ACS Photonics

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

Interferometric scattering (iSCAT) microscopy combines label-free detection with nanometer-scale motion sensitivity. It captures temporal signal fluctuations from sub-diffraction movements of scatterers in live cells, including macromolecules and cell membranes. Here, we investigate label-free mapping of subcellular dynamics by calculating the power spectral density (PSD) of the iSCAT time series. Using a wide-field iSCAT, we observe an inverse-power-law relationship S(f) = {beta}f- over 30 - 1250 Hz in multiple cell types. We compute pixel-wise PSDs and obtain a color-coded spectral exponent map based on the fitted spectral exponent () and amplitude ({beta}), which indicates the characteristics and strength of subcellular movements. We also incorporate the goodness-of-fit as color saturation to enhance the contrast of the spectral exponent map. Following this approach, we demonstrate label-free imaging of live cells while effectively distinguishing between benign and malignant thyroid cancer subtypes, mitotic and interphase cells, as well as live and apoptotic cells. Dynamic imaging using wide-field iSCAT provides an intrinsic, label-free marker of cellular states and mechanical properties, which may benefit studies in mechanobiology, cancer diagnostics, and stem cell therapies.

Fluorescence microscopy is a cornerstone of biological research. However, fluorescent labeling is challenging in live cells and is constrained by photobleaching and phototoxicity. Label-free methods allow cells to be studied in their native state, but most techniques have poor contrast, lack 3D capability, rely on complex optics, and fail to provide structural information. We present broadband backscattering confocal microscopy (BBCM), which employs a broadband supercontinuum laser and collects backscattered light in confocal geometry using a photomultiplier tube. Broadband illumination averages out size-dependent oscillations that confound monochromatic backscattering. This eliminates blind spots and intensity ambiguities, allowing all scatterers to be visible, with the signal increasing approximately linearly with scatterer size. BBCM is easy to retrofit to standard confocal microscopes, requires no specialized optics, and is straightforward for nonspecialists. It enables high-contrast, label-free 3D imaging of live cells with size sensitivity to subcellular structures without employing custom optics or complex data processing.

Label-free and slide-free imaging is highly desired in clinical pathology because it holds the potential to avoid time- and labor-intensive tissue processing and chemical staining while preserving molecular information for downstream analyses. Deep-ultraviolet (UV) microscopy offers high-resolution, label-free molecular contrast via short wavelengths and intrinsic biomolecular absorption, but prior implementations have been limited to the analysis of thin sections and cell monolayers. Here, we present a fast, low-cost, LED-based, epi-illumination deep-UV microscope (epi-DUV) for label- and slide-free imaging of fresh, thick tissues. Using 255 nm and 280 nm absorption images, and tryptophan autofluorescence, the method yields quantitative maps of nucleic acid mass, protein mass, and quantum yield. Moreover, H&E-like contrast can be generated using native 255-nm absorption images. The system achieves 0.5 {micro}m lateral resolution with an effective slice thickness of [~]6 {micro}m across a 707 {micro}m x 707 {micro}m field of view and uses [~]330-ms exposure. To the best of our knowledge, this is the first demonstration of quantitative deep-UV molecular imaging of fresh, unlabeled thick tissues. Epi-DUV has significant potential to streamline the histopathology workflow while adding objective molecular readouts, enabling point-of-care assessment of unprocessed specimens (e.g., rapid intraoperative evaluation).

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The thereafter relaxation heats up the surrounding and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity. One-Sentence SummaryWe demonstrate a new spectroscopic imaging method that improves the signal intensity by >500-fold.

Extracting biological information from awake and unrestrained mice is imperative to in vivo basic and pre-clinical research. Accordingly, imaging methods which preclude invasiveness, anesthesia, and/or physical restraint enable more physiologically relevant biological data extraction by eliminating these extrinsic confounders. In this article we discuss the recent development of shortwave infrared (SWIR) fluorescent imaging to visualize peripheral organs in freely-behaving mice, as well as propose potential applications of this imaging modality in the neurosciences.

Bessel beam has long been utilized in physics for its ability to maintain lateral confinement during propagation. When used for two-photon fluorescence microscopy, Bessel foci have enabled high-speed volumetric imaging of the brain. At high numeric aperture (NA), however, the substantial energy in the side rings of Bessel foci reduces image contrast. Therefore, a compromise between resolution and contrast has to be made, limiting Bessel foci in microscopy to low NA. Here, we describe a method of generating axially extended Bessel-droplet foci with much suppressed side rings. Shaping the excitation wavefront with novel phase patterns, we generated Bessel-droplet foci of variable NAs at high power throughput and scanned them interferometrically along the axial direction for continuous volume imaging. More resistant to optical aberrations than Bessel foci, Bessel-droplet foci enabled high-resolution and high-contrast volumetric imaging of synaptic anatomy and function as well as lymphatic circulation in the mouse brain in vivo.

Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cells morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.\n\nStatement of significanceActively patterned illumination in fluorescence microscopy can reduce bleaching and phototoxicity as well as actively manipulate proteins in optogenetic applications. Matching the coordinate system of the camera and the light patterning device such as digital mirror arrays (DMA) remains a challenge. We developed a fast and easy calibration procedure that determines and corrects for the transformation between the camera and DMA coordinate system with ~50 nm precision. Using this approach, we develop spatial feedback photoactivation for Single Molecule Localization Microscopy (SMLM) to photoswitch only intracellular regions containing photoswitchable fluorophores. Our results show a 44% improvement in the possible data acquisition time before phototoxic effects become detectable and a 28% increase in detected localizations. Spatial feedback photoactivation thus significantly improves SMLM experiments.

Recently-developed kilopixel single-photon avalanche diode (SPAD) arrays with in-pixel timing hold great promise for fluorescence lifetime imaging microscopy of dynamic samples, thanks to their widefield single-photon time-of-arrival imaging capabilities. However digitally-scanned light-sheet microscope (DSLM) and two-photon microscope systems present significant technical barriers which have to date prevented full and efficient use of the capabilities of SPAD arrays. Because the 12.4 kHz frame-rate of our array camera is faster than achievable DSLM scan rates, most pixels would be sitting idle most of the time. We present a new optical design based around astigmatic imaging optics, enabling rapid and efficient acquisition of fluorescence lifetime imaging data. We demonstrate our system with both one- and two-photon excitation sources, validate performance with lifetime reference beads, and demonstrate separation of similar fluorescence emission spectra in biological samples via lifetime contrast.

Despite the easier use of multiple tissue clearing techniques in recent years, poor access to adequate light-sheet fluorescence microscopy remains a major obstacle for biomedical end users. Here, we propose a solution by developing descSPIM (desktop-equipped SPIM for cleared specimens) as a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practically substantial do-it-yourself light-sheet microscopy. Academically open-sourced (https://github.com/dbsb-juntendo/descSPIM), descSPIM allows routine three-dimensional imaging of cleared samples in minutes.

We have demonstrated 30-Hz three-photon imaging using a single 24-MHz mode-locked Cr:forsterite oscillator with a center wavelength at 1260 nm. By managing the dispersion distribution in the resonator using double-chirped mirrors, we have produced 32-fs pulses with 22-nJ pulse energy. Using the oscillator as a driving source, we have realized multi-color three-photon images using a GFP-labeled Drosophila brain and an AF647-labeled mouse brain. To demonstrate the capability of deep-tissue imaging, we have obtained a 10-times higher SBR from the three-photon images than the two-photon results at different depths in a GFP-labeled Drosophila brain dissection. Furthermore, we have shown the impact of excitation pulse width on three-photon deep-tissue imaging. Our results indicate the superiority of using shorter pulses for deeper-tissue imaging, especially in the Drosophila brain. In addition, we have recorded the three-photon calcium imaging in vivo from the Drosophila mushroom body in response to external electric shocks. We believe our demonstration provides a robust approach for high-speed three-photon microscopy applications, especially for intravital investigations in the Drosophila brain.

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.\n\nHere we combine dual color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that STED-ICCS provides not only a value of colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the Proliferating cell nuclear antigen (PCNA) protein have a high level of proximity and are correlated at a nanometer distance which is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of nanoscale distribution of functional sites in the nucleus.\n\nStatement of significanceSeveral methods are available to quantify the proximity of two labeled molecules from dual color images. Among them, image cross-correlation spectroscopy (ICCS) is attractive as it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. Here, we combine for the first time ICCS with superresolution stimulated emission depletion (STED) microscopy (STED-ICCS) to quantify the spatial distribution of functional sites in the nucleus. Our results show that STED-ICCS, in addition to quantifying the colocalized fraction, detects characteristic nanometer distances associated to correlated nuclear sites. This work shows that STED-ICCS can be a powerful tool to quantify the nanoscale distribution of functional sites in the nucleus.

Phytoplankton are a major source of primary production. Their photosynthetic fluorescence uniquely reports on their type, physiological state and response to environmental conditions. Changes in phytoplankton photophysiology are commonly monitored by bulk fluorescence spectroscopy, where gradual changes are reported in response to different perturbations such as light intensity changes. What is the meaning of such trends in bulk parameters if their values report ensemble averages of multiple unsynchronized cells? To answer this, we developed an experimental scheme that enables acquiring multiple fluorescence parameters, from multiple excitation sources and spectral bands. This enables tracking fluorescence intensities, brightnesses and their ratios, as well as mean photon nanotimes equivalent to mean fluorescence lifetimes, one cell at a time. We monitored three different phytoplankton species during diurnal cycles and in response to an abrupt increase in light intensity. Our results show that we can define specific subpopulations of fluorescence parameters for each of the phytoplankton species and in response to varying light conditions. Importantly, we identify the cells undergo well-defined transitions between these subpopulations that characterize the different light behaviors. The approach shown in this work will be useful in the exact characterization of phytoplankton cell states and parameter signatures in response to different changes these cells experience in marine environments, which will be useful in monitoring marine-related effects of global warming. Significance StatementUsing three representatives of red-linage phytoplankton we demonstrate distinct photophysiological behaviors at the single cell level. The results indicate cell wide coordination into discrete cell states. We test cell state transitions as a function of light acclimation during diurnal cycle and in response to large intensity increases, which stimulate distinct photoprotective response mechanisms. The analysis was made possible through the development of flow-based confocal detection at multiple excitation and emission wavelengths monitoring both pigment composition and photosynthetic performance. Our findings show that with enough simultaneously recorded parameters per each cell, the detection of multiple phytoplankton species at their distinct cell states is possible. This approach will be useful in examining the response of complex natural marine populations to environmental perturbations.

The highest level of three-dimensional (3D) resolution in stimulated emission depletion (STED) nanoscopy involves harnessing a 4Pi architecture using two opposing objectives. This protocol describes the construction and alignment of a 4Pi-STED nanoscope, commonly referred to as an isoSTED nanoscope. It guides interested researchers through assembling optomechanical components, configuring electronic and control devices, aligning the optical beam path, and assessing the instruments performance. Designed for adept optical instrument builders, this protocol offers a detailed roadmap for constructing an isoSTED nanoscope with adaptive optics (AO) in approximately 12 months. With this finely calibrated instrument, researchers can achieve 3D biological images with isotropic sub-50-nm resolution in thick samples up to 35 {micro}m in depth.

Phase contrast electron microscopy is fundamental for visualizing unstained biological specimens. Advances in electron detection have not yet overcome the low contrast caused by weak scattering. Here, we demonstrate that an orthogonal pulsed laser-electron beam interaction produces a pronounced peak phase shift of 430 radians through ponderomotive defocusing, leading to a maximum angular deflection of 45 {micro}rad. Experiments encompassing a variety of probe pulse energies and pump positions verified the properties of the electron pulses in a range of pulse durations from 5.8 {+/-} 1.9 ps to 13.4 {+/-} 0.9 ps and a width of 15.0 {+/-} 2.6 {micro}m at the interaction region. The stability of the beam was also tested across 10 hours of cumulative acquisition time, with only small variations in laboratory conditions resulting in a gradually shifting baseline measurement. Pulsed laser lensing of the electron beam offers the potential for refinement in phase shift and electron beam shaping with careful consideration to the overlap between laser and electron pulses. Calculations of phase shifts across a wide experimental envelope show that poorly chosen laser parameters can generate large incoherent distributions at both 30 keV and 300 keV. Thus, a delicate balance between laser and electron widths and pulse durations must be struck to adequately achieve uniform phase shifts, particularly when singling out specific beamlets in the back-focal-plane.

Light-sheet imaging of 3D objects with high spatial resolution remains an open challenge because of the trade-off between field-of-view (FOV) and axial resolution originating from the diffraction of light. We developed acoustic light-sheet microscopy (acoustic LSM), which actively manipulates the light propagation inside a large sample to obtain wide-field microscopic images deep inside a target. By accurately coupling a light-sheet illumination pulse into a planar acoustic pulse, the light-sheet can be continuously guided over large distances. We imaged a fluorescence-labeled transparent mouse brain for the FOVs of 19.3 x 12.4 mm2 and 9.7 x 5.9 mm2 with resolved microstructures and single cells deep inside the brain. Acoustic LSM creates new opportunities for the application of light-sheet in the field of industry to basic science. One Sentence SummaryAn acoustic-optical method overcomes a trade-off between field-of-view and axial resolution in light-sheet microscopy.

AO_SCPLOWBSTRACTC_SCPLOWSuper-resolution fluorescence microscopy has transformed the study of biological structures and functions beyond the diffraction limit. Unlike fluorescence methods, label-free chemical imaging, mostly based on Raman and infrared spectroscopy, provides intrinsic molecular contrast, enabling the study of biomolecules, nanostructures, drug molecules, and metabolites that cannot be easily tagged. However, while a wide range of fluorescence-based super-resolution techniques are well-established, extending super-resolution to label-free chemical imaging has remained challenging due to low signal levels and limited resolution improvement. Super-resolution stimulated Raman scattering microscopy (SRS) is most promising due to its high sensitivity and imaging speed. Similar to fluorescence, existing SRS super-resolution approaches are mostly based on either photoswitching/saturation of molecular labels or sample expansion, which suffers from poor sensitivity due to limitations in labeling density or signal dilution, respectively. Moreover, axial resolution is typically much worse than lateral resolution, yet most super-resolution SRS techniques focused on improving lateral resolution. In this work, we combine stimulated Raman scattering (SRS) with 4Pi-interferometry to significantly improve the axial resolution by nearly 7-fold. We report on the characterization of improvements in imaging sensitivity and axial resolution using 80 nm polystyrene beads. Harnessing the improved axial resolution, we demonstrate super-resolution 4Pi-SRS imaging in resolving small lipid droplet structures in mammalian cells and lipid membranes in E. coli cells. Because 4Pi-SRS uses interferometry to improve axial resolution, it is completely orthogonal to all previous super-resolution SRS techniques, including visible excitation, photoswitching, sample expansion, and computational approaches, thus it is straightforward to combine them to achieve much higher resolution chemical imaging than currently possible.

Discerning two or more identical and constantly scattering point sources using freely propagating waves is thought to be limited by diffraction. Here we show both theoretically and experimentally that by employing a diffraction minimum rather than a maximum for resolution, a given number of point scatterers can be discerned at tiny fractions of the employed wavelength. Specifically, we identify an 8 nm distance between two constantly emitting (non-blinking, non-switchable) fluorescent molecules, corresponding to 1/80 of the wavelength. Moreover, we show that contrary to naive expectations, the measurement precision improves with decreasing distance between the scatterers and with increased scatterer density, thus opening up the prospect of resolving clusters of (optical) point scatterers at tiny fractions of the wavelength.

Nonlinear optical imaging modalities, such as two-photon microscopy and stimulated Raman scattering (SRS) microscopy, make use of pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we investigated the short and long-term effects of pulsed laser induced phototoxicity. We used bulk RNA sequencing, quantitative measurement of cell proliferation, and measurement of the generation of reactive oxygen species (ROS) to assess phototoxic effects, at different time scales, for a range of laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant ROS generation, differential gene expression, or change in proliferation rates of mouse Neuro2A cells. Changes in proliferation rate and ROS generation were observed under imaging conditions with an excitation intensity of over 600 mW/m2. Repeated imaging of the same field of view at this excitation intensity of over 600 mW/m2 resulted in visual damage to N2A cells. Laser induced perturbations in live cells may impact downstream measurements of cell state including subsequent imaging or molecular measurements. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

The organism-wide effects of viral infection SARS-CoV-2 are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. Two-photon (2P) imaging combined with a proper subcellular staining technique has been an effective tool for studying mechanisms at such resolutions and organelle levels. Herein, we report the development of a novel calcium sensor molecule along with a 2P-technique for identifying imaging patterns associated with cellular correlates of infection damage within the cells. The method works as a cell viability assay and also provides valuable information on how the calcium level and intracellular distribution are perturbed by the virus. Moreover, it allows the quantitative analysis of infection dynamics. This novel approach facilitates the study of the infection progression and the quantification of the effects caused by viral variants and viral load.