Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal... more Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal resolution. However, the creation of thin light-sheets for high axial resolution is challenging, as the thickness of the sheet, field of view and confinement of the excitation need to be carefully balanced. Some of the thinnest light-sheets created so far have found little practical use as they excite too much out-of-focus fluorescence. In contrast, the most commonly used lightsheet for subcellular imaging, the square lattice, has excellent excitation confinement at the cost of lower axial resolving power. Here we leverage the recently discovered Field Synthesis theorem to create light-sheets where thickness and illumination confinement can be continuously tuned. Explicitly, we scan a line beam across a portion of an annulus mask on the back focal plane of the illumination objective, which we call it C-light-sheets. We experimentally characterize these light-sheets and their application on biological samples.
bioRxiv (Cold Spring Harbor Laboratory), Jun 13, 2022
Fast volumetric imaging of large fluorescent samples with high-resolution is required for many bi... more Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial sampling, respectively. In this Letter, we introduce a novel dual-axis scan unit for OPM that enables rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables imaging of model organisms, such as zebrafish embryos, with subcellular resolution. Furthermore, we combined this microscope with a real-time and multi-perspective projection imaging technique to increase the volumetric interrogation rate to more than 10 Hz.
We introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered li... more 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, thereby providing uncompromised biological imaging with decreased rates of photobleaching.
We introduce a cost-effective and easily implementable scan unit that converts any camera-based m... more We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on-the-fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet, and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.
ABSTRACT The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cell... more ABSTRACT The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated β-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of β-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of β-catenin. This article has an associated First Person interview with the first authors of the paper.
bioRxiv (Cold Spring Harbor Laboratory), Jun 9, 2021
Metastasis is the primary cause of cancer-related deaths [1]. The ability of a metastatic cancer ... more Metastasis is the primary cause of cancer-related deaths [1]. The ability of a metastatic cancer cell to arrive, survive and thrive in diverse tissue microenvironments (MEs) is a testament to their adaptability. Despite decades of research, our understanding of the mechanisms of metastasis has many remaining gaps, making this the single biggest obstacle to successful cancer treatment [1, 2]. Therefore, a key technical and conceptual challenge in cancer research is to identify and probe relevant functional differences between cancer cells as they relate to adaptation to specific tissue microenvironments. Historically, cancer research has predominantly relied on the mouse for investigation of the progression of metastasis [3]. However, for the task of probing functional adaptation of cancer cells in the secondary tissue environment, this model has several limitations. Cellular responses are likely very dynamic and heterogeneous, and meaningful functional plasticity is superimposed by random cell-to-cell variations that need to be filtered from the phenotype distributions. The mouse provides neither easy access to most metastatic sites nor the throughput necessary to fulfill this requirement. As an alternative, 2D and 3D cell culture have been exploited for studying cancer cell heterogeneity [4, 5], but for the understanding functional adaptation they lack the relevant micro-environmental complexity of an organism. Here, we propose human cancer cell xenografts in zebrafish larvae as the currently best compromise between the competing quests for environmental diversity, experimental access, and sufficient throughput to acquire meaningful descriptions of cancer cell adaptation [5-8]: (1) Zebrafish represents a living organism with various complex tissue microenvironments, with tissue mechanics, physiology, and disease susceptibility that is highly conserved with those of humans [6, 9] (2) The optical transparency of zebrafish grants easy experimental access to any metastatic site by highresolution imaging, and (3) a single experimental session can yield hundreds of successful xenografts, allowing for high throughput within a single comparative study.
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microsc... more Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combined with lower photo-toxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1Hz.
Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores... more Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future
Signal transduction and cell function are governed by the spatiotemporal organization of membrane... more Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any o...
Tissue microenvironments affect the functional states of cancer cells, but determining these infl... more Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specifi...
Fast volumetric imaging of large fluorescent samples with high-resolution is required for many bi... more Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe...
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microsc... more Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combine...
Figure S4. Validation of the image based assay for identifying fibroblasts in 3D culture. HFF1-H2... more Figure S4. Validation of the image based assay for identifying fibroblasts in 3D culture. HFF1-H2BeGFP cells in 3D collagen were treated with RMIC as a function of concentration and pixels positive for GFP were compared to pixels positive for Hoechst to determine the overlap between GFP cells and Hoechst positive cells. (PNG 33 kb)
Figure S1. Cell profiler pipeline for image analysis Image analysis using cell profiler involved ... more Figure S1. Cell profiler pipeline for image analysis Image analysis using cell profiler involved thresholding, followed by segmentation to identify pixels to identify the total image area occupied for each channel. The intersection between EtHd and Apopxin was used to exclude pixels positive for both markers from being counted twice. (PNG 138 kb)
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2017
Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation pr... more Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are d...
We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, ... more We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, that uses a bespoke glass-tipped tertiary objective and improves the resolution, field of view, usability, and stability over previous variants. Owing to its high numerical aperture optics, this microscope achieves the highest lateral resolution in light-sheet fluorescence microscopy, and its axial resolution is similar to that of Lattice Light-Sheet Microscopy. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and natural killer cell-mediated cell death. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through a confined space within a microfluidic device, photoactivation of PI3K, and diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz.
Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal... more Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal resolution. However, the creation of thin light-sheets for high axial resolution is challenging, as the thickness of the sheet, field of view and confinement of the excitation need to be carefully balanced. Some of the thinnest light-sheets created so far have found little practical use as they excite too much out-of-focus fluorescence. In contrast, the most commonly used lightsheet for subcellular imaging, the square lattice, has excellent excitation confinement at the cost of lower axial resolving power. Here we leverage the recently discovered Field Synthesis theorem to create light-sheets where thickness and illumination confinement can be continuously tuned. Explicitly, we scan a line beam across a portion of an annulus mask on the back focal plane of the illumination objective, which we call it C-light-sheets. We experimentally characterize these light-sheets and their application on biological samples.
bioRxiv (Cold Spring Harbor Laboratory), Jun 13, 2022
Fast volumetric imaging of large fluorescent samples with high-resolution is required for many bi... more Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial sampling, respectively. In this Letter, we introduce a novel dual-axis scan unit for OPM that enables rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables imaging of model organisms, such as zebrafish embryos, with subcellular resolution. Furthermore, we combined this microscope with a real-time and multi-perspective projection imaging technique to increase the volumetric interrogation rate to more than 10 Hz.
We introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered li... more 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, thereby providing uncompromised biological imaging with decreased rates of photobleaching.
We introduce a cost-effective and easily implementable scan unit that converts any camera-based m... more We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on-the-fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet, and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.
ABSTRACT The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cell... more ABSTRACT The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated β-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of β-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of β-catenin. This article has an associated First Person interview with the first authors of the paper.
bioRxiv (Cold Spring Harbor Laboratory), Jun 9, 2021
Metastasis is the primary cause of cancer-related deaths [1]. The ability of a metastatic cancer ... more Metastasis is the primary cause of cancer-related deaths [1]. The ability of a metastatic cancer cell to arrive, survive and thrive in diverse tissue microenvironments (MEs) is a testament to their adaptability. Despite decades of research, our understanding of the mechanisms of metastasis has many remaining gaps, making this the single biggest obstacle to successful cancer treatment [1, 2]. Therefore, a key technical and conceptual challenge in cancer research is to identify and probe relevant functional differences between cancer cells as they relate to adaptation to specific tissue microenvironments. Historically, cancer research has predominantly relied on the mouse for investigation of the progression of metastasis [3]. However, for the task of probing functional adaptation of cancer cells in the secondary tissue environment, this model has several limitations. Cellular responses are likely very dynamic and heterogeneous, and meaningful functional plasticity is superimposed by random cell-to-cell variations that need to be filtered from the phenotype distributions. The mouse provides neither easy access to most metastatic sites nor the throughput necessary to fulfill this requirement. As an alternative, 2D and 3D cell culture have been exploited for studying cancer cell heterogeneity [4, 5], but for the understanding functional adaptation they lack the relevant micro-environmental complexity of an organism. Here, we propose human cancer cell xenografts in zebrafish larvae as the currently best compromise between the competing quests for environmental diversity, experimental access, and sufficient throughput to acquire meaningful descriptions of cancer cell adaptation [5-8]: (1) Zebrafish represents a living organism with various complex tissue microenvironments, with tissue mechanics, physiology, and disease susceptibility that is highly conserved with those of humans [6, 9] (2) The optical transparency of zebrafish grants easy experimental access to any metastatic site by highresolution imaging, and (3) a single experimental session can yield hundreds of successful xenografts, allowing for high throughput within a single comparative study.
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microsc... more Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combined with lower photo-toxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1Hz.
Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores... more Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future
Signal transduction and cell function are governed by the spatiotemporal organization of membrane... more Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any o...
Tissue microenvironments affect the functional states of cancer cells, but determining these infl... more Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specifi...
Fast volumetric imaging of large fluorescent samples with high-resolution is required for many bi... more Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe...
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microsc... more Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combine...
Figure S4. Validation of the image based assay for identifying fibroblasts in 3D culture. HFF1-H2... more Figure S4. Validation of the image based assay for identifying fibroblasts in 3D culture. HFF1-H2BeGFP cells in 3D collagen were treated with RMIC as a function of concentration and pixels positive for GFP were compared to pixels positive for Hoechst to determine the overlap between GFP cells and Hoechst positive cells. (PNG 33 kb)
Figure S1. Cell profiler pipeline for image analysis Image analysis using cell profiler involved ... more Figure S1. Cell profiler pipeline for image analysis Image analysis using cell profiler involved thresholding, followed by segmentation to identify pixels to identify the total image area occupied for each channel. The intersection between EtHd and Apopxin was used to exclude pixels positive for both markers from being counted twice. (PNG 138 kb)
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2017
Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation pr... more Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are d...
We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, ... more We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, that uses a bespoke glass-tipped tertiary objective and improves the resolution, field of view, usability, and stability over previous variants. Owing to its high numerical aperture optics, this microscope achieves the highest lateral resolution in light-sheet fluorescence microscopy, and its axial resolution is similar to that of Lattice Light-Sheet Microscopy. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and natural killer cell-mediated cell death. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through a confined space within a microfluidic device, photoactivation of PI3K, and diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz.
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Papers by Bo-Jui Chang