Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca2+ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca2+ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca2+ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

Single-molecule localization microscopy (SMLM) has redefined optical imaging by enabling imaging beyond the diffraction limit, allowing nanoscale investigation into cellular architecture and molecular dynamics. Light sheet illumination enhances SMLM through optical sectioning of the sample, which drastically improves the signal-to-background ratio and reduces photobleaching and photodamage. Lattice light sheet (LLS) microscopy, in which a 2D optical lattice is implemented for light sheet illumination, can provide exceptional sectioning and extended imaging depth when imaging in scattering samples. However, its conventional dual-objective design poses challenges for certain applications. Here, we present an imaging platform which implements LLS illumination with a reflective single-objective geometry (soLLS) inside a microfluidic chip, enabling the use of a single high numerical aperture objective for both illumination and detection, mitigating constraints of a dual-objective setup. We provide quantitative characterization of the propagation properties of the soLLS and demonstrate that it outperforms conventional Gaussian light sheets in terms of useful field of view and sectioning when propagating through scattering samples. Next, by combining soLLS with point spread function engineering, we demonstrate the platform for improved 3D single-molecule super-resolution imaging of multiple targets across multiple cells. The soLLS imaging platform thus expands investigations of nanoscale cellular and intercellular structures and mechanisms into more challenging samples for a wide range of applications in biology and biomedicine.

We present a novel method used to implement tiling lattice light sheets (LLS) in lattice light sheet microscopy (LLSM) on regular LLS microscopes without changing the LLS microscope hardware. A LLS is tiled by applying binary phase maps acquired from off-center cross-sections of the corresponding optical lattice to the binary SLM used in LLS microscopes, by which a thin LLS can be tiled to image large specimens while maintaining the 3D imaging ability in the entire field of view. We investigate the method via numerical simulations and experiments, and demonstrate the method by imaging fluorescent particles embedded in agarose gel and expanded cells in the dithered mode of LLSM.

The data acquisition speed of point scanning microscopy techniques at sub-cellular resolution limits imaging of large samples, as sample stability and focus drift are becoming an issue. Therefore, light sheet fluorescent microscopy (LSFM) has become the method of choice for imaging cleared samples. However, this method still suffers from a trade-off between imaging depth and resolution, due to diffraction of the illuminating beam limiting the achievable field of view. After improvement of the latter with non-diffracting Bessel beams, lattice light sheet microscopy has substantially reduced the illumination point-spread-function (PSF). Here, we propose further improvement by generation of structured light sheets via phased arrays implemented as silicon nitride photonic integrated circuits (PICs). Beam generation with PICs results in much higher power efficiency than beam forming with conventional liquid crystal based spatial phase modulators, as it does not require the use of narrow blocking apertures. Moreover, this approach enables increased control over the generated field profile. Modeling of concrete PIC concepts indicates that sub-cellular resolution with mm scale imaging depths can be concomitantly achieved. Maintaining a small PSF along the axis perpendicular to the direction of light propagation is sacrificed in order to maintain it over increased imaging depth along the beam propagation axis. Rapid lateral scanning of the illumination beam inside the plane of the light sheet is then obtained by scanning of the laser wavelength within the excitation spectrum of the target fluorescent protein, allowing for a wide bidirectional field-of-view with high resolution.

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d26882e143">Over the past 30 years, therapeutic monoclonal antibodies (mAbs) have been used successfully as anti-cancer agents [ <a class="xref-link" href="#r1">1</a>]. Specific binding to target antigens induces cytotoxicity by promoting immune responses. Cluster of differentiation 20 (CD20) is a cell-surface receptor expressed in most B cells. Rituximab (RTX) was the first approved anti-CD20 therapeutic mAb for the treatment of B-cell malignancies and autoimmune disorders [ <a class="xref-link" href="#r2">2</a>]. In the meantime, RTX is complemented by human and humanized mAbs such as ofatumumab (OFA) and obinutuzumab (OBZ), respectively. Binding of these mAbs to B cells alter the nanoscale organization of CD20, consequently activating a cytotoxic pathway. However, molecular details on the binding mechanism of mAbs and binding-induced reorganization of endogenous CD20 on the membrane and how this activates the immune system to kill B cells remains poorly understood. To decode the molecular interplay of endogenous CD20 on B cells with therapeutic mAbs, 3D volumetric super-resolution fluorescence imaging methods are needed that provide a high spatiotemporal resolution. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d26882e151">In this work, we have introduced a speed-optimized variant of DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) [ <a class="xref-link" href="#r3">3</a>]. Using standard imager probes with two identical fluorophores ( <a class="xref-link" href="#fg001">Fig. 1a</a>) that form non-fluorescent H-dimers in the unbound state enables the use of higher probe concentrations and consequently ~15-fold faster 2D imaging, compared to classical DNA-PAINT probes [ <a class="xref-link" href="#r4">4</a>]. For implementing fast 3D imaging of whole cells at virtually molecular resolution, we combined two-dye imager (TDI)-DNA-PAINT with lattice light-sheet (LLS) microscopy ( <a class="xref-link" href="#fg001">Fig. 1b</a>) [ <a class="xref-link" href="#r5">5</a>]. Furthermore, we exploited LLS imaging to elucidate the real time binding interactions of mAbs and CD20 in live B cells in 4D. <div class="fig panel" id="fg001"> <a class="named-anchor" id="fg001"> <!-- named anchor --> </a> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/857f59d2-151c-4143-a6bf-7ea96473d0fd/ScienceOpen/image/292_6725fecdb0b33-ArindamGhoshAPMC2025_fig1.jpg"/> </div> <div class="panel-content"> <div class="label">Fig. 1</div> <div class="caption" id="d26882e172"> <p class="first" dir="auto" id="d26882e173"> <b>a</b>, TDI-DNA-PAINT workflow with dual-labeled imager probes (left). Exemplary fast super-resolved reconstruction of cellular microtubule networks after 5 minutes (right) of data acquisition. Scale 1 μm. <b>b,</b> LLS imaging principle. Created with BioRender. </div> </div> </div>

We introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered light-sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. This user-friendly and modular approach offers a drastically simplified optical design, higher light-throughput, simultaneous multicolor illumination, and a 100% spatial duty cycle that provides biological imaging with low rates of photobleaching.

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Microscopy is advancing at a rapid pace, enabling high-speed, high-resolution analyses to be conducted in a wide range of cellular contexts. For example, the capacity to quickly capture high-resolution images from multiple optical sections over multiple channels with confocal microscopy has allowed researchers to gain deeper understanding of tissue morphology via techniques such as three-dimensional rendering, as have more recent advances such as lattice light sheet microscopy and superresolution structured illumination microscopy. With this, though, comes the challenge of storing, curating, analysing and sharing data. While there are ways in which this has been attempted previously, few approaches have provided a central repository in which all of these different aspects of microscopy can be seamlessly integrated. Here, we describe a web-based storage and analysis platform called Microndata, that enables relatively straightforward storage, annotation, tracking, analysis and multi-user access to micrographs. This easy to use tool will simplify and harmonise laboratory work flows, and, importantly, will provide a central storage repository that is readily accessed, even after the researcher responsible for capturing the images has left the laboratory. Microndata is open-source software, available at http://www.microndata.net/.

Large brain tissue sections are imaged with nanoscale resolution using expansion and lattice light sheet microscopy.

Spatiotemporal Co-Dependency between Macrophages and Exhausted CD8 &lt;sup&gt;+&lt;/sup&gt; T Cells in Cancer

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

The axial resolving power of a light-sheet microscope is determined by the thickness of the illumination beam and the numerical aperture of its detection optics. Bessel-beam based optical lattices have generated significant interest owing to their reportedly narrow beam waist and propagation-invariant characteristics. Yet, despite their significant use in lattice light-sheet microscopy and recent incorporation into commercialized systems, there are very few quantitative reports on their physical properties and how they compare to standard Gaussian illumination beams. Here, we measure the beam properties in the transmission of dithered square lattices, which is the most commonly used variant of lattice light-sheet microscopy, and Gaussian-based light-sheets. After a systematic analysis, we find that square lattices are very similar to Gaussian-based light-sheets in terms of thickness, confocal parameter, propagation length and overall imaging performance.

Lattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

The project was supported by the UK Engineering and Physical Sciences Research Council, RS MacDonald Charitable Trust, SULSA, and the St. Andrews 600th anniversary BRAINS appeal. K. D. is a Royal Society Wolfson Merit Award holder.