Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit

Nieves, Daniel J.; Baker, Matthew A. B.

doi:10.1042/bst20200746

Cited by 5 publications

(3 citation statements)

References 56 publications

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“…Similar to several advanced fluorescence imaging methods [ 5 , 20 , 21 ], SXT data provide a near-isotropic resolution which allows for the accurate 3D reconstruction of the whole cell. The advantage of observing the cell using its natural contrast is that it does not require labeling of specific structures.…”

Section: Advantages Of Using Sxtmentioning

confidence: 99%

The use of soft X-ray tomography to explore mitochondrial structure and function

Loconte

White

2022

Molecular Metabolism

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Background Mitochondria are cellular organelles responsible for energy production, and dysregulation of the mitochondrial network is associated with many disease states. To fully characterize the mitochondrial network's structure and function, a three-dimensional whole cell mapping technique is required. Scope of review This review highlights the use of soft X-ray tomography (SXT) as a relatively high-throughput approach to quantify mitochondrial structure and function under multiple cellular conditions. Major conclusions The use of SXT opens the door for mapping cellular rearrangements during critical processes such as insulin secretion, stem cell differentiation, or disease progression. SXT provides unique information such as biochemical compositions or molecular densities of organelles and allows for unbiased, label-free imaging of intact whole cells. Mapping mitochondria in the context of the near-native cellular environment will reveal more information regarding mitochondrial network functions within the cell.

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Section: Advantages Of Using Sxtmentioning

confidence: 99%

The use of soft X-ray tomography to explore mitochondrial structure and function

Loconte

White

2022

Molecular Metabolism

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“…Advances in fluorescence microscopy continue to push the limits of our knowledge of cellular and molecular biology. In particular, with roughly 25 years of steady improvements to super-resolution fluorescence microscopy, light microscopy is now able to reveal many of the finest details of the microscopic world [1][2][3][4]. Conventional fluorescence microscopy is fundamentally hindered by diffraction, limiting the resolution to hundreds of nanometers [5].…”

Section: Introductionmentioning

confidence: 99%

Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy

Nino

Milstein

2021

Preprint

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In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photoswitching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.

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“…achieves light absorption measurement and acoustic detection simultaneously. [2][3][4] While the resolution and imaging depth of optical imaging modalities such as confocal microscopy, two-photon microscopy, and optical-coherence tomography are limited by the diffraction barrier and optical diffusion limit, respectively, 1,5 PACT overcomes the diffraction and diffusion limits of these imaging modalities to enable imaging with deep penetration, high spatiotemporal resolution, and rich contrast. [1][2][3][4] Currently, the biomedical applications of PACT include both preclinical research and clinical applications.…”

mentioning

confidence: 99%

Application of photoacoustic computed tomography in biomedical imaging: A literature review

Sun

Wang

et al. 2022

Bioengineering & Transla Med

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Photoacoustic computed tomography (PACT) is a hybrid imaging modality that combines optical excitation and acoustic detection techniques. It obtains high‐resolution deep‐tissue images based on the deep penetration of light, the anisotropy of light absorption in objects, and the photoacoustic effect. Hence, PACT shows great potential in biomedical sample imaging. Recently, due to its advantages of high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth, PACT has received increasing attention in preclinical and clinical practice. To date, there has been a proliferation of PACT systems designed for specific biomedical imaging applications, from small animals to human organs, from ex vivo to in vivo real‐time imaging, and from simple structural imaging to functional and molecular imaging with external contrast agents. Therefore, it is of great importance to summarize the previous applications of PACT systems in biomedical imaging and clinical practice. In this review, we searched for studies related to PACT imaging of biomedical tissues and samples over the past two decades; divided the studies into two categories, PACT imaging of preclinical animals and PACT imaging of human organs and body parts; and discussed the significance of the studies. Finally, we pointed out the future directions of PACT in biomedical applications. With the development of exogenous contrast agents and advances of imaging technique, in the future, PACT will enable biomedical imaging from organs to whole bodies, from superficial vasculature to internal organs, from anatomy to functions, and will play an increasingly important role in biomedical research and clinical practice.

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Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit

Cited by 5 publications

References 56 publications

The use of soft X-ray tomography to explore mitochondrial structure and function

The use of soft X-ray tomography to explore mitochondrial structure and function

Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy

Application of photoacoustic computed tomography in biomedical imaging: A literature review

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