In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Confocal microscopy offers high spatial resolution for imaging fluorescent molecules in cells.
• Total internal reflection microscopy (TIRM) further enhances resolution, enabling imaging of sub-diffraction limit volumes.
• Single-molecule spectroscopy in situ avoids ensemble averaging and studies proteins in native cellular environments.

Purpose of the Study:

• To introduce a novel variation of TIRM, focused TIRM (fTIRM), for significantly reduced detection volumes.
• To demonstrate the capability of fTIRM for in situ single-molecule fluorescence spectroscopy.
• To investigate the application of fTIRM in studying protein dynamics within muscle tissue.

Main Methods:

• Development and implementation of focused total internal reflection microscopy (fTIRM).
• Experimental verification of attoL volume elements using Brownian motion of fluorescent nanospheres.
• Application of fTIRM for in situ single-molecule fluorescence spectroscopy of myosin in muscle fibers.

Main Results:

• fTIRM reduces the imaging volume element to approximately 3 attoL.
• The technique successfully isolates single proteins for in situ spectroscopic analysis.
• fTIRM detected that over 75% of bleachable fluorescence in muscle fibers originated from five chromophore-labeled myosins.

Conclusions:

• fTIRM represents a significant advancement in achieving ultra-small detection volumes for microscopy.
• The technique enables high-resolution, in situ single-molecule studies of protein function and dynamics.
• fTIRM provides novel insights into protein behavior within the native cellular environment, exemplified by myosin studies.