High-speed hyperspectral microscopy: Fourier transform spectroscopy vs. liquid crystal tunable filters

Area of Science:

• Biophotonics
• Microscopy
• Spectroscopy

Background:

• Hyperspectral fluorescence microscopy enables multiplexed imaging of living biological samples.
• Conventional spectral filters (LCTF, AOTF) cause photon loss, limiting imaging performance.
• Photon loss in traditional filters hinders the imaging of low-signal biological targets.

Purpose of the Study:

• To evaluate Fourier transform spectroscopy (FTS) as a high-speed hyperspectral microscopy platform.
• To compare the performance of FTS against conventional liquid crystal tunable filters (LCTFs).
• To assess the signal-to-noise ratio (SNR) and imaging capabilities of FTS under low photon budgets.

Main Methods:

• A head-to-head comparison of LCTF and FTS platforms was conducted using identical cameras and optical setups.
• Twelve distinct CD marker-fluorophore bead conjugates were used to test performance across varying signal strengths.
• Exposure constraints were matched between LCTF and FTS systems for a fair comparison.

Main Results:

• FTS captures all emission wavelengths simultaneously, resulting in a lower noise floor and spectral uncertainty compared to LCTF.
• FTS demonstrated a significantly higher signal-to-noise ratio (SNR) than LCTF across all tested biomarker combinations.
• FTS achieved a 42% higher SNR for equivalent peak intensities and a 2.5-fold to 8.5-fold SNR boost for the same targets, enabling visualization of previously invisible dim targets.

Conclusions:

• Fourier transform spectroscopy (FTS) provides a superior architecture for high-speed hyperspectral microscopy.
• FTS overcomes the photon loss limitations of conventional filters, offering improved SNR and imaging of low-signal specimens.
• FTS is a promising enabling platform for advanced biological imaging applications requiring high spectral resolution and speed.