Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision
Preprint posted on 5 December 2018 https://www.biorxiv.org/content/early/2018/12/05/487728
Super resolution microscopy has revolutionized imaging of biological samples by improving (lateral) resolution by an order of magnitude, from a few hundred nanometers to tens of nanometers, under optimized conditions, winning its inventors the 2014 Nobel Prize in Chemistry. Even higher resolutions are theoretically possible, however, current approaches suffer from hardware-induced problems.
In particular, drift-induced artefacts are problematic, because to obtain super-resolution, many images have to be captured of one and the same field of view. In every image, only a small sub-population of fluorescently labelled molecules are lit up, enabling precise localization of each molecule, using the knowledge that one and only one molecule is responsible for a given fluorescent spot. By combining the knowledge of all the molecules’ localizations, one obtains the final image, often by combining tens of thousands of sparsely lit frames. Evidently, it is crucial that the absolute position of molecules stays the same between frames. Any drift will have a severe impact on resolution, because one molecule could be mapped to different positions.
Key Technological Improvements
This preprint aims to overcome the current hardware limitations of super-resolution microscopy by introducing three different hardware-based correction mechanisms. In a first step, the authors use a feedback loop between the stage and fiducial markers close to the sample to realign the stage such that the same area of the sample is captured in the field of view at all times. Second, the light path, which also forms a major error source, is kept well-aligned by an independent feedback loop. Here, a piezo-electric mirror is used to repeatedly realign the optical path. Third, by imaging a nano-fabricated array of holes with precisely known positions, systematic errors of the camera and chromatic and optical aberrations can be reduced. Taken together, these three correction mechanisms increase precision and enable longer acquisitions.
Among several use cases highlighted by the preprint, particularly impressive is the ability to perform relatively precise measurements of the spatial separation of molecules on the scale of a few nanometers. According to the authors, similar measurements are not possible using other current technologies such as FRET. As proof of principle the nearest neighbor distance between pCD3ζ and CD45, two molecules involved in initiation of T cell receptor signaling, is measured, answering a so far unresolved question.
One thing missing from a non-expert’s perspective is a concise account of how difficult this setup is to implement in practice. Also, from a practical point of view, making the code available in a public repository and not ’on request’ would be a nice addition. In any case, feedback single-molecule localization microscopy seems like a potential game-changer, possibly being able to replace FRET and allowing imaging at unprecedented spatial resolution.
Posted on: 14 January 2019Read preprint
Also in the biophysics category:
Learning a conserved mechanism for early neuroectoderm morphogenesis
Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2
Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom
Also in the immunology category:
Prenatal inflammation reprograms hyperactive ILC2s that promote allergic lung inflammation and airway dysfunction
NAD+ metabolism is a key modulator of bacterial respiratory epithelial infections
TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling
preListsbiophysics category:in the
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
|preLights peer support
66th Biophysical Society Annual Meeting, 2022
Preprints presented at the 66th BPS Annual Meeting, Feb 19 - 23, 2022 (The below list is not exhaustive and the preprints are listed in no particular order.)
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
Biophysical Society Meeting 2020
Some preprints presented at the Biophysical Society Meeting 2020 in San Diego, USA.
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
|Madhuja Samaddar et al.
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
Preprints related to the application and development of biomolecular NMR spectroscopy
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
|Joseph Jose Thottacherry
Also in the immunology category:
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
|Sandra Malmgren Hill
Antimicrobials: Discovery, clinical use, and development of resistance
Preprints that describe the discovery of new antimicrobials and any improvements made regarding their clinical use. Includes preprints that detail the factors affecting antimicrobial selection and the development of antimicrobial resistance.
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.