Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact...

Area of Science:

• Microscopy and imaging techniques
• Biophysics and cell biology
• Optical engineering

Background:

• Standard microscopy limits axial resolution at the cell-substrate interface.
• Total internal reflection fluorescence microscopy (TIRFM) offers improved surface sensitivity.
• Variable-angle TIRFM (VA-TIRFM) allows control over evanescent wave penetration depth.

Purpose of the Study:

• To develop and characterize a novel compact illumination device for VA-TIRFM.
• To achieve high axial resolution for studying cell-substrate interactions.
• To demonstrate the device's utility in quantitative biological measurements.

Main Methods:

• Integration of a compact illumination device replacing the standard condenser in an upright microscope.
• Delivery of laser light via monomode fiber to the sample at variable angles.
• Excitation of fluorophores using an evanescent wave with controlled penetration depth.
• Analysis of fluorescence signals using a four-layer model and Gaussian intensity profile.

Main Results:

• The device enables precise localization of fluorophores with nanometre axial resolution.
• Accurate determination of distances between the glass substrate and plasma membrane (0-30 nm in focal contacts, 100-300 nm elsewhere).
• Successful examination of cytoplasmic and plasma membrane markers (calcein, laurdan) in endothelial cells.

Conclusions:

• The novel VA-TIRFM illumination device provides high axial resolution for cell-substrate interface studies.
• It is suitable for quantitative measurements, cell topology analysis, and studying dynamic cellular processes like endocytosis/exocytosis.
• The device's compatibility with advanced techniques (FLIM, FRET) broadens its application scope.