Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection...

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Evanescent field intensity decay at dielectric interfaces is angle-dependent.
• Total internal reflection fluorescence microscopy (TIRFM) is limited in 3D reconstruction.
• Accurate measurement of submicroscopic distances in biological samples is challenging.

Purpose of the Study:

• To develop and validate computer-automated multiple angle-TIRFM (MA-TIRFM) for 3D fluorophore distribution analysis.
• To establish MA-TIRFM for nanometer-scale resolution of cell-substrate contact geometry.
• To compare cell-substrate contact in different cell types using MA-TIRFM.

Main Methods:

• Constructed computer-automated MA-TIRFM instrumentation using an F2 glass prism and microstepper motors.
• Acquired TIRFM images at multiple incident angles (>15 angles, ~0.5 degree steps).
• Developed theory for z-distribution computation via inverse Laplace transform and corrected for angle-dependent intensity.

Main Results:

• Instrument performance validated by accurately mapping a thin film's thickness, reproducing a spherical surface.
• MA-TIRFM successfully compared nanometer-scale cell-substrate contact geometry for 3T3 fibroblasts and MDCK epithelial cells.
• Demonstrated MA-TIRFM's capability for measuring submicroscopic distances between fluorescent probes and cell membranes.

Conclusions:

• MA-TIRFM provides precise 3D reconstruction of fluorophore distribution by leveraging angle-dependent evanescent field decay.
• The developed MA-TIRFM system offers nanometer resolution for studying biological interfaces and cell-substrate interactions.
• MA-TIRFM is established as a valuable tool for quantitative analysis of submicroscopic distances in cell biology.