Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

Diekmann, Robin; Kahnwald, Maurice; Schoenit, Andreas; Deschamps, Joran; Matti, Ulf; Ries, Jonas

doi:10.1038/s41592-020-0918-5

Cited by 101 publications

(143 citation statements)

References 31 publications

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“…Incorporating this uncertainty in the density and spatial distribution of the fluorophores into this counting procedure is highly non-trivial and outside the scope of this paper. However, recent optimisation strategies (Cohen et al, 2019; Diekmann et al, 2020) suggest that a sparse imaging environment designed to minimise the number of fluorophores simultaneously in the On state, and therefore the number of PSFs per frame, can exponentially reduce this effect and maximise data quality. Furthermore, recent developments in localisation algorithms (e.g.…”

Section: Discussionmentioning

confidence: 99%

Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Patel

Owen

Cohen

2019

Preprint

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Many recent advancements in single molecule localization microscopy exploit the stochastic photo-switching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging. Modeling the photoswitching behavior of a fluorophore as a continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photo-switching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a dSTORM experiment involving an arbitrary number of molecules. We demonstrate that when training data is available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations.

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Section: Discussionmentioning

confidence: 99%

Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Patel

Owen

Cohen

2019

Preprint

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“…The range of dyes that can be induced to blink is limited, however, and each dye has its own optimal buffer and blinking characteristics, which complicates multicolour imaging. Photobleaching can also reduce image quality as fluorophores that bleach before they are imaged are not detected 10 . These problems are largely avoided by an alternative labelling approach called DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), in which the protein of interest is labelled with a docking oligonucleotide (the docking strand) and then imaged using the complementary oligonucleotide (the imager strand) fused to a fluorescent probe 11 .…”

Section: Introductionmentioning

confidence: 99%

A method for single molecule localization microscopy of tissues reveals non-random distribution of nuclear pores in Drosophila

Cheng

Allgeyer

Richens

et al. 2021

Preprint

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Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

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“…Moreover, ultrashort laser pulses have broad spectral bandwidth, broader than most of trivalent lanthanide doped-material absorption linewidth, and thus the spectroscopic study must be performed taking this regime into account, thereby adding further complexity to the light-matter interaction study. Nonetheless, considering the tremendous progress in fundamental understanding of the luminescent properties of lanthanide ions in complexes and host matrices [6][7][8][9] as well as UCNPs [10][11][12][13], together with the importance of UCNPs in basic science [14,15] and applications [16][17][18], understanding light-matter interactions involving femtosecond lasers and UCNPs may be useful in studies requiring higher energies, for example, in realtime applications and faster super-resolution bioimaging [19].…”

Section: Introductionmentioning

confidence: 99%

Femtosecond Laser Induced Luminescence in Hierarchically Structured Nd3+,Yb3+,Er3+ Co-Doped Upconversion Nanoparticles: Light-Matter Interaction Mechanisms from Experiments and Simulations

Oliveira¹,

Ferreira²,

Ferbonink³

et al. 2020

Preprint

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Fundamental studies of light-matter interactions are important for basic knowledge and in applications. Thanks to advances in experimental and theoretical methods, nowadays it is possible to perform such studies in a broad dynamic range, covering timescales from that of elementary interactions to real time. In the present work, we perform an experimental-theoretical study of light intensity-dependent femtosecond and CW-laser induced frequency upconversion in hierarchically structured core-multishell nanoparticles co-doped with NdIII, YbIII, and ErIII. Upconversion spectra recorded with CW and femtosecond excitation are qualitatively similar whereas the intensity dependence of upconversion depends on excitation mode (CW or femtosecond). To further assess the observed intensity dependence, we perform light-matter interaction simulations in the dynamic range from 100 fs to 3 ms for 18-level system describing the UCNPs, including 9 NdIII levels, 2 YbIII, and 7 ErIII levels and a classical model for the excitation source. The calculated time- and intensity-dependent energy level population are compared with measured spectra to understand CW vs femtosecond laser-induced upconversion. To further discuss the differences between CW and femtosecond laser-induced light-matter interactions for the systems studied here, we perform semi-classical pulse propagation simulations and ultrafast pump-probe measurements to study how the light source bandwidth, relative to the absorption linewidth, influence light absorption and transmission and further connect these results with the intensity dependence. Overall, we report our progress toward mechanistic studies of light-matter interaction and photophysical pathways following femtosecond excitation and UCNPs.

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Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

Cited by 101 publications

References 31 publications

Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

A method for single molecule localization microscopy of tissues reveals non-random distribution of nuclear pores in Drosophila

Femtosecond Laser Induced Luminescence in Hierarchically Structured Nd3+,Yb3+,Er3+ Co-Doped Upconversion Nanoparticles: Light-Matter Interaction Mechanisms from Experiments and Simulations

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