Volumetric Single-Molecule Tracking Inside Subcellular Structures

Area of Science:

• Cellular Biology
• Microscopy Techniques
• Biophysics

Background:

• Cellular functions rely on molecular motion in 3D.
• Existing 3D-SMLM methods offer high resolution but often lose cellular context.
• Need for advanced microscopy to study molecular dynamics within cellular structures.

Purpose of the Study:

• To develop and validate a correlative imaging approach combining SMLFM and widefield Fourier light-field microscopy.
• To improve the sensitivity of measurements for molecular organization, chemical environment, and diffusion.
• To enable volumetric sub-cellular segmentation for enhanced analysis.

Main Methods:

• Integration of single-molecule light-field microscopy (SMLFM) with widefield Fourier light-field microscopy.
• Correlative volumetric organelle imaging for instantaneous acquisition of subcellular volumes.
• Application of volumetric sub-cellular segmentation for data analysis.

Main Results:

• Demonstrated measurement of molecular organization of nuclear-localized HaloTag protein relative to cell nuclei.
• Characterized molecular diffusion of calreticulin in ${\alpha }_1$ -antitrypsin deficiency.
• Revealed increased heterogeneous motion of calreticulin within endoplasmic reticulum inclusions.

Conclusions:

• The combined SMLFM and Fourier light-field microscopy approach provides enhanced cellular context for molecular measurements.
• This technique is valuable for studying molecular organization and dynamics in various cellular compartments.
• The findings offer insights into molecular diffusion changes associated with specific cellular conditions like ${\alpha }_1$ -antitrypsin deficiency.