Source: http://aoot.osa.org/boe/abstract.cfm?uri=boe-10-4-1905
Timestamp: 2019-04-25 03:58:55+00:00

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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.
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Fig. 1 (a) Measured spectra of the laser source operating at 1450 nm and 1700 nm. (b) Transmission data for the three emission filters used for fluorescein (520/15 nm), Texas Red (615/20 nm) and Alexa Fluor 647 (711/25 nm).
Fig. 2 Dependence of three-photon-excited fluorescence on excitation power for (a) fluorescein excited at 1450 nm, (b) Texas Red excited at 1450 nm, (c) Texas Red excited at 1700 nm, and (d) Alexa Fluor 647 excited at 1700 nm. The blue diamonds are the measured data, and the red lines are linear fits to the experimental results. The slope is indicated in each figure.
Fig. 3 Three-photon fluorescence images of the brain vasculature labeled by fluorescein in (a) and (c), and by Texas Red in (b) and (d). Images in (a) to (d) were acquired by using 1450 nm excitation. Three-photon fluorescence images of the brain vasculature labeled by Texas Red in (e) and (g), and by Alexa Fluor 647 in (f) and (h). Images in (e) to (h) were acquired by using 1700 nm excitation. The depths are indicated in the images. All the images are shown with the same contrast setting. Scale bars, 50 µm.
Fig. 4 Simultaneous imaging of fluorescein and Texas Red using 1450 nm excitation. (a) Normalized fluorescence signal of fluorescein and Texas Red as a function of depth. (b) Ratio of the normalized Texas Red signal and the normalized fluorescein signal at each depth.
Fig. 5 Simultaneous imaging of Texas Red and Alexa Fluor 647 using 1700 nm excitation. (a) Normalized fluorescence signal of Texas Red and Alexa Fluor 647 as a function of depth. (b) Ratio of the normalized Alexa Fluor 647 signal and the normalized Texas Red signal at each depth.
Fig. 6 (a) Ratio of the normalized fluorescence of Texas Red and fluorescein averaged every 100 μm depth interval in 10 different mice. Each color/marker represents a different mouse. (b) Ratio of the normalized fluorescence of Alexa Fluor 647 and Texas Red averaged every 100 μm depth interval in 7 different mice. Each color/marker represents a different mouse.
Fig. 7 Variations between sequential imaging sessions. (a) Normalized fluorescein signal excited at 1450 nm as a function of depth. (b) Normalized Texas Red signal excited at 1450 nm as a function of depth. (c) Normalized Texas Red signal excited at 1700 nm as a function of depth. (d) Normalized Alexa Fluor 647 signal excited at 1700 nm as a function of depth.
Fig. 9 Simultaneous imaging before and after channel swapping. (a) Normalized fluorescence signal of fluorescein and Texas Red as a function of depth for the two simultaneous imaging sessions before and after channel swapping. (b) Normalized fluorescence signal of Texas Red and Alexa Fluor 647 as a function of depth for the two simultaneous imaging sessions before and after channel swapping.
Fig. 10 Diffusion theory of emission light transport at different wavelengths for mouse brain in vivo. (a) Effective attenuation length of emission light calculated by the modified solution of diffusion theory, Eq. (2). (b) The collected fluorescence at the brain surface from a depth of 600 μm, 800 μm, 1 mm, and 1.6 mm based on the calculated effective attenuation length. The transmission data for the three emission filters used in imaging are also shown.
Fig. 11 Beer’s law calculations of emission light transmission from a depth of 600 μm, 800 μm, 1 mm, and 1.6 mm as well as the emission light transmission through a 50-μm-diameter vessel.
Table 1 Ratio of the normalized fluorescence of Texas Red and fluorescein for all 10 mice at 100 μm depth interval.
Table 2 Ratio of the normalized fluorescence of Alexa Fluor 647 and Texas Red for all 7 mice at 100 μm depth interval.
Table 3 Summary of the experimental results and theoretical calculations with different blood concentrations.
Table 4 Summary of the fluorescence transmission ratios for fluorescein (FL), Texas Red (TR) and Alexa Fluor 647 (AF647) with 40-nm bandwidth filters and with no filters at all (full emission spectrum). The blood volume concentration is assumed to be 3%.
Ratio of the normalized fluorescence of Texas Red and fluorescein for all 10 mice at 100 μm depth interval.
Ratio of the normalized fluorescence of Alexa Fluor 647 and Texas Red for all 7 mice at 100 μm depth interval.
Summary of the experimental results and theoretical calculations with different blood concentrations.
Summary of the fluorescence transmission ratios for fluorescein (FL), Texas Red (TR) and Alexa Fluor 647 (AF647) with 40-nm bandwidth filters and with no filters at all (full emission spectrum). The blood volume concentration is assumed to be 3%.

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