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Related Experiment Video

Updated: Aug 31, 2025

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

Published on: December 5, 2019

In situ single particle classification reveals distinct 60S maturation intermediates in cells.

Bronwyn A Lucas^1,2, Kexin Zhang^1,2, Sarah Loerch²

¹RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States.

|August 25, 2022

Summary

This study introduces a new method using signal-to-noise ratio (SNR) in two-dimensional template matching (2DTM) to accurately classify related molecular complexes, like ribosome biogenesis intermediates, within cryo-electron microscopy images.

Keywords:

2DTM FIB-milling S. cerevisiae cell biology in situ cryo-EM molecular biophysics nucleus particle classification ribosome biogenesis structural biology

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Area of Science:

Structural biology
Molecular imaging
Cell biology

Background:

High-resolution template matching (2DTM) previously localized ribosomes in cryo-EM images.
Differentiating closely related molecular complexes in cellular images remains challenging due to noise variations.

Purpose of the Study:

To develop and validate a novel method for classifying related molecular complexes using signal-to-noise ratio (SNR) in 2DTM.
To apply this method to distinguish intermediate states of ribosome biogenesis in yeast cells.

Main Methods:

Comparing SNRs from 2DTM with different templates against cellular targets to correct for local noise.
Utilizing a maximum likelihood approach to determine classification confidence.
Applying the method to nuclear pre-60S and cytoplasmic mature 60S ribosome populations in Saccharomyces cerevisiae.

Main Results:

The SNR-based 2DTM approach effectively corrects for local noise variations in cryo-EM images.
Related molecular populations, including intermediate states of 60S ribosome biogenesis, were successfully separated.
Subcellular localization was used to validate the classification of pre-60S and mature 60S ribosomes.

Conclusions:

SNR-based 2DTM offers a robust method for classifying related molecular complexes without requiring 3D reconstructions.
This technique enables classification even with limited numbers of target particles.
The method is valuable for studying dynamic molecular processes like ribosome biogenesis.