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Proceedings of the National Academy of... Nov 2020The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end,...
The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.
Topics: Algorithms; Cell Line; Electron Microscope Tomography; Frozen Sections; Gold Colloid; Humans; Image Enhancement; Imaging, Three-Dimensional; Microscopy, Electron, Scanning Transmission
PubMed: 33077585
DOI: 10.1073/pnas.2000700117 -
Physical Review Letters Jul 2022In this Letter, we propose a new quantitative phase imaging methodology named Fourier optical spin splitting microscopy (FOSSM). FOSSM relies on a metasurface located at...
In this Letter, we propose a new quantitative phase imaging methodology named Fourier optical spin splitting microscopy (FOSSM). FOSSM relies on a metasurface located at the Fourier plane of a polarized microscope to separate the object image into two replicas of opposite circularly polarized states. The bias retardation between the two replicas is tuned by translating the metasurface or rotating the analyzer. Combined with a polarized camera, FOSSM can easily achieve single-shot quantitative phase gradient imaging, which greatly reduces the complexity of current phase microscope setups, paving the way for the next generation high-speed real-time multifunctional microscopy.
Topics: Microscopy
PubMed: 35867452
DOI: 10.1103/PhysRevLett.129.020801 -
Viruses Jan 2021During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in... (Review)
Review
During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.
Topics: Cryoelectron Microscopy; Electron Microscope Tomography; Image Processing, Computer-Assisted; Intracellular Membranes; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; RNA Viruses; RNA, Viral; Viral Nonstructural Proteins; Viral Replication Compartments; Virus Replication
PubMed: 33525547
DOI: 10.3390/v13020197 -
Journal of Cell Science Jul 2022Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and...
Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes.
Topics: Cell Nucleus; Chromatin; Microscopy
PubMed: 35748225
DOI: 10.1242/jcs.259009 -
Journal of Biomedical Optics Apr 2022There have been numerous academic and commercial efforts to develop high-resolution in vivo microscopes for a variety of clinical use cases, including early disease...
SIGNIFICANCE
There have been numerous academic and commercial efforts to develop high-resolution in vivo microscopes for a variety of clinical use cases, including early disease detection and surgical guidance. While many high-profile studies, commercialized products, and publications have resulted from these efforts, mainstream clinical adoption has been relatively slow other than for a few clinical applications (e.g., dermatology).
AIM
Here, our goals are threefold: (1) to introduce and motivate the need for in vivo microscopy (IVM) as an adjunctive tool for clinical detection, diagnosis, and treatment, (2) to discuss the key translational challenges facing the field, and (3) to propose best practices and recommendations to facilitate clinical adoption.
APPROACH
We will provide concrete examples from various clinical domains, such as dermatology, oral/gastrointestinal oncology, and neurosurgery, to reinforce our observations and recommendations.
RESULTS
While the incremental improvement and optimization of IVM technologies should and will continue to occur, future translational efforts would benefit from the following: (1) integrating clinical and industry partners upfront to define and maintain a compelling value proposition, (2) identifying multimodal/multiscale imaging workflows, which are necessary for success in most clinical scenarios, and (3) developing effective artificial intelligence tools for clinical decision support, tempered by a realization that complete adoption of such tools will be slow.
CONCLUSIONS
The convergence of imaging modalities, academic-industry-clinician partnerships, and new computational capabilities has the potential to catalyze rapid progress and adoption of IVM in the next few decades.
Topics: Artificial Intelligence; Forecasting; Intravital Microscopy; Microscopy
PubMed: 35478042
DOI: 10.1117/1.JBO.27.4.040601 -
Bioscience Reports Aug 2017Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of... (Review)
Review
Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.
Topics: Animals; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 28694303
DOI: 10.1042/BSR20170031 -
Anatomical Science International Jul 2023Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale... (Review)
Review
Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale information within crowded intracellular organelles of tissues provide more direct implications when compared to analyses of cells in culture or isolation. However, there are some difficulties in detecting individual shape using light microscopy, including super-resolution microscopy. Transmission electron microscopy (TEM), wherein the ultrastructure can be imaged at the membrane level, cannot determine the whole structure, and analyze it quantitatively. Volume EM, such as focused ion beam/scanning electron microscopy (FIB/SEM), can be a powerful tool to explore the details of three-dimensional ultrastructures even within a certain volume, and to measure several parameters from them. In this review, the advantages of FIB/SEM analysis in organelle studies are highlighted along with the introduction of mitochondrial analysis in injured motor neurons. This would aid in understanding the morphological details of mitochondria, especially those distributed in the cell bodies as well as in the axon initial segment (AIS) in mouse tissues. These regions have not been explored thus far due to the difficulties encountered in accessing their images by conditional microscopies. Some mechanisms of nerve regeneration have also been discussed with reference to the obtained findings. Finally, future perspectives on FIB/SEM are introduced. The combination of biochemical and genetic understanding of organelle structures and a nanoscale understanding of their three-dimensional distribution and morphology will help to match achievements in genomics and structural biology.
Topics: Mice; Animals; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Motor Neurons; Volume Electron Microscopy; Organelles; Imaging, Three-Dimensional
PubMed: 37071350
DOI: 10.1007/s12565-023-00720-y -
Scientific Reports Nov 2022We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces...
We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10 to 23 × 10 lipid droplets/µm after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.
Topics: Lipid Droplets; Second Harmonic Generation Microscopy; Microscopy; Fungi; Nitrogen
PubMed: 36335164
DOI: 10.1038/s41598-022-23502-4 -
Advanced Drug Delivery Reviews Jan 2024Despite the enormous potential of nanomedicines to shape the future of medicine, their clinical translation remains suboptimal. Translational challenges are present in... (Review)
Review
Despite the enormous potential of nanomedicines to shape the future of medicine, their clinical translation remains suboptimal. Translational challenges are present in every step of the development pipeline, from a lack of understanding of patient heterogeneity to insufficient insights on nanoparticle properties and their impact on material-cell interactions. Here, we discuss how the adoption of advanced optical microscopy techniques, such as super-resolution optical microscopies, correlative techniques, and high-content modalities, could aid the rational design of nanocarriers, by characterizing the cell, the nanomaterial, and their interaction with unprecedented spatial and/or temporal detail. In this nanomedicine arena, we will discuss how the implementation of these techniques, with their versatility and specificity, can yield high volumes of multi-parametric data; and how machine learning can aid the rapid advances in microscopy: from image acquisition to data interpretation.
Topics: Humans; Nanomedicine; Nanoparticles; Nanostructures; Microscopy; Optical Imaging
PubMed: 37980951
DOI: 10.1016/j.addr.2023.115138 -
Cell Nov 2011
Topics: Light; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence
PubMed: 22118471
DOI: 10.1016/j.cell.2011.11.015