Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo

: Visualization of lymphatic vessels is key to the understanding of their structure, function, and dynamics. Multiphoton microscopy (MPM) is a potential technology for imaging lymphatic vessels, but tissue scattering prevents its deep penetration in skin. Here we demonstrate deep-skin MPM of the lymphatic vessels in mouse hindlimb in vivo, excited at the 1700 nm window. Our results show that with contrast provided by indocyanine green (ICG), 2-photon fluorescence (2PF) imaging enables noninvasive imaging of lymphatic vessels 300 µ m below the skin surface, visualizing both its structure and contraction dynamics. Simultaneously acquired second-harmonic generation (SHG) and third-harmonic generation (THG) images visualize the local environment in which the lymphatic vessels reside. After removing the surface skin layer, 2PF and THG imaging visualize finer structures of the lymphatic vessels: most notably, the label-free THG imaging visualizes lymphatic valves and their open-and-close dynamics in real time. MPM excited at the 1700-nm window thus provides a promising technology for the study of lymphatic vessels.


Introduction
Lymphatic vessels are the transporting vessels of the lymphatic system. They collect extravasated fluid, macromolecules and cells from the interstitium and return them to the venous arm of the circulation [1,2]. Lymphatic vessels are also involved in cancer progression, as the cancer cells may enter into them which leads to lymph node metastases. They carry clear lymph and contain few cells, so optical imaging of lymph vessels typically uses exogenous fluorescent dyes to generate image contrast [1].
In order to visualize lymphatic vessels, different imaging technologies have been demonstrated, which is summarized in a recent review paper [1]. Optical imaging of lymphatic vessels has the advantages of fast acquisition speed and high spatial resolution. Among the various optical imaging modalities, single-photon fluorescence imaging has been the most widely used: Sevick and Kwon imaged the deep conducting lymphatic vessels propelling lymph from the inguinal lymph node to the axillary lymph node in mice [3]; Nakajima et al investigated chronic hindlimb lymphedema in mice [4]; Unno et al measured transition times of dye from injection to knees and inguinal region [5]; Sevick and Rasmussen imaged the pumping of deep lymphatic vessels to lymph nodes in humans [6]; John C. Rasmussen et al assessed tumor draining lymphatic basins in cancer patients to guide the resection of first draining lymph node [7]. ICG is taken up by lymph and provides contrast for visualizing the lumen of lymphatic vessels using its singlephoton fluorescence in both animal models and human subjects [3][4][5][6][7]. However, single-photon fluorescence imaging lacks depth resolving power in highly scattering tissue such as skin, as both in-focus and out-of-focus will be acquired simultaneously by the large-area detectors. Besides, for most of the single-photon fluorescence imaging of lymphatic vessels, it only captures the vessels but cannot simultaneously capture other structures next to or even within the vessel.
Multiphoton microscopy (MPM) is a nonlinear optical imaging technology that provides both high spatial and temporal resolutions, intrinsic 3D sectioning capability, and simultaneous imaging of multiple structures when different modalities of MPM are simultaneously performed, which makes them an indispensable tool for visualizing and studying the lymphatic system: Hagerling et al imaged lymphatic vessel growth and behavior during mouse fetal development and in adult mice [2]; Padera et al performed microlymphangiography in the tail of nude mice [8]; Hoshida et al imaged lymphatic valves in peritumor lymphatics in mouse ear and lymphatic metastasis in the exposed cervical lymph node [9]; Sabine et al investigated formation of lymphatic valves in transgenic mice [10]; in terms of dynamics, Bouta et al imaged lymphatic vessels and measured its flow speed [11]. All these experiments were exclusively in the MPM modality of 2-photon fluorescence (2PF) microscopy. In terms of structure resolution, MPM is capable of resolving various skin structures such as corneocytes [12][13][14], collagen [13,14], sebaceous glands [15,16], adipocytes [16,17], blood vessels [18,19], as well as lymphatic vessels. In terms of wavelength selection, so far, most MPM of lymphatic vessels use excitation at the 800-nm window (corresponding to the output wavelength of Ti: Sapphire mode-locked laser), which suffer from dramatic scattering from the multilayered skin structure. Besides, few attempts in MPM have been made to visualize lymphatic vessel structures in a label-free manner in vivo.
It is well-known that shifting the wavelength to longer excitation windows is an efficient way of reducing tissue scattering. Both the 1300-and 1700-nm window have been demonstrated to be suitable for deep-tissue imaging in both brain and skin samples in vivo [14,[19][20][21][22][23][24][25]. These two excitation windows are also low-water-absorption windows. Consequently, the resultant attenuation of excitation light is smaller than other excitation windows, and potentially enable larger MPM depths in both brain and skin.
Recently, we have demonstrated deep-skin MPM excited at the 1700-nm window in mouse skin in vivo. Labeled by structure-specific dyes, elastic fibers [21] and myelin sheaths [22] in mouse dorsal and digital skin can be imaged through 3-photon fluorescence (3PF) microscopy. Furthermore, sebaceous glands, adipocytes and collagen fibers can also be visualized by the label-free THG and SHG imaging technologies excited at this window in mouse ear [26]. All these skin MPM results excited at the 1700-nm lay the basis for imaging the lymphatic vessels deep in the skin in vivo.
Our main aim of this paper is to provide a novel nonlinear optical imaging technology to visualize both the structure and dynamics of lymphatic vessels in the mouse skin in vivo. Using indocyanine green (ICG), which generates 2PF upon excitation at the 1700-nm window [19], we demonstrate noninvasive 2PF imaging of lymphatic vessels 300 µm below the mouse skin in vivo. Besides, time-lapse 2PF imaging captures the contraction of the lymphatic vessels. After removing the surface layer of the skin to improve spatial resolution, we demonstrate that both the structure and the open-and-close dynamics of the lymphatic valves can be imaged using label-free THG imaging. In this exposed-skin preparation, we also demonstrate THG imaging of some unidentified particles flowing in the lymphatic vessel.

Experimental setup
Our laser source was based on the soliton self-frequency shift (SSFS) technique ( Fig. 1(A)) [27]. A 1-MHz, 500-fs, 1550-nm fiber laser (FLCPA-02CSZU, Calmar) was coupled into a 44-cm photonic-crystal rod (SC-1500/100-Si-ROD, NKT Photonics) to generate optical solitons at 1620 nm, which was suitable for exciting 2PF from ICG (HY-D0711, MedChemExpress) [19,28]. The soliton pulse width was 80 fs. A 1635-nm long-pass filter (1635lpf, Omega Optical) with angle tuning was used to remove the residual pump. Solitons were then sent into a laser scanning microscope (MOM, Sutter) for MPM. Images from two photomultiplier (PMT) channels were simultaneously acquired. One was a GaAsP PMT (H7422p-40; Hamamatsu) with a 540-80 bandpass filter (FF01-540/80-25, Semrock) to detect THG signals with both a 630/92-nm bandpass filter (FF01-630/92-25, Semrock) and a 593-nm longpass filter (FF01-593/LP-25, Semrock) to acquire 3PF images. The other was a GaAs PMT (H7422p-50, Hamamatsu). For noninvasive imaging within the intact skin, SHG and 2PF images were acquired with an 855-210 bandpass filter (FF01-855/210-25, Semrock) in front of the GaAs PMT. And for imaging in the exposed-skin preparation, 2PF images were acquired with an 850 longpass filter (FELH0850, Thorlabs) in front of the GaAs PMT. A water immersion objective lens (XLPLN25XWMP2, NA=1.05, Olympus) with 2-mm working distance (WD) was used. The objective lens was overfilled to achieve the maximum spatial resolution. D 2 O was used as the immersion medium. The maximum optical power on the sample was 38 mW and was only used for imaging the deepest structures. On the sample surface, 3 mW optical power was used, corresponding to an average and peak intensity of 5.5 × 10 3 W/mm 2 and 6.9 × 10 10 W/mm 2 , respectively. No tissue damage was observed. The maximum frame rate was 0.6 s/frame, which was limited by the galvo mirrors (6215H, Cambridge) used in our experiments. Image acquisition and processing were performed using ScanImage (Vidrio Technologies, Ashburn, Virginia) and ImageJ (NIH, Bethesda, Maryland), respectively. Pixel size was 512 × 512 for each image.

Animal procedures
Animal procedures were reviewed and approved by Shenzhen University. Adult BALB/c mice were all from Guangdong Medical Laboratory Animal Center, aging between 10 and 16 weeks. Mice were anesthetized with isoflurane using a gaseous anesthesia system (Matrx VIP 3000, Midmark). Body temperature was kept at ∼37°C with a heating pad. For noninvasive imaging preparation, the mouse hindlimb was depilated and sealed between a cover slip and a glass slide with dental cement (Fig. 1(B)). The imaging position (indicated by yellow arrow in Fig. 1(B)) is before the popliteal lymphatic node [4]. Saline was added between the cover slip and the hindlimb. For the exposed-skin preparation, the surface layer of skin was removed with scalpel before sealing with the cover slip ( Fig. 1(C)). ICG was administered to provide contrast: 20-µL 645-µM ICG was injected into the foot pad [3] through a 34G ultra-thin wall nanoneedle (JBP3404, Japan Bio Products). To label blood vessels, 150-µL quantum dots (W-01-640, Beida Jubang Science & Technology) were retro-orbitally injected. Imaging was performed immediately after injection

Noninvasive, deep-skin MPM of the mouse lymphatic vessels
First, we demonstrate noninvasive MPM of the lymphatic vessels in the intact skin of the mouse hindlimb in vivo. Simultaneously acquired and merged THG and 2PF/SHG images at different depths below the skin surface are shown in Fig. 2. Corneocytes (Fig. 2(A)), sebaceous gland ( Fig. 2(B)) and adipocytes (Fig. 2(C)-2(G)) can be resolved in THG images, while collagen fibers can be resolved in SHG images at various depths. Most notably, the ICG-contrasted lymphatic vessels can be clearly resolved in 2PF images 300 µm below the skin surface. These are the deepest lymphatic vessels, to the best of our knowledge, that can be resolved in the intact mouse skin through MPM. In our imaged lymphatic vessels in the hindlimb, they are embedded in adipocytes, which is better illustrated in Fig. 2(G). Apparently, blood vessels are also tubular structures that resemble lymphatic vessels. In order to differentiate the two and ascertain what we imaged were not blood vessels, we performed simultaneous 3PF imaging of blood vessels, which were labeled by quantum dots [24], and 2PF imaging of ICG-contrasted lymphatic vessels. The merged images (Fig. 3) show clear distinction between the two kinds of vessels deep in the skin. We note that in terms of dye properties, the wide separation of the emission peaks between ICG (emission peaks>800 nm) and quantum dots (emission peak<650 nm) facilitates this distinction.
Lymphatic vessels contract to drive fluid, which is vital for the normal functioning of the entire lymphatic system. In order to capture this contraction dynamics, we performed noninvasive, time-lapsing 2PF imaging in the intact mouse skin in vivo. The recorded 2PF imaging video (Visualization 1) shows that, during an imaging span of 598.8 s, the imaged lymphatic vessel contracted 8 times. In order to quantify this contraction frequency, we measured the timedependent average THG signals along the yellow lines (Fig. 4(A-C)) at the same position which corresponds to the diameter of the lymphatic vessel. The measured THG signals show local minimum during contraction (dips in Fig. 4(D)). This is because during contraction, the lymphatic vessel occupies less pixels due to shrinkage of its diameter along the line, so the average THG signal drops. We also verify that the dips in Fig. 4(D) correspond to contraction in the recorded 2PF imaging video (Visualization 1). During this imaging span of 598.8 s, the average contraction frequency of the imaged lymphatic vessel is 0.8 times per minute, similar to that reported in the  Figure 4 shows 2PF images excerpted from the video, which shows representative images of lymphatic vessel contraction before ( Fig. 4(A)), during ( Fig. 4(B)), and after ( Fig. 4(C)) contraction. For these 8 contractions, each last from 7.2 s to 12 s. Using noninvasive 2PF imaging excited at the 1700-nm window, together with exogenous contrast by ICG, we can thus visualize both the structure and contraction dynamics of lymphatic vessels in their native environment.

MPM of the lymphatic vessels after removal of the surface skin layer
Although MPM excited at the 1700-nm window enables us to image lymphatic vessels in the skin noninvasively, the multilayered skin imposes notable aberration onto the excitation light. The aberration degrades spatial resolution and image quality, which makes the resolution of fine lymphatic vessel structures difficult. In order to improve spatial resolution, we first removed the surface layer of the skin (Fig. 1(C)) to better expose the lymphatic vessel, and then performed 2PF and THG imaging. In this exposed-skin preparation, we can clearly resolve the ICG-contrasted lymphatic vessel embedded in adipocytes (Fig. 5). From both the THG image ( Fig. 5(B)) as well as the merged 2PF and THG image (Fig. 5(C)), we can also identify lymphatic vessel walls lining the ICG-contrasted lymphatic vessel (indicated by blue arrow in Fig. 5(B)). Since lymphatic vessels are filled with clear lymph and few cells [1,16] and homogenous medium with normal dispersion does not generate THG signals [30,31], the majority of lymphatic vessel in the THG images appears black. However, inside the lymphatic vessel, the most notable structure that can be resolved in THG image are valves (indicated by white arrow in Fig. 5(B)), which yield discernible THG signals. The lymphatic valves comprise extracellular matrix lined by endothelial cells [32], whose composition are different from that of the flowing lymph. Neither the valves nor the lymphatic endothelial cells are homogeneous medium, so they generate THG signals and provide image contrast for these two structures. The ICG provides 2PF contrast for the lumen of the lymphatic vessel. The valves recorded by THG imaging at different depths below the exposed skin surface are further shown in Fig. 6.
The function of valves are to ensure unidirectional lymph flow through the lymphatic vessel [32]. Their typical dynamical behavior is opening and closing. Using time-lapse THG imaging, we can capture this open-and-close dynamics in real time (Fig. 7(A), 7(B) and Visualization 2): during a total imaging span of 179.4 s, the valves closed twice (Visualization 2). Interestingly, in THG images ( Fig. 7 and Visualization 2), we can also visualize particles flowing in lymphatic vessels. From these images we can measure the size of these particles. The full-width-at-half-maximum (FWHM) lateral sizes of the particles in Fig. 7(B) and 7(E) are 1.3 µm (Fig. 7(C)) and 2.7 µm ( Fig. 7(F)), respectively. The measured FWHM of the lateral 3PF point spread function (PSF) is 0.48 µm in our microscope. After deconvolution with this PSF, the particle size is 1.21 µm and 2.66 µm for Fig. 7(C) and 7(F), respectively. Besides, in the time-lapse THG imaging (Fig. 7(B) and Visualization 2), we can see that a particle is temporarily trapped near the valve and the lymphatic vessel. The particles could also be visualized in THG images without injecting ICG, in the exposed-skin preparation. The exact nature of these particles is unknown. Lymphatic vessels can transport exosomes, vesicles and cells [33,34]. Judging by the size of these particles (>1 µm), they are less likely to be exosomes, since exosomes typically have size below 100 nm.

Signal-to-background ratio (SBR) calculation
In MPM, in order for the structures at the focal plane to be resolved, the signal-to-background ratio (SBR) should be larger than unity [20]. Using the same method in [20], we calculated SBRs for the structures of different imaging modalities used in this paper. Representative results from 2PF images of ICG-contrasted lymphatic vessels are shown in Fig. 8, yielding SBR=15:1 in the intact skin (Fig. 8(A), B) and SBR=197:1 in the exposed-skin preparation (Fig. 8(C), D). For other imaging modalities used in this paper, the calculated SBRs are: for SHG imaging SBR=14:1 and 47:1 in the intact and exposed-skin preparation, for 3PF imaging SBR=57:1 and 180:1 in the intact and exposed-skin preparation, and for THG imaging SBR=47:1 and 78:1 in the intact and exposed-skin preparation. These SBRs facilitate both the lymphatic vessels and other structures to be resolved in different modalities of MPM.

Discussion and conclusion
Here we demonstrate MPM excited at the 1700-nm window is a promising technology for imaging the structure and dynamics of lymphatic vessels in animal models in vivo. Targeting different needs in lymphatic research, our demonstrated imaging technology have the following capabilities: (1) Noninvasive imaging of lymphatic vessels and their contraction. With ICG contrast, 2PF imaging excited at the 1700-nm window enables visualization of lymphatic vessels 300 µm deep in the mouse skin in vivo, without the need to remove the surface skin layer. Simultaneously acquired THG and SHG images further reveal the local environment of the lymphatic vessels. For example, in our experiments on the mouse hindlimb, we have found that lymphatic vessels are surrounded by adipocytes.
Besides noninvasive structural imaging, we demonstrate time-lapse 2PF imaging can also capture the contraction of lymphatic vessels in real time. The highly heterogenous and scattering nature of skin also lead to degradation of the spatial resolution of MPM in the intact skin. For example, we could not resolve the lymphatic vessel wall or the valves in THG imaging.
(2) Label-free imaging of lymphatic vessel structures and their dynamics. To improve optical resolution, we removed the surface layer of the skin. Using label-free THG imaging excited at the 1700-nm window, we can resolve lymphatic walls lining the lymphatic vessels by simultaneous THG imaging and 2PF imaging. Furthermore, we can resolve valves in THG images, which to the best of our knowledge, the first demonstration of label-free MPM of such structures.
Besides label-free structural imaging of the valves, our time-lapse THG imaging can also capture the open-and-close dynamics of valves. Unexpectedly, we can also image unknown particles within lymphatic vessels in THG images. Their rich dynamical behavior (e.g., flowing and temporarily trapped near the valve and the wall) may help us better understand the dynamics of lymphatic flow. Currently our maximum imaging speed (0.6 s/frame) is limited by the maximum scanning speed of galvo mirrors, which prevents us from capturing the fast-flowing particles for speed measurement. This could be potentially solved by using a resonant scanner which typically acquires at much higher frame rate (<0.1 s/frame). Besides, the exact nature of these particles remains unknown. Future study may focus on both the nature and dynamics of these particles. Besides. the focused laser intensity is high on the sample, which could cause heating to the sample. Due to the small size of the focus, currently we cannot measure temperature rise at the focus caused by the laser. We expect development of temperature measurement technology for tiny focused laser beams may solve this problem. Disclosures. The authors declare that there are no conflicts of interest related to this article. Data availability. Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.