Abstract
Tissue scattering and absorption impact the excitation and emission light in different ways for multiphoton imaging. The collected fluorescence includes both ballistic photons and scattered photons whereas multiphoton excited signal within the focal volume is mostly generated by ballistic photons. The impact of excitation wavelengths on multiphoton imaging has been extensively investigated before; however, experimental data is lacking to evaluate the impact of emission wavelengths on fluorescence attenuation in deep imaging. Here we perform three-photon imaging of mouse brain vasculature in vivo using green, red, and near-infrared emission fluorophores, and compare quantitatively the attenuation of the fluorescence signal in the mouse brain at the emission wavelengths of 520 nm, 615 nm and 711 nm. Our results show that the emission wavelengths do not significantly influence the fluorescence collection efficiency. For the green, red and near-infrared fluorophores investigated, the difference in fluorescence collection efficiency is less than a factor of 2 at imaging depths between 0.6 and 1 mm. The advantage of long wavelength dyes for multiphoton deep imaging is almost entirely due to the long excitation wavelengths.
Highlights
Multiphoton microscopy (MPM) utilizes nonlinear excitation to confine the fluorescence generation within the focal volume, which allows the use of efficient, large-area detectors for the fluorescence collection to improve the signal-to-noise ratio (SNR) for deep imaging in scattering biological tissue [1,2,3,4,5,6,7]
Two imaging sessions were performed on the dye-labeled blood vessels and the normalized fluorescence signal is plotted as a function of depth for fluorescein excited at 1450 nm (Fig. 7(a)), Texas Red excited at 1450 nm (Fig. 7(b)), Texas Red excited at 1700 nm (Fig. 7(c)), and Alexa Fluor 647 excited at 1700 nm (Fig. 7(d))
Based on the experimental results, we found that, within 1 mm depth in the mouse brain, the attenuation of the green fluorescence (~520 nm) is slightly higher than the red fluorescence (~615 nm) fluorescence, with the difference being 1.34-1.43 times for imaging depths of 0.6 to 0.8 mm
Summary
Multiphoton microscopy (MPM) utilizes nonlinear excitation to confine the fluorescence generation within the focal volume, which allows the use of efficient, large-area detectors for the fluorescence collection to improve the signal-to-noise ratio (SNR) for deep imaging in scattering biological tissue [1,2,3,4,5,6,7]. Multiphoton excited signal within the focal volume is mostly generated by ballistic photons that are not scattered along the excitation path. Because of the wide-field geometry for fluorescence collection in MPM, both ballistic and scattered fluorescence photons contribute to the image formation [8]. Understanding the transport of light in biological tissue is important in selecting the optimum excitation and emission wavelengths for MPM. The advantage of using long excitation wavelengths for MPM has been extensively explored [10,11,12,13,14,15], the impact of emission wavelengths on deep imaging has not been systematically investigated before
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
