Ratiometric widefield imaging with spectrally balanced detection

Area of Science:

• Optics and Photonics
• Microscopy Techniques
• Biophysical Measurements

Background:

• Ratiometric imaging is crucial for quantitative microscopy, enabling detection of FRET, anisotropy, and spectral shifts.
• It measures local physical and chemical variations like pH, ion composition, and electric potential using nano-scale optical probes.
• Existing methods may lack spectral tunability for complex, spectrally heterogeneous samples.

Purpose of the Study:

• To propose and demonstrate a novel scheme for widefield ratiometric imaging.
• To enable continuous tuning of the cutoff wavelength between spectral detection channels.
• To facilitate ratiometric imaging of spectrally heterogeneous samples.

Main Methods:

• The proposed scheme utilizes angle-tuning of an image splitting dichroic beamsplitter.
• This method is analogous to tunable interference filters.
• It allows for spectral tunability within the detection path.

Main Results:

• Demonstration of a widefield ratiometric imaging scheme with tunable spectral channels.
• Successful implementation of angle-tuning for continuous wavelength cutoff adjustment.
• Validation of the method for spectrally heterogeneous sample imaging.

Conclusions:

• The developed scheme provides continuous spectral tunability for ratiometric imaging.
• This advancement is particularly beneficial for analyzing spectrally heterogeneous samples.
• The technique enhances quantitative microscopy by allowing spectrally balanced ratiometric detection.