Quantitative two-photon imaging of fluorescent biosensors

Area of Science:

• Neuroscience
• Biomedical Optics
• Molecular Imaging

Background:

• Two-photon microscopy is widely used for imaging fluorescent biosensors in intact tissues, including brain slices and living animal models.
• Current applications primarily focus on temporal variations (e.g., calcium transients) rather than precise quantification of analyte levels.
• Challenges in quantitative measurements include in situ sensor calibration and comparing optical signals between in vivo and in vitro settings.

Purpose of the Study:

• To address the limitations of quantitative fluorescent biosensor measurements in intact tissues.
• To explore alternative microscopy techniques for accurate analyte level determination.
• To evaluate fluorescence lifetime imaging microscopy (FLIM) as a method for calibrated biosensor measurements.

Main Methods:

• Utilized two-photon microscopy for imaging fluorescent biosensors in biological tissues.
• Investigated the challenges associated with ratiometric measurements due to laser power fluctuations and tissue-dependent optical attenuation.
• Employed fluorescence lifetime imaging microscopy (FLIM) for quantitative analysis of biosensor occupancy.

Main Results:

• Demonstrated that traditional methods struggle with accurate in situ calibration and inter-environment signal comparison.
• Identified variations in laser power and wavelength-dependent tissue attenuation as significant hurdles for ratiometric imaging.
• Showcased FLIM's capability to provide well-calibrated measurements of analyte levels, independent of optical variations.

Conclusions:

• Quantitative analysis of fluorescent biosensors in vivo remains a significant challenge.
• FLIM presents a robust alternative for obtaining calibrated measurements of analyte concentrations in biological tissues.
• This technique holds promise for advancing precise molecular imaging and analyte quantification in neuroscience and beyond.