Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

Coelho, Simao; Baek, Je Hyun; Graus, Matthew S.; Halstead, James M.; Nicovich, Philip R.; Feher, Kristen; Gandhi, Hetvi; Gooding, J. Justin; Gaus, Katharina

doi:10.1126/sciadv.aay8271

Cited by 64 publications

(49 citation statements)

References 46 publications

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“…The second dichroic filter can be a 775 nm short pass dichroic. Alternatively, if bead fiducials can be introduced, the laser can be replaced with an IR LED above the sample to limit the drift to below 1 nm across all three dimensions [124][125][126][127]. (D) Another camera is added so that two or more fluorophores can be imaged at the same time [128][129][130][131].…”

Section: Example Microscope Configurations (A)mentioning

confidence: 99%

Squid: Simplifying Quantitative Imaging Platform Development and Deployment

Krishnamurthy

et al. 2020

Preprint

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With rapid developments in microscopy methods, highly versatile, robust and affordable implementations are needed to enable rapid and wide adoption by the biological sciences community. Here we report Squid, a quantitative imaging platform with a full suite of hardware and software components and configurations for deploying facility-grade widefield microscopes with advanced features like flat field fluorescence excitation, patterned illumination and tracking microscopy, at a fraction of the cost of commercial solutions. The open and modular nature (both in hardware and in software) lowers the barrier for deployment, and importantly, simplifies development, making the system highly configurable and experiments that can run on the system easily programmable. Developed with the goal of helping translate the rapid advances in the field of microscopy and microscopy-enabled methods, including those powered by deep learning, we envision Squid will simplify roll-out of microscopy-based applications - including at point of care and in low resource settings, make adoption of new or otherwise advanced techniques easier, and significantly increase the available microscope-hours to labs.

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Section: Example Microscope Configurations (A)mentioning

confidence: 99%

Squid: Simplifying Quantitative Imaging Platform Development and Deployment

Krishnamurthy

et al. 2020

Preprint

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“…The single-cell imaging of cancer cells has been achieved in monolayer cultures with simple microscopy. Current techniques in super-resolution microscopy can even distinguish single-molecule dynamics at a nanometer resolution [ 74 , 75 ]. However, the acquisition of single-cell resolution images in in vivo samples, live or fixed, is far more challenging.…”

Section: Single-cell Imagingmentioning

confidence: 99%

Dive into Single, Seek Out Multiple: Probing Cancer Metastases via Single-Cell Sequencing and Imaging Techniques

2021

Cancers

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Metastasis is the cause of most cancer deaths and continues to be the biggest challenge in clinical practice and laboratory investigation. The challenge is largely due to the intrinsic heterogeneity of primary and metastatic tumor populations and the complex interactions among cancer cells and cells in the tumor microenvironment. Therefore, it is important to determine the genotype and phenotype of individual cells so that the metastasis-driving events can be precisely identified, understood, and targeted in future therapies. Single-cell sequencing techniques have allowed the direct comparison of the genomic and transcriptomic changes among different stages of metastatic samples. Single-cell imaging approaches have enabled the live visualization of the heterogeneous behaviors of malignant and non-malignant cells in the tumor microenvironment. By applying these technologies, we are achieving a spatiotemporal precision understanding of cancer metastases and clinical therapeutic translations.

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“…Coelho et al recently presented a new principle, active stabilization, that allows the direct measurement distance between molecules within the 1–20 nm scale in intact cells. 72 The authors presented an actively stabilized microscope utilizing 3D real-time drift corrections, resulting in a stabilization of <1 nm and ultrahigh localization precision of ∼1 nm. The newly developed microscope can be used to reveal a 4 to 7 nm difference in spatial separation between signaling T cell receptors and phosphatases (CD45) in active and resting T cells.…”

Section: Super-resolution Microscopy For Imaging Of Single Cells and mentioning

confidence: 99%