Optical alignment device for two-photon microscopy.

Area of Science:

• Neuroscience
• Optical Microscopy
• Biophysics

Background:

• Two-photon excitation fluorescence microscopy offers high resolution and penetration depth for in vivo brain studies.
• Optical access to the brain often involves skull implantation of glass windows, introducing spherical aberrations.
• Collar-correction objectives are used to correct aberrations, but their efficiency is tilt-angle dependent.

Purpose of the Study:

• Characterize the effect of glass window tilt angle on fluorescence signal in thicker windows.
• Compare performance with and without collar-correction objectives.
• Develop and validate a device and procedure for precise in vivo optical alignment.

Main Methods:

• Experimental characterization of fluorescence signal as a function of tilt angle using double cover slides.
• Design and implementation of a novel optical alignment device for in vivo microscopy.
• Development of a tilt-correction procedure for rapid and accurate alignment.

Main Results:

• Significant impact of tilt angle on collected fluorescence signal, especially with thicker windows.
• Demonstration of a simple alignment device achieving <0.25° tilt angle in vivo.
• Validation of a procedure enabling accurate alignment (<0.2° resolution) in few iterations.

Conclusions:

• Minimizing glass window tilt is crucial for optimizing two-photon microscopy in vivo.
• The developed device and procedure significantly improve imaging quality by enabling precise optical alignment.
• This work provides a practical solution for enhancing neural structure investigation in live subjects.