Selective imaging of surface fluorescence with very high aperture microscope objectives

Area of Science:

• Microscopy
• Biophysics
• Optical Imaging

Background:

• Selective surface fluorescence detection is crucial for studying biological processes at interfaces.
• Previous methods using lower NA objectives had limitations in resolution and sensitivity.
• The advent of high NA objectives (1.45 and 1.65) offers new possibilities for enhanced surface imaging.

Purpose of the Study:

• To describe three novel approaches for selective surface fluorescence detection.
• To demonstrate the utility of high NA objectives in these techniques.
• To provide practical guidance for implementing these advanced imaging methods.

Main Methods:

• Elaboration of prismless total internal reflection fluorescence (TIRF) using laser and arc lamp illumination with high NA objectives.
• A third approach utilizing high NA objectives to capture near-field emission from surface-bound fluorophores.
• Experimental validation and schematic illustrations of all described techniques.

Main Results:

• High NA objectives significantly enhance TIRF performance for biological cells compared to older 1.4 NA objectives.
• The near-field collection method offers a distinct, non-TIRF approach for surface fluorescence detection.
• All three methods provide selective surface detection capabilities.

Conclusions:

• Extremely high NA objectives are enabling new frontiers in selective surface fluorescence detection.
• These advanced techniques, including enhanced TIRF and near-field collection, offer superior performance for biological imaging.
• The described methods and practical suggestions facilitate the adoption of these powerful imaging tools.