AI Article Synopsis
- Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) allows for detailed 3D imaging of molecular structures in living specimens.
- The study explores the signal-to-noise ratio (SNR) of frequency-domain MPM-FLIM, focusing on how many photons are needed to achieve optimal imaging quality.
- Findings reveal that two-photon FD-FLIM can use 50% fewer photons compared to traditional one-photon FLIM to reach the same SNR, with additional enhancements possible by utilizing nonlinear optical mixing techniques.
Article Abstract
Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) has enabled three-dimensional quantitative molecular microscopy in vivo. The signal-to-noise ratio (SNR), and thus the imaging rate of MPM-FLIM, which is fundamentally limited by the shot noise and fluorescence saturation, has not been quantitatively studied yet. In this paper, we investigate the SNR performance of the frequency-domain (FD) MPM-FLIM with two figures of merit: the photon economy in the limit of shot noise, and the normalized SNR in the limit of saturation. The theoretical results and Monte Carlo simulations find that two-photon FD-FLIM requires 50% fewer photons to achieve the same SNR as conventional one-photon FLIM. We also analytically show that the MPM-FD-FLIM can exploit the DC and higher harmonic components generated by nonlinear optical mixing of the excitation light to improve SNR, reducing the required number of photons by an additional 50%. Finally, the effect of fluorophore saturation on the experimental SNR performance is discussed.
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| http://dx.doi.org/10.1364/JOSAA.33.0000B1 | DOI Listing |
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