observation by two-wavelength excitation and deep learning-based processing. A U-Net-based convolutional neural network is adapted for fluorophore depth maps from the ratiometric information of the two-wavelength excitation fluorescence. The proposed method offers depth map estimation from wide-field fluorescence images with rapid processing. The feasibility of the proposed method was demonstrated experimentally by estimating the depth map of protoporphyrin IX, a recognized cancer biomarker, at different depths within an optical phantom.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Photon-noise: is a single-pixel camera better than point scanning? A signal-to-noise ratio analysis for Hadamard and Cosine positive modulation

In a single-pixel camera, an unknown object is sequentially illuminated by intensity patterns. The total reflected or transmitted intensity is summed in a single-pixel detector from which the object is computationally reconstructed. In the situation where the measurements are limited by photon-noise, it is questionable whether a single-pixel camera performs better or worse than simply scanning the object with a focused intensity spot—a modality known as point raster scanning and employed in many laser scanning systems. Here, we solve this general question and report that positive intensity modulation based on Hadamard or Cosine patterns does not necessarily improve the single-to-noise ratio (SNR) of single-pixel cameras, as compared to point raster scanning (RS). Instead, we show that the SNR is only improved on object pixels at least k times brighter than the object mean signal , where k is a constant that depends on the modulation scheme (modulation matrix, number of detectors, etc). The constant k is derived for several widespread cases and has important consequences on the choice of the optical deign. This fundamental property is demonstrated theoretically, numerically, and is experimentally confirmed in the spatial domain (widefield fluorescence imaging) and in the spectral domain (spontaneous Raman spectral measurements). Finally, we provide user-oriented guidelines that help decide when and how multiplexing under photon-noise should be used instead of point RS.

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity.

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.

Abstract P6-01-01: Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors

Abstract Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. While combinations of chemotherapies and targeted therapies show potential improvements in TNBC clinical outcomes, patient stratification and prediction of treatment response is critical. Spatio-temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter minimum residual disease (MRD) and recur. Currently, there are limited tools to temporally evaluate heterogeneous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. Here, we utilized an optical imaging-based platform to identify in vivo, longitudinal differences in metabolic reprogramming between a resistant and sensitive tumor model at high resolutions along three metabolic axes of TNBC chemoresistance (oxidative phosphorylation, glycolysis, and fatty acid oxidation). Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen once the tumor reached a volume ~150mm3. MDA-MB-231 xenografts were resistant to PTX, defined as an initial response to PTX, a period of minimal residual disease, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). HCC-1806 xenografts were sensitive to PTX, defined as an initial response to PTX in all mice and a complete cure in 7/10 mice. A separate cohort of mice for each tumor line was implanted with window chambers and imaged longitudinally at distinct stages of the tumor’s lifecycle with previously validated fluorophores 2-NBDG, TMRE, and Bodipy to directly report on glucose uptake, mitochondrial membrane potential, or fatty acid uptake, respectively. Wide field fluorescence imaging of MDA-MB-231 mice showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p< 0.05), a significant decrease in 2-NBDG as early as two days after the 5th PTX dose (n=5, p< 0.05) and no significant changes in bodipy uptake. This increase in non-glucose-driven mitochondrial respiration was sustained during MRD. An increase in heterogeneity of TMRE uptake was seen during disease regression, MRD, and recurrence (n=5, p< 0.05). HCC-1806 tumors showed increased glucose uptake, decreased fatty acid uptake, and no significant changes in mitochondrial membrane potential during acute treatment. Metabolic changes were transient, with no significant changes in probe uptakes after drug withdrawal and during MRD. Unlike the MDA-MB-231 tumors, no significant changes in the heterogeneity of TMRE uptake were seen following paclitaxel withdrawal in HCC-1806 tumors (n=5, p>0.05). Our results point towards a metabolic switch from glycolysis to non-glucose, non-fat-driven mitochondrial respiration in MDA-MB-231 mice, possibly suggesting amino acid catabolism as a fuel during MRD. In the sensitive HCC-1806 line, we observed changes in glucose and fatty acid uptake during acute treatment, but no significant changes in metabolism during MRD. Consistent with the literature, our results point toward an increase in metabolic plasticity and heterogeneity in a chemo-resistant model compared to a chemo-sensitive one. Together, our results show the potential of using metabolic changes following therapy as a biomarker of therapy response, while highlighting the importance of tracking the change (or lack of change) in metabolism longitudinally. We aim to use this system to visualize and exploit early, in vivo metabolic vulnerabilities of disease regression that accompany local and distal recurrences. Citation Format: Enakshi D. Sunassee, Elizabeth Maydew, Brian Crouch, Megan Madonna, Gregory M Palmer, Nimmi Ramanujam. Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-01.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

Three-dimensional wide-field fluorescence microscopy for transcranial mapping of cortical microcirculation

Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm3 imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.

Aligned silver nanowires for plasmonically-enhanced fluorescence detection of photoactive proteins in wet and dry environment

We developed a method of aligning silver nanowires in a microchannel and fixing them to glass substrates via appropriate functionalization. The attachment of nanowires to the substrate is robust with no variation of their angles over minutes. Specific conjugation with photoactive proteins is observed using wide-field fluorescence imaging in real-time for highly concentrated protein solution, both in a microchannel and in a chip geometry. In the latter case we can detect the presence of the proteins in the dropcasted solution down to single proteins. The results point towards possible implementation of aligned silver nanowires as geometrically defined plasmonic fluorescence sensing platforms.