Robust scan synchronized force-fluorescence imaging

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Simultaneous atomic force microscopy (AFM) and confocal fluorescence microscopy provide insights into protein dynamics in live cells.
• Current limitations in synchronously scanning AFM and confocal microscope stages hinder visualization of force-induced protein redistribution.

Purpose of the Study:

• To develop an integrated AFM-confocal fluorescence microscope platform with synchronous scanning capabilities.
• To overcome artifacts associated with piezo motion ramping in dual-mode microscopy.
• To enable visualization of force-induced changes in fluorescent protein distribution within live cells.

Main Methods:

• An integrated AFM-confocal fluorescence microscope platform was constructed.
• A synchronous scanning method was implemented for both microscope piezo stages.
• The platform was used to apply mechanical force to single points on live cells while collecting scanned images.

Main Results:

• The synchronous scanning method eliminated image artifacts from piezo motion ramping.
• Accurate pixel binning was achieved, enabling precise data collection.
• The redistribution of fluorescent E-cadherin in response to applied mechanical force was successfully monitored in live cells.

Conclusions:

• The developed integrated AFM-confocal platform offers a robust technique for studying force-induced cellular responses.
• This technology facilitates mechanistic and structural insights into protein dynamics under mechanical stress.
• The platform enables precise visualization of transmembrane protein redistribution, such as E-cadherin, upon force application.