Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Deep learning enables fast, gentle STED microscopy.

STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that restoring STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

Deep learning enables fast, gentle STED microscopy.

STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that denoising STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

Reduced Cell-ECM Interactions in the EpiSC Colony Center Cause Heterogeneous Differentiation.

Mechanoregulation of cell-extracellular matrix (ECM) interactions are crucial for dictating pluripotent stem cell differentiation. However, not all pluripotent cells respond homogeneously which results in heterogeneous cell populations. When cells, such as mouse epiblast stem cells (EpiSCs), are cultured in clusters, the heterogeneity effect during differentiation is even more pronounced. While past studies implicated variations in signaling pathways to be the root cause of heterogeneity, the biophysical aspects of differentiation have not been thoroughly considered. Here, we demonstrate that the heterogeneity of EpiSC differentiation arises from differences in the colony size and varying degrees of interactions between cells within the colonies and the ECM. Confocal imaging demonstrates that cells in the colony periphery established good contact with the surface while the cells in the colony center were separated by an average of 1-2 µm from the surface. Traction force measurements of the cells within the EpiSC colonies show that peripheral cells generate large tractions while the colony center cells do not. A finite element modeling of EpiSC colonies shows that tractions generated by the cells at the colony periphery lift off the colony center preventing the colony center from undergoing differentiation. Together, our results demonstrate a biophysical regulation of heterogeneous EpiSC colony differentiation.

2.5D microscopy with polarization independent SLM for enhanced detection efficiency and aberration correction.

Fast, volumetric imaging by fluorescence microscopy is essential in studying biological phenomena and cellular functions. Recently, single-shot 2.5D microscopy showed promising results for high-throughput quantitative subcellular analysis via extended depth of field imaging without sequential z-scanning; however, the detection efficiency was limited and it lacked depth-induced aberration correction. Here we report that a spatial light modulator (SLM) in a polarization insensitive configuration can significantly improve the detection efficiency of 2.5D microscopy, while also compensating for aberrations at large imaging depths caused by the refractive index mismatch between the sample and the immersion medium. We highlight the improved efficiency via quantitative single-molecule RNA imaging of mammalian cells with a 2-fold improvement in the fluorescence intensity compared to a conventional SLM-based microscopy. We demonstrate the aberration correction capabilities and extended depth of field by imaging thick specimens with fewer z-scanning steps.

Incoherent superposition of polychromatic light enables single-shot nondiffracting light-sheet microscopy.

We demonstrate single-shot nondiffracting light-sheet microscopy by the incoherent superposition of dispersed polychromatic light sources. We characterized our technique by generating a Bessel light-sheet with a supercontinuum light-source and a C-light-sheet using a diode laser, and demonstrated its applicability to fluorescence microscopy. We emphasize that our method is easily implementable and compatible with the requirements of high-resolution microscopy.

2.5D microscopy: Fast, high-throughput imaging via volumetric projection for quantitative subcellular analysis.

Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2×2 mm2 region within an imaging depth of ~5 µm for mammalian cells in <10 min and immunofluorescence imaging at a >30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multi-color imaging, depth control and super-resolution imaging.

Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.

Optimizing the performance of multiline-scanning confocal microscopy.

Line-scanning confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional line-scanning systems have only used a single excitation line and slit, and thus have not fully exploited benefits of parallelization. Here we investigate the optical performance of multi-line scanning confocal microscopy (mLS) by employing a digital micro-mirror that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as scanning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single line-scanning confocal microscopy.

Instantaneous non-diffracting light-sheet generation by controlling spatial coherence.

We demonstrate single-shot non-diffracting light-sheet generation by controlling the spatial coherence of light. A one-dimensional coherent beam, created by either increasing the spatial coherence of an LED or decreasing the spatial coherence of a laser, makes it unnecessary to scan non-diffracting beams to generate light-sheets. We theoretically and experimentally demonstrate the equivalence between our method and a scanned light-sheet, and investigate the characteristics of the light-sheet in detail. Our method is easily implementable and universally applicable for high-resolution multicolor light-sheet fluorescence imaging.

Low-photobleaching line-scanning confocal microscopy using dual inclined beams.

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line-scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2-fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single-molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three-dimensional single-molecule RNA imaging in mammalian cells.

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

Nuclear speckle fusion via long-range directional motion regulates speckle morphology after transcriptional inhibition.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar long-range speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.

Facile single-molecule pull-down assay for analysis of endogenous proteins.

The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab')2 or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.

Recent advancement of light-based single-molecule approaches for studying biomolecules.

Recent advances in single-molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single-molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single-molecule applications with a focus over the last few years of development. The advancements covered will mainly include light-based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods.

Fluorescence imaging with tailored light.

Fluorescence microscopy has long been a valuable tool for biological and medical imaging. Control of optical parameters such as the amplitude, phase, polarization and propagation angle of light gives fluorescence imaging great capabilities ranging from super-resolution imaging to long-term real-time observation of living organisms. In this review, we discuss current fluorescence imaging techniques in terms of the use of tailored or structured light for the sample illumination and fluorescence detection, providing a clear overview of their working principles and capabilities.

Flat-field illumination for quantitative fluorescence imaging.

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay.

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.