Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures [preprint]

Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs [preprint]

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 μm thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data [preprint]

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs [preprint]

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 μm thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues

CRISPR-Cas9 nuclear dynamics and target recognition in living cells

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from \u3e3 h to as low as time

Vision Beyond Optics: Standardization, Evaluation and Innovation for Fluorescence Microscopy in Life Sciences

Fluorescence microscopy is an essential tool in biomedical sciences that allows specific molecules to be visualized in the complex and crowded environment of cells. The continuous introduction of new imaging techniques makes microscopes more powerful and versatile, but there is more than meets the eye. In addition to develop- ing new methods, we can work towards getting the most out of existing data and technologies. By harnessing unused potential, this work aims to increase the richness, reliability, and power of fluorescence microscopy data in three key ways: through standardization, evaluation and innovation. A universal standard makes it easier to assess, compare and analyze imaging data – from the level of a single laboratory to the broader life sciences community. We propose a data-standard for fluorescence microscopy that can increase the confidence in experimental results, facilitate the exchange of data, and maximize compatibility with current and future data analysis techniques. Cutting-edge imaging technologies often rely on sophisticated hardware and multi-layered algorithms for reconstruction and analysis. Consequently, the trustworthiness of new methods can be difficult to assess. To evaluate the reliability and limitations of complex methods, quantitative analyses – such as the one present here for the 3D SPEED method – are paramount. The limited resolution of optical microscopes prevents direct observation of macro- molecules like DNA and RNA. We present a multi-color, achromatic, cryogenic fluorescence microscope that has the potential to produce multi-color images with sub-nanometer precision. This innovation would move fluorescence imaging beyond the limitations of optics and into the world of molecular resolution

An automated Bayesian pipeline for rapid analysis of single-molecule binding data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein\u27s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites

Cri Spr-Cas9 Nuclear Dynamics And Target Recognition In Living Cells

The bacterial CRI SPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRI SPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRI SPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from \u3e 3 h to as low as \u3c 2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRI SPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time