Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biophysics

Background:

• Total internal reflection fluorescence microscopy (TIRFM) is crucial for studying surface-bound structures in cell biology.
• Conventional TIRFM typically requires complex setups like prisms or specialized objectives for illumination.
• Existing methods face limitations in simplicity and accessibility for routine surface analysis.

Purpose of the Study:

• To present a new, simplified approach to total internal reflection fluorescence microscopy (TIRFM).
• To demonstrate the use of a high-index upconverting glass-ceramic coverslip for TIRFM.
• To enable efficient excitation of surface-bound fluorescent species using a novel method.

Main Methods:

• Samples were mounted on a coverslip made from a high-index upconverting glass-ceramic.
• Excitation was achieved using a low-cost near-infrared laser diode.
• The coverslip generated visible emission (violet, green, red) upon excitation, creating an evanescent wave.

Main Results:

• The novel coverslip generated intense, narrow-band visible emission, enabling TIRFM.
• The method allowed excitation of a wide range of fluorescent labels.
• Fluorescence from the sample could be detected in spectral regions with minimal direct coverslip emission.

Conclusions:

• The developed method offers a simple and effective alternative for total internal reflection fluorescence microscopy.
• Upconverting glass-ceramic coverslips provide a versatile platform for TIRFM applications.
• This technique holds promise for advancing the study of surface-bound structures in cell biology.