Flat-field illumination for quantitative fluorescence imaging

Khaw, Ian; Croop, Benjamin; Tang, Jialei; Moehl, Anna; Fuchs, Ulrike; Han, Kyu Young

doi:10.1101/297242

Cited by 5 publications

(5 citation statements)

References 30 publications

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“…As a field-of-view (FOV) becomes larger, uniform illumination is increasingly important. High precision optical systems have demonstrated a flat-field illumination with high efficiency (Figure 2a), which will facilitate quantitative single-molecule imaging (Khaw et al, 2018).…”

Section: High-throughput and High-content Single-molecule Assaymentioning

confidence: 99%

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

Croop

Zhang

Lim

et al. 2019

WIREs Mechanisms of Disease

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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

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Section: High-throughput and High-content Single-molecule Assaymentioning

confidence: 99%

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

Croop

Zhang

Lim

et al. 2019

WIREs Mechanisms of Disease

Self Cite

View full text Add to dashboard Cite

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“…We used an objective type total internal reflection fluorescence (TIRF) microscope equipped with lasers emitting 561 nm and 638 nm (Cobolt), and the beam shaping component (TopShape, asphericon GmbH) to generate flat-field illumination [30]. The excitation intensities were 10 W cm −2 measured after the microscope objective.…”

Section: Image Acquisition and Analysismentioning

confidence: 99%

“…Twenty fields-of-view (FOV; 82 × 82 μm 2 ) were recorded for each sample. Single-molecule spots were identified and intensity distributions were generated using custom MATLAB scripts [30].…”

Section: Image Acquisition and Analysismentioning

confidence: 99%

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

Croop¹,

Han²

2019

Phys. Biol.

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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 preincubation 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.

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“…Thus, several recent studies have aimed at obtaining a uniform excitation over a large FOV. Classical solutions revolve around beam-reshapers [20][21][22][23] , multimode fibers [24][25][26] and Spatial light modulators (piSMLM 27 ). These methods illuminate the whole field at once, so provide larger FOVs under the premise of using higher laser power.…”

Section: Introductionmentioning

confidence: 99%

Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

Mau

Friedl

Leterrier

et al. 2020

Preprint

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Quantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussianshaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical (up to 200 µm x 200 µm). This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2spectrin along the axons of neurons. ASTER's sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.

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Flat-field illumination for quantitative fluorescence imaging

Cited by 5 publications

References 30 publications

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

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

Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

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