Single‐molecule localization microscopy goes quantitative

Scalisi, Silvia; Pisignano, Dario; Zanacchi, Francesca Cella

doi:10.1002/jemt.24281

Cited by 4 publications

(2 citation statements)

References 86 publications

(147 reference statements)

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“…This review provides a comprehensive view of confinement-based ultrasensitive optical assays (Table 1), enabling the (digital) measurement of analytes without relying on advanced imaging systems such as total internal reflection fluorescence (TIRF) 43,44 microscopy or photo-activated localization microscopy (PALM). 128,129 As the investigation of novel and uncommon disease markers expands, along with the need to decipher multifactorial diseases, there is a rising demand for the development of unsophisticated, ultrasensitive, and multiplexed detection platforms to address these complex challenges. Luminescent systems, either through self-illuminating compounds or through the products of enzymatically induced luminescence or color systems, can be confined to internal or external spaces.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

Confinement-guided ultrasensitive optical assay with artificial intelligence for disease diagnostics

Zhang,

Lu,

et al. 2023

TIME

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<p>The necessity for ultrasensitive detection is becoming increasingly apparent as it plays a pivotal role in disease early diagnostics and health management, particularly when it comes to detecting and monitoring low-abundance biomarkers or precious samples with tiny volumes. In many disease cases, such as cancer, infectious disease, autoimmune disorder, and neurodegenerative disease, low-abundant target biomarkers like circulating tumor cells (CTCs), extracellular vesicle (EV) subpopulations, and post-translational modified proteins (PTMs) are commonly existing and can be served as early indicators of disease onset or progression. However, these biomarkers often exist in ultra-low quantities in body fluids, surpassing the detection limits of conventional diagnostic tools like enzyme-linked immunosorbent assay (ELISA). This leads to the inability to probe disease evolution at a very early stage from molecular pathology perspective. In such regard, ultrasensitive optical assays have emerged as a solution to overcome these limitations and have witnessed significant progress in recent decades. This review provides a comprehensive overview of the recent advancements in ultrasensitive optical detection for disease diagnostics, particularly focusing on the conjunction of confinement within micro-/nano-structures and signal amplification to generate distinguishable optical readouts. The discussion begins with a meticulous evaluation of the advantages and disadvantages of these ultra-sensitive optical assays. Then, the spotlight is turned towards the implementation of artificial intelligence (AI) algorithms. The ability of AI to process large volumes of visible reporter signal and clinical data has proven invaluable in identifying unique patterns across multi-center cohort samples. Looking forward, the review underscores future advancements in developing convergent biotechnology (BT) and information technology (IT) toolbox, especially optical biosensors for high-throughput biomarker screening, point-of-care (PoC) testing with appropriate algorithms for their clinical translation are highlighted.</p>

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Section: Conclusion and Perspectivementioning

confidence: 99%

Confinement-guided ultrasensitive optical assay with artificial intelligence for disease diagnostics

Zhang,

Lu,

et al. 2023

TIME

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“…When higher resolution is needed, especially to observe individual proteins, conventional light‐microscopy methods are not applicable due to their resolution limit. In these cases, transmission electron microscopy (TEM; Petralia & Wang, 2021; Piludu et al., 2018) and other super‐resolution techniques, such as high‐resolution scanning electron microscopy (SEM; Goldberg, 2008; Ou et al., 2015), stochastic optical reconstruction microscopy (STORM; Fish, 2022; Scalisi et al., 2023), photoactivated localization microscopy (PALM; Butler et al., 2022), total internal reflection fluorescence microscopy (TIRF; Fish, 2022), stimulated emission depletion (STED; Sahl & Hell, 2019), and so on, can be used to obtain more detailed information.…”

Section: Introductionmentioning

confidence: 99%

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface

Kormos,

Daróczi,

Szöllősi

et al. 2024

Current Protocols

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The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical‐point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image‐processing algorithm, we implemented pair correlation and pair cross‐correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sample preparation and transmission electron micrographyAlternate Protocol: Direct cell labeling for transmission electron micrographyBasic Protocol 2: Analysis of TEM images to detect immunogold‐labeled proteins

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Fortunate molecules boost signal to background ratio and localization precision in correlation based single molecule localization microscopy

Zanacchi,

Mondal

2024

Commun Biol

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Single-molecule localization microscopy (SMLM) can decipher fine details that are otherwise impossible using diffraction-limited microscopy. Often, the reconstructed super-resolved images suffer from noise, strong background and are prone to false detections that may impact quantitative imaging. To overcome these limitations, we propose a technique (corrSMLM) that recognizes and detects fortunate molecules (molecules with long blinking cycles) from the recorded data. The method uses correlation between two or more consecutive frames to identify and isolate fortunate molecules that blink longer than the standard blinking period of a molecule. The corrSMLM is based on the fact that random fluctuations (noise) do not last longer (usually limited to a single frame). In contrast, fortunate molecules consistently fluoresce for extended periods and hence appear on more than one frame. Accordingly, strongly correlated spots (representing fortunate molecules) are compared in the consecutive frames, followed by data integration to determine their position and localization precision. The technique addresses two significant problems that plague existing SMLM : (1) false detection due to random noise that contributes to a strong background and (2) poor localization leading to overall low resolution. To demonstrate, corrSMLM is used for imaging fixed NIH3T3 cells (transfected with Dendra2-Actin, Dendra2-Tubulin, and mEos-Tom20 plasmid DNA). The super-resolved images show a significant reduction in background noise ( > 1.5 fold boost in SBR) and > 2-fold improvement in localization precision as compared to standard SMLM. Intensity analysis based on the number of molecules suggests that corrSMLM better corroborates the raw data and preserves finer features (e.g., edges), which are wiped out in standard SMLM. Overall, an improvement is noted in the localization precision and spatial resolution. The proposed technique is anticipated to advance SMLM and is expected to contribute to a better understanding of single-molecule dynamics in a cellular environment.

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Single‐molecule localization microscopy goes quantitative

Cited by 4 publications

References 86 publications

Confinement-guided ultrasensitive optical assay with artificial intelligence for disease diagnostics

Confinement-guided ultrasensitive optical assay with artificial intelligence for disease diagnostics

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface

Fortunate molecules boost signal to background ratio and localization precision in correlation based single molecule localization microscopy

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