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Updated: Nov 30, 2025

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for

Partha Pratim Mondal¹

¹Nanobioimaging Lab, Department of Instrumentation & Applied Physics, Indian Institute of Science, Bangalore, INDIA.

|November 16, 2020

Summary

This summary is machine-generated.

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy enhances resolution by selecting bright molecules. This technique reveals detailed molecular clusters crucial for understanding disease progression.

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Super-resolution Imaging of the Bacterial Division Machinery

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Published on: January 21, 2013

Area of Science:

Biomedical Imaging
Molecular Biology
Cell Biology

Background:

Traditional single-molecule localization microscopy (SMLM) faces resolution limits due to variable molecule sizes and overlapping point spread functions (PSFs).
Understanding molecular cluster dynamics, density, and size is vital for disease progression research.

Purpose of the Study:

To introduce and validate the Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy technique.
To achieve ultra-superresolution imaging by selectively reconstructing data from bright single molecules.

Main Methods:

POSSIBLE microscopy utilizes a narrow Gaussian probability distribution to filter and select single molecules based on their size and brightness.
The technique processes datasets to identify and map 'fortunate' molecules, discarding 'unfortunate' ones to reconstruct high-resolution images.
Demonstrated reconstruction of single-molecule images with average PSF sizes of 15 ± 10 nm, 30 ± 2 nm, and 50 ± 2 nm.

Main Results:

POSSIBLE microscopy achieved significantly better-resolved Dendra2-HA clusters with higher density in NIH3T3 fibroblast cells compared to traditional SMLM.
Cluster analysis revealed densely packed HA molecules, HA-HA interactions, and an increased number of HA molecules per cluster 24 hours post-transfection.
The technique provides new insights into influenza biology and molecular interactions.

Conclusions:

POSSIBLE microscopy offers a pathway to ultra-superresolution imaging, potentially achieving resolution close to the actual size of single molecules.
The technique enhances the study of molecular clusters, providing critical data for disease biology, oncology, and biomedical imaging.
This advanced imaging method promises significant applications in understanding complex biological systems and disease mechanisms.