Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging.

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

Large-scale expanded sample imaging with tiling lattice lightsheet microscopy.

The ability to observe biological nanostructures forms a vital step in understanding their functions. Thanks to the invention of expansion microscopy (ExM) technology, super-resolution features of biological samples can now be easily visualized with conventional light microscopies. However, when the sample is physically expanded, the demand for deep and precise 3D imaging increases. Lattice lightsheet microscopy (LLSM), which utilizes a planar illumination that is confined within the imaging depth of high numerical aperture (NA=1.1) detection objective, fulfils such requirements. In addition, optical tiling could be implemented to increase the field of view (FoV) by moving the lightsheet without mechanically moving the samples or the objective for high-precision 3D imaging. In this review article, we will explain the principle of the tiling lattice lightsheet microscopy (tLLSM), which combines optical tiling and lattice lightsheet, and discuss the applications of tLLSM in ExM.

Identification of two pathways mediating protein targeting from ER to lipid droplets.

Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.

Optogenetic manipulation of cell migration with high spatiotemporal resolution using lattice lightsheet microscopy.

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.

Microfluidic channel integrated with a lattice lightsheet microscopic system for continuous cell imaging.

In this study, a continuous cell-imaging system with subcellular resolution was developed by integrating a microfluidic platform with lattice lightsheet microscopy (LLSM). To reduce aberrations of the lightsheet propagating into the device, a microfluidic channel sealed with a water refractive index-matched thin film was fabricated. When the lightsheet emerged from the water-immersed objectives and penetrated through the water refractive-matched thin film into the microfluidic channel at an incident angle, less light scattering and fewer aberrations were found. Suspended cells flowed across the lattice lightsheet, and an imaging system with the image plane perpendicular to the lightsheet was used to sequentially acquire cell images. By applying a thinner lattice lightsheet, higher-resolution, higher-contrast images were obtained. Furthermore, three-dimensional cell images could be achieved by reconstructing sequential two-dimensional cell images.

Rapid single-wavelength lightsheet localization microscopy for clarified tissue.

Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System.

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.

Lightsheet localization microscopy enables fast, large-scale, and three-dimensional super-resolution imaging.

Recent advances in super-resolution microscopy allow the localization of single molecules within individual cells but not within multiple whole cells due to weak signals from single molecules and slow acquisition process for point accumulation to reconstruct super-resolution images. Here, we report a fast, large-scale, and three-dimensional super-resolution fluorescence microscope based on single-wavelength Bessel lightsheet to selectively illuminate spontaneous blinking fluorophores tagged to the proteins of interest in space. Critical parameters such as labeling density, excitation power, and exposure time were systematically optimized resulting in a maximum imaging speed of 2.7 × 104 µm3 s-1. Fourier ring correlation analysis revealed a reconstructed image with a lateral resolution of ~75 nm through the accumulation of 250 image volumes on immobilized samples within 15 min. Hence, the designed system could open new insights into the discovery of complex biological structures and live 3D localization imaging.