In Vivo Deep-Brain Structural and Hemodynamic Multiphoton

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In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots Hongji Liu,†,§ Xiangquan Deng,†,§ Shen Tong,†,§ Chen He,† Hui Cheng,† Ziwei Zhuang,† Mengyao Gan,† Jia Li,† Weixin Xie,‡ Ping Qiu,*,† and Ke Wang*,† †

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Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China ‡ College of Electronics and Information Engineering, Shenzhen University, Shenzhen, 518060, China S Supporting Information *

ABSTRACT: Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4−5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths. KEYWORDS: Multiphoton microscopy, 1700 nm window, quantum dots, brain imaging circuits,7,8 and identifying brain disease,9,10 to name just a few examples. In comparison with other 3D optical microscopy technology such as confocal microscopy, the most notable merit of MPM is its superb deep penetration capability. Among the various modalities of MPM using different excitation wavelengths, it has been demonstrated that three-photon microscopy (3PM) excited at the 1700 nm window (covering roughly 1600−1840 nm) enables the largest brain imaging depth in vivo so far.2,11−16 The underlying principles are2 (1) excitation within the 1700 nm window suffers from least attenuation of excitation light due to brain scattering and absorption, resulting in higher signal level deep in the brain; (2) 3PM better suppresses background fluorescence generated from the

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rain is the most complex biological tissue, the function and mechanism of which are far from full comprehension. It embraces a variety of biological structures such as vasculature and different types of cells and the related functions such as blood flow and neuronal firing. In terms of brain research, the prerequisite is the ability to see the structures to be investigated. Optical microscopy has been widely adopted for brain imaging. Among the various modalities in optical microscopy, multiphoton microscopy (MPM)1 is especially suitable for both structural and functional brain imaging due to the following reasons: (1) intrinsic 3D sectioning, (2) noninvasiveness, (3) subcellular resolution, (4) minimization of out-of-focus photobleaching in multiphoton fluorescence microscopy, (5) hemodynamic and cellular function tracking, and (6) deep-tissue penetration. As a result, MPM has been applied to visualizing brain vasculature and cells,2−4 measuring blood flow speed,5,6 mapping brain © XXXX American Chemical Society

Received: April 25, 2019 Revised: June 21, 2019 Published: July 3, 2019 A

DOI: 10.1021/acs.nanolett.9b01708 Nano Lett. XXXX, XXX, XXX−XXX

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Nano Letters

Figure 1. Characterization of three-photon excitation properties. (a) Measured fluorescence signals (symbols) vs excitation power in Qtracker655 at three different excitation wavelengths of 1600, 1700, and 1800 nm plotted on log scales. The lines are fitting results, with fitted slopes indicated in the figure. (b) Measured ησ3 for quantum dots (Qtracker655, Qtracker705, Qtracker800, and Beida630), Texas Red, and SR101 from 1600 to 1840 nm.

such as fluorescein).18 It can be expected that quantum dots may also dramatically boost the signal level in 3PM excited at the 1700 nm window and enable larger imaging depths. However, the following undetermined parameters hinder their application: (1) What is the ησ3 for quantum dots, especially when compared with the commonly used Texas Red dextran and SR101? (2) What is the three-photon emission spectrum of quantum dots in the vasculature in vivo? It has been demonstrated that the emission spectrum of quantum dots depends on environmental parameters such as temperature.25 For a complex biological environment such as the vasculature in the mouse brain in vivo, the three-photon emission spectrum may be different from prepared solutions. The photodetector selection for efficient detection (boosting φ) of the three-photon signals in the deep brain should be based on the measured three-photon emission spectrum in vivo. Targeting deep-brain structural and hemodynamic MPM, here we demonstrate characterization of the three-photon action cross section and emission spectrum of red quantum dots Qtracker655 excited at the 1700 nm window. This commercially available product has been demonstrated to be suitable for both cell and tissue 2PM.25,26 These results, to the best of our knowledge, are the first characterization of these parameters related to deep-brain imaging. On the basis of these measured results, we further demonstrate 3PM of both vasculature and measurement of blood flow speed on the basis of hemodynamic 3PM in the deep mouse brain in vivo. The 2100 μm vasculature imaging depth and 1600 μm hemodynamic imaging depth, to the best of our knowledge, set record depths among all MPM modalities. In order to verify the fluorescence generated from Qtracker655 excited at the 1700 nm window had its threephoton origin, first we characterized the dependence of fluorescence signal counts on excitation power at three excitation wavelengths of 1600, 1700, and 1800 nm within this excitation window. The measured results plotted on log scales (Figure 1a) show that the slopes after linear fitting are 2.97, 3.03, and 3.33 for excitations at 1600, 1700, and 1800 nm, respectively. These results verify that three-photon fluorescence can indeed be excited from Qtracker655. We also note that, toward longer excitation wavelength (e.g., 1800 nm), the slightly larger slope than 3 (measured slope = 3.33) is supposedly from the contribution of four-photon fluorescence. Next, we measured wavelength-dependent ησ3 for the fluorophores covering the entire 1700 nm excitation window

surface of the brain. Using this technique, brain structures below the highly scattering white matter layer in the mouse brain were clearly imaged in vivo for the first time, without damaging the overlying layers.2 According to theory,2 dictated by the signal-to-background ratio (SBR), the depth limit for structural 3PM in brain is ∼3700 μm assuming imaging in the neocortex (aka gray matter) only. Experimentally, the largest imaging depth using this technique is only 1620 μm below the surface of the brain.12 The huge gap between theoretical and experimental imaging depth is entirely due to three-photon signal depletion in the deep regions of the brain. In the application of hemodynamic MPM to measure the blood flow speed, so far the largest imaging depth is 900 μm, limited to the neocortical and white matter layer.6,11 Since 3PM excited at the 1700 nm window enables the largest imaging depth, the key to overcoming the current depth limit in both structural and hemodynamic MPM is thus to drastically boost three-photon signal levels. The collected three-photon fluorescence signal F3P is given by17−20 F3P = C*ησ3*φ*LPar, where C is the fluorophore concentration, ησ3 is the three-photon action cross section (product of quantum efficiency η and three-photon absorption cross section σ3) of the fluorophore, φ is the collection efficiency dependent on parameters such as the photosensitivity of the photodetector, and LPar is a parameter determined by the excitation laser. Various techniques for increasing the laser-dependent parameter LPar have been demonstrated.12,21−24 However, from the material perspective, there has been no investigation or effort to optimize the fluorophore-dependent parameter ησ3, whose influence on the resultant three-photon signal is manifested by its linear proportionality. Commonly used exogenous fluorophores suitable for 3PM excited at the 1700 nm window are Texas Red dextran2,12 and sulforhodamine 101 (SR101), both of which are red due to the long one-photon excitation wavelength (533−580 nm) corresponding to the 1700 nm window. Texas Red dextran is more suitable for imaging vasculature on a long-time basis, as SR101 will be cleared out faster due to its small molecule nature.3 Quantum dots are exceptionally bright nanocrystals suitable for labeling biological tissues. Their successful applications to two-photon microscopy (2PM) are critically dependent on their exceptionally large two-photon action cross section (∼3 orders of magnitude larger than conventional fluorophores B

DOI: 10.1021/acs.nanolett.9b01708 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 2. 3PM-guided in vivo three-photon emission spectrum measurement of Qtracker655 in the circulating blood in the mouse brain in vivo. (a) Three-photon fluorescence image of the blood vessel labeled by Qtracker655. (b) Zoomed-in area in part a for measuring the three-photon emission spectrum. (c) Measured three-photon emission spectrum of Qtracker655 corresponding to part b in the circulating blood in vivo (red) and in saline ex vivo (black).

Figure 3. Deep-brain structural 3PM of the Qtracker655-labeled vasculature in adult mouse in vivo. (a) 3D reconstruction of 3PM images of the mouse brain in vivo. Red, vasculature labeled by Qtracker655; green, THG signals delineating the white matter (WM) layer extending from 860 to 1000 μm below the surface of the brain. “0” denotes the brain surface. (b−e) 2D images from the 3D stack showing blood vessels at depths of 900 μm (b, in white matter), 1400 μm (c, in hippocampus), 1800 μm (d, in hippocampus), and 2100 μm (e, in hippocampus) below the surface of the brain. Scale bars: 50 μm. Pixel size: 512 × 512. (f−i) The fluorescence profiles of the lines across the vessels corresponding to parts b−e are displayed in semilogarithmic plots, which are used for SBR calculation. The calculated SBRs are indicated in each figure. C

DOI: 10.1021/acs.nanolett.9b01708 Nano Lett. XXXX, XXX, XXX−XXX

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Nano Letters using a home-built system and measured ησ3 for SR101 excited at 1680 nm17 as a reference (Supporting Information). The superb three-photon signal brightness of quantum dots is best illustrated by quantitative comparison of the measured wavelength-dependent ησ3 among Qtracker655, Texas Red dextran, and SR101, shown in Figure 1b. Within the 1700 nm window, ησ3 of Qtracker655 is 4−5 orders of magnitude higher than those of both Texas Red dextran and SR101. Specifically, the maximum ησ3 of Qtracker655 is 7.1 × 10−78 cm6 s2/ photon2, 5.9 × 104 times larger than the maximum ησ3 of Texas Red dextran (1.2 × 10−82 cm6 s2/photon2). This indicates that, for the same concentration of fluorophores and excitation parameters of the laser, three-photon fluorescence signals generated from Qtracker655 will be 5.9 × 104 times higher than that from Texas Red dextran, which lays the material basis for our deep-brain 3PM with quantum dots labeling. We have also measured ησ3 for several other commercially available quantum dots. The results in Figure 1b show that Qtracker655 has the largest ησ3 at the 1700 nm window among these quantum dots. This is the reason why we selected Qtracker655 to label the vasculature. Photodetector selection for efficient fluorescence signal detection especially in the deep brain is sensitively dependent on the emission spectrum of the fluorophores. For example, different photodetectors may show a difference by a factor of 2 or more in detected signals.23 The three-photon emission spectrum can be conveniently measured in solutions ex vivo; however, it may not be the same as the measured result in vivo. Both solvent (blood plasma) and body temperature (different from room temperature) may lead to a discrepancy in the measured spectra ex vivo and in vivo. In order to measure the in vivo three-photon emission spectrum from Qtracker655, we integrated an EMCCD spectrometer into our multiphoton microscope and performed 3PM-guided emission spectrum measurement in the mouse brain in vivo (Supporting Information). Zooming in on the Qtracker655-labeled blood vessel (Figure 2a,b), we were able to measure the three-photon emission spectrum of Qtracker655 in the circulating blood (Figure 2c, red line). In comparison with the ex vivo measured result (Figure 2c, black line), the measured three-photon emission spectrum in the circulating blood shows a slight red shift (2.5 nm) and broadening. It has been demonstrated that an elevated temperature could lead to the red shift of the twophoton emission spectrum for these same quantum dots.25 Here the observed red shift of the three-photon emission shift can at least be partly due to the higher temperature relative to the environment. The mechanisms of red shift and spectral broadening were explained in ref 27 and the references therein. Another contributing factor could be the different solvent (blood plasma for in vivo vs saline for ex vivo). The measured in vivo emission spectrum falls within the optimal detection bandwidth of the GaAsP photomultiplier tube,23 which we chose for subsequent deep-brain 3PM of the Qtracker655labeled vasculature. Having characterized the three-photon excitation and emission properties of the quantum dots, next we performed deep-brain structural 3PM of Qtracker655-labeled vasculature in the mouse brain in vivo, excited by a home-built femtosecond laser operating at the 1700 nm window. The reconstructed 3D stack is shown in Figure 3. In order to delineate the anatomical layers of the mouse brain, we performed simultaneous dual-channel 3PM (Supporting Information): three-photon fluorescence imaging (red in

Figure 3a) which reveals the Qtracker655-labeled vasculature and third harmonic generation (THG) imaging (green in Figure 3a). In brain MPM, it has been demonstrated that strong THG signals arise from myelinated axons, such as the white matter layer.2,28 In our experiment, the white matter layer spans from 860 to 1000 μm below the surface of the brain, above and below which are the neocortex and hippocampus, respectively. From both the 3D stack (Figure 3a) and the individual 2D image (Figure 3e), blood vessels 2100 μm below the surface of the brain can be clearly imaged using Qtracker655. This is, to the best of our knowledge, the largest brain imaging depth among all MPM modalities at all excitation wavelengths. The frame rate for each image in the whole stack is 2 s/frame (given by 2 ms/line, 512 × 512 pixels, two-frame average). It is heuristic to compare imaging depths and speeds using different MPM modalities excited at different wavelengths: 2PMs excited at 775, 925, and 1280 nm are restricted by the signal-to-background (SBR) limit2,29 to imaging depths of 700,11 1000,13 and 1280 μm (for the same imaging position as in this work, i.e., 2 mm lateral and posterior to the bregma point),30 respectively. 3PM excited at the 1700 nm window was first demonstrated to break the above records by reaching an imaging depth of 1350 μm2 and then pushed even deeper to 1620 μm by optimizing the laser parameter LPar,12 both of which using Texas Red dextran labeling. The frame rates for acquiring the deepest images are 20 s/frame in ref 2 and 8 s/ frame in ref 12. From this comparison, we can readily see that the exceptionally large three-photon action cross section of quantum dots is key to the notable increase in both imaging depth (by ∼500 μm) and speed (by 4 times). In order to quantify SBR, we followed previous procedures2 by plotting three-photon fluorescence line profiles across blood vessels at different depths (Figure 3f−i) and calculated the ratio between the maximum signal and the background (average value over the line profile without a signal). The measured SBRs range from 43 to 423, indicating that, even down to the imaging depth of 2100 μm, three-photon fluorescence imaging has not run into the SBR limit. In 2PM, in line with the SBR limit for structural imaging, blood flow speed measurement was confined to the neocortex and white matter only.6,11 In previously demonstrated 3PM with Texas Red dextran labeling, blood flow speed measurement in the deep brain was hindered by the slow frame rate as discussed above. Here, through structural 3PM, we expect this depth limit for hemodynamic MPM can be overcome. Experimentally, we used the standard line scan technique in our hemodynamic 3PM for measuring the blood flow speed.6 Figure 4 shows representative 2D and corresponding line scan images of the blood vessels in the white matter (Figure 4a,b) and hippocampus (Figure 4c−f), respectively. The line scan images are acquired along the dashed lines in each 2D image at 2 ms/line, and the vertical direction denotes time as indicated in the images. The line scan images show tilted stripes, a consequence of unlabeled blood cells flowing by. The blood flow speed is derived by calculating the slope of the stripes. The measured results are 0.94, 0.84, and 1.13 mm/s for the blood vessels 950, 1330, and 1600 μm below the surface of the brain. To the best of our knowledge, this is the deepest hemodynamic imaging and extraction of blood flow speed using MPM, which provides a novel methodology for relevant hemodynamic research in the hippocampus in vivo. We note that, although we could get clear 2D images of the blood D

DOI: 10.1021/acs.nanolett.9b01708 Nano Lett. XXXX, XXX, XXX−XXX

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quantum dots that sense calcium or membrane voltage in the brain.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01708. Detailed experimental setup and methods for quantum dot characterization, multiphoton microscopy, sample preparation, and animal procedures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ping Qiu: 0000-0002-2114-5909 Ke Wang: 0000-0002-9445-2168 Author Contributions §

H.L., X.D., S.T.: These authors contributed equally to this work. Funding

This work was supported by National Natural Science Foundation of China (NSFC) (61775143, 61605120, 61475103), (Key) Project of Department of Education of Guangdong Province (2017KZDXM073), and China Postdoctoral Science Foundation (2019M653025). Notes

The authors declare no competing financial interest.

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Figure 4. Deep-brain hemodynamic 3PM for measuring blood flow speed in vivo. (a, c, e) 2D images of Qtracker655-labeled blood vessels (red) 950 μm (a), 1330 μm (c), and 1600 μm (e) below the surface of the mouse brain. The green signals in part a show the myelinated axons in the white matter layer. (b, d, f) Line scan images along the dashed lines corresponding to parts a, c, and e, respectively, for measuring blood flow speed. Pixel size: 512 × 512.

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DOI: 10.1021/acs.nanolett.9b01708 Nano Lett. XXXX, XXX, XXX−XXX