Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

Area of Science:

• Biophysics
• Optical Microscopy
• Biomedical Engineering

Background:

• Conventional microscopy generates light signals above and below the focal plane, reducing image clarity.
• Multiphoton microscopy offers improved spatial resolution by confining signal generation to the focal point.
• Low emitted light intensity in biological samples often limits imaging quality.

Purpose of the Study:

• To enhance photon collection efficiency in multiphoton microscopy.
• To improve signal intensity for epi-detected signals without forward detection.
• To provide a practical solution for imaging challenging biological specimens.

Main Methods:

• A simple aluminum-coated coverslip mirror was placed directly beneath the sample.
• The method was tested with fluorescent test slides, collagen gels, and intact mouse skeletal muscles.
• Quantitative analysis measured signal intensity increases using epi-detection.

Main Results:

• Second-harmonic generation signal in skeletal muscle increased ninefold.
• Third-harmonic generation signal in skeletal muscle increased sevenfold.
• Fluorescent signal in test slides increased up to twofold.

Conclusions:

• Placing a mirror under the sample significantly increases collected photon fraction for epi-detection.
• This method enhances signal intensity in multiphoton microscopy, particularly when forward detection is not feasible.
• The technique offers a valuable improvement for intravital microscopy and imaging complex biological structures.