Dataset Information

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

ABSTRACT: Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off ("blink"), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.

SUBMITTER: Nahidiazar L

PROVIDER: S-EPMC4938622 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

Nahidiazar Leila L Agronskaia Alexandra V AV Broertjes Jorrit J van den Broek Bram B Jalink Kees K

PloS one 20160708 7

Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off ("blink"), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and l ...[more]

PMID: 27391487

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Project description:Over the last decade, single-molecule localization microscopy (SMLM) has developed into a set of powerful techniques that have improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features down to a few tens of nanometers. We introduce a single molecule-based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Along with epi-illumination, the system employs a scanning-based 4f detection for volume imaging. The 4f system comprises a combination of an electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of an electrically-tunable lens (ETL) in a 4f detection system, the selectivity of the axial object plane is achieved, for which the image forms in the image/detector plane. So, in principle, one can scan the object volume by just altering the aperture of ETL. Two schemes were adopted to carry out volume imaging: cyclic scan and conventional scan. The cyclic scheme scans the volume in each scan cycle, whereas plane-wise scanning is performed in the conventional scheme. Hence, the cyclic scan ensures uniform dwell time on each frame during data collection, thereby evenly distributing photobleaching throughout the cell volume. With a minimal change in the system hardware (requiring the addition of an ETL lens and related electronics for step-voltage generation) in the existing SMLM system, volume scanning (along the z-axis) can be achieved. To calibrate and derive critical system parameters, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to visualize the architecture of actin-filaments and the distribution of Meos-Tom20 molecules on the mitochondrial membrane. The technique is further exploited to understand the clustering of Hemagglutinin (HA) protein single molecules in a transfected cell for studying Influenza-A disease progression. The system, for the first time, enabled 3D visualization of HA distribution that revealed HA cluster formation spanning the entire cell volume, post 24 hrs of transfection. Critical biophysical parameters related to HA clusters (density, the number of HA molecules per cluster, axial span, fraction of clustered molecules, and others) are also determined, giving an unprecedented insight into Influenza-A disease progression at the single-molecule level.

Dataset Information

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

Publications

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

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