Around-the-objective total internal reflection fluorescence microscopy.

Area of Science:

• Biophysics
• Optical Microscopy
• Fluorescence Imaging

Background:

• Total internal reflection fluorescence (TIRF) microscopy illuminates fluorophores near a surface using an evanescent field.
• Existing TIRF methods, like epi-illumination and prism-based, have limitations such as background autofluorescence or inability to collect near-field emission.

Purpose of the Study:

• To develop a novel TIRF microscopy technique that overcomes the limitations of existing methods.
• To enhance signal-to-noise ratio and improve near-field emission collection.

Main Methods:

• Developed "around-the-objective" TIRF microscopy.
• Conditioned the exciting laser beam to propagate through the gap between the objective and coverslip.
• Utilized the evanescent field generated at the interface.

Main Results:

• The new method effectively eliminates background light from microscope optics.
• Achieved selective illumination of fluorophores within approximately 50 nm of the interface.
• Successfully collected near-field emission from excited dipoles.

Conclusions:

• "Around-the-objective" TIRF microscopy offers a superior approach for high-resolution fluorescence imaging near interfaces.
• This technique enhances sensitivity and reduces artifacts in TIRF applications.
• Provides a valuable tool for studying biological processes at interfaces with improved optical performance.