1Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
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Moxifloxacin: Clinically compatible 
contrast agent for multiphoton 
imaging
Taejun Wang1,*, Won Hyuk Jang1,*, Seunghun Lee2,*, Calvin J. Yoon1,*, Jun Ho Lee2, 
Bumju Kim1, Sekyu Hwang3, Chun-Pyo Hong1, Yeoreum Yoon2, Gilgu Lee2, Viet-Hoan Le1, 
Seoyeon Bok1, G-One Ahn1, Jaewook Lee4, Yong Song Gho4, Euiheon Chung5, Sungjee Kim3, 
Myoung Ho Jang6, Seung-Jae Myung7, Myoung Joon Kim8, Peter T. C. So9 & Ki Hean Kim1,2
Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for 
cellular imaging of thick tissues and live animals in biological studies. However, MPM application to 
human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous 
probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-
labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue 
penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in 
various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is 
promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on 
endogenous fluorescence.
Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique based on multiphoton (MP) 
excitation1,2. MPM is the technique of choice for fluorescence microscopy of thick tissue and live animals due to 
its high imaging depths and reduced photodamage compared to other fluorescence-based methods. MPM ena-
bles molecular imaging by using either exogenous markers or endogenous signals. Endogenous signals may be 
produced by autofluorescence (AF) or by other intrinsic nonlinear mechanism such as second harmonic genera-
tion (SHG) and third harmonic generation (THG). MPM has been applied to various biological studies including 
neurobiology3,4, immunology5, cancer biology6, and pharmacology. MPM based on AF and other intrinsic con-
trasts have been used extensively as a label-free imaging method in pre-clinical or clinical research fields includ-
ing dermatology7–10, ophthalmology10,11 and cancer biology12–15. However, AF signal is generally weak, thereby 
necessitating high excitation laser power or extension of pixel duration time15,16. We report the application of 
moxifloxacin as new contrast agent for in vivo cell-labeling in order to overcome the limitations of AF-based MP 
tissue imaging.
Moxifloxacin hydrochloride, hereafter referred to as moxifloxacin, is a fourth-generation fluoroquinolone 
antibiotic that prevents DNA replication of both Gram-positive and Gram-negative bacteria by inhibiting gyrase 
and topoisomerase IV activity17. Moxifloxacin is used in the clinic to treat or prevent a range of bacterial infections 
1Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, 77 
Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Rep. of Korea. 2Department of Mechanical Engineering, 
Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Rep. of 
Korea. 3Department of Chemistry, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, 
Gyeongbuk 37673, Rep. of Korea. 4Department of Life Sciences, Pohang University of Science and Technology, 
77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Rep. of Korea. 5Department of Biomedical Science and 
Engineering, and School of Mechanical Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-
gwagiro, Buk-gu, Gwangju 61005, Rep. of Korea. 6Academy of Immunology and Microbiology, Institute for Basic 
Science, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Rep. of Korea. 7Department of Gastroenterology, 
University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro, 43-gil, Songpa-gu, Seoul 05505, Rep. 
of Korea. 8Department of Ophthalmology, Asan University of Ulsan College of Medicine, Asan Medical Center, 88 
Olympic-ro, 43-gil, Songpa-gu, Seoul 05505, Rep. of Korea. 9Department of Mechanical Engineering and Biological 
Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. *These 
authors contributed equally to this work. Correspondence and requests for materials should be addressed to K.H.K. 
(email: kiheankim@postech.ac.kr)
Received: 18 December 2015
accepted: 16 May 2016
Published: 10 June 2016
OPEN
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2Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
throughout the body, including the eye, skin, and respiratory tract, by topical, intravenous, and oral administra-
tion. Pharmacokinetic studies of moxifloxacin have shown good tissue penetration, high cellular-to-extracellular 
and tissue-to-serum concentration ratios compared to other fluoroquinolone antibiotics, attributed to its higher 
lipophilicity and aqueous solubility18,19. Moxifloxacin has intrinsic fluorescence with peak excitation and emission 
wavelengths at 290 nm and 500 nm, respectively, and its concentration in tissues is usually measured by high per-
formance liquid chromatography (HPLC) of fluid extracts via fluorescence detection. Using moxifloxacin oph-
thalmic solution, we found that moxifloxacin can be readily excited via two-photon absorption at 700–800 nm20 
and has a high two-photon cross-section approximately 8 times smaller than that of fluorescein (pH 7.4) at 780 nm 
(Supplementary Fig. 1). MPM of moxifloxacin-treated mouse cornea showed the spatial distribution of moxiflox-
acin, where cells in the cornea were visible due to the high intracellular concentration of moxifloxacin, therefore 
yielding much stronger signal than AF contrast20. Intracellular concentration of moxifloxacin in human neutro-
phils and tissue-cultured epithelial cells was reported to be up to 10 times greater than that of the extracellular21. 
Therefore, these suggested the possibility of utilizing moxifloxacin as a nonspecific cell-labeling agent for MP 
tissue imaging in clinical applications. Here we demonstrate the applications of moxifloxacin as a nonspecific 
cell-labeling agent for MP imaging.
Results
To investigate the cellular labeling properties of moxifloxacin, we imaged by MPM a variety of in vitro cell lines of 
mice and human before and after treatment of 10 μ M moxifloxacin (Fig. 1a). Moxifloxacin-treated cell lines were 
detectable by approximately 7–14 times greater intracellular signal than that of AF at the same excitation laser 
power of 2.52 mW (Fig. 1b). In conjunction with the report that intracellular moxifloxacin concentration in cells is 
higher than that of the extracellular21, we concluded that moxifloxacin is capable of cellular labeling and detection 
by MPM. We imaged a freshly extracted, moxifloxacin-treated rat cornea by both MPM and reflectance confocal 
microscopy (RCM), a standard method to examine the cornea at the cellular level (Supplementary Videos 1, 2). 
Figure 1. In vitro multiphoton microscopy (MPM) images of cell lines and intracellular moxifloxacin 
fluorescence intensities. MPM and reflectance confocal microscopy (RCM) images of a freshly extracted rat 
cornea. All MPM and RCM images are based on moxifloxacin fluorescence. (a) Cultured cell lines: mouse 
embryonic fibroblast (NIH3T3), human normal colon (CCDTr841), and human colon carcinoma (HT29). 
Representative MPM images of cell lines are presented as maximum intensity projection (MIP) images with a 
stepwise increment of 1 μ m in the z direction. Scale bars, 50 μ m. (b) Quantification of intracellular moxifloxacin 
fluorescence intensities. Intracellular signal was enhanced significantly by approximately 10 times after 
moxifloxacin labeling. (c) MPM images of various layers of rat cornea comprising of superficial epithelium, 
basal epithelium, stroma, and endothelium. Corresponding RCM images of the same regions in the respective 
MPM images. Scale bars, 50 μ m.
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In both, we were able to detect individual cells in the epithelium, stroma, and endothelium, confirming that mox-
ifloxacin allowed for the visualization of cells by cellular labeling (Fig. 1c).
AF-based MPM provides cellular information of the skin down to the dermis at sufficiently high excitation 
laser powers7,8,10. To investigate moxifloxacin fluorescence-based MP imaging of skin in vivo using compara-
tively reduced excitation laser power, we first topically treated the skin of mouse hind limb with moxifloxacin 
(12.4 mM) after mild tape stripping to allow efficient delivery, then imaged with MPM and compared the results 
with AF-based MP imaging (Fig. 2a). While the layers of the epidermis were clearly visible by AF (Supplementary 
Video 3), the application of moxifloxacin significantly enhanced the visualization of cellular structures in the 
dermis, permitting the reduction of excitation laser power from 19 mW to 5 mW (Supplementary Fig. 2 and 
Supplementary Video 4). Fluorescence intensities from the epidermis and dermis were quantified under the same 
condition of constant excitation laser power of 5 mW along the z-axis (Fig. 2b). Fluorescence intensity, espe-
cially from the cellular structures in moxifloxacin-labeled dermis, was approximately 10 times higher than that 
of unlabeled dermis. In order to determine whether the signals captured in the dermis originated from intra-
cellular moxifloxacin fluorescence, we counterstained with Hoechst 33342 (b2261, Sigma-Aldrich, Saint Louis, 
US) to locate nuclei within these structures. Additionally, to identify the vascular microstructures, we intra-
venously injected tetramethylrhodamine (TAMRA, D-7139, ThermoFisher Scientific, Waltham, US). Indeed, 
within each structure labeled by moxifloxacin, a nucleus was present, confirmed by spectral unmixing (Fig. 2c 
and Supplementary Figs 3 and 4). In the epidermis, cellular morphology and distribution of the cells of the 
stratum spinosum with their counterstained nuclei were visible. Below the epidermis, thin fibrous structures 
branching from cell bodies were visible. Based on morphology and location, these fibrous structures could pos-
sibly be nerves22. We also identified capillary endothelial cells by their narrow and elongated morphology run-
ning parallel with the TAMRA signal. Other cells in the dermis made visible by moxifloxacin could include 
Figure 2. In vivo MPM images of mouse hind limb skin. (a) 3D rendered hind limb skin images based on 
autofluorescence (AF) and moxifloxacin fluorescence. Cellular structures in the dermis were captured by 
approximately 4 times higher laser power (19 mW) than that used for moxifloxacin-treated skin (5 mW).  
(b) Quantification of epidermis and dermis fluorescence intensities. The junction in between the epidermis 
and dermis was divided along the basal cell layer. (c) MPM hind limb skin images at different depths based on 
topically treated moxifloxacin (green), Hoechst 33342 (blue), and intravenously injected tetramethylrhodamine 
(TAMRA, red) for identification of cells, their nuclei, and blood vessels, respectively. The stratum spinosum 
is shown in the first MIP MPM image (2–6 μ m). Thin fibrous structures (yellow arrowhead) branching from 
dermal cell bodies (red arrowhead) and capillary endothelial cells (white arrowhead) with blood vessel are 
shown in subsequent MIP MPM images (32–40 μ m and 48–60 μ m, respectively). Scale bars, 50 μ m. (d) Mobile 
cell tracking in time-lapse imaging of the dermis after topical treatment of moxifloxacin, in vivo. Mobile cell 
(orange arrowhead) passing by a static cell (black arrowhead) is shown. Total elapsed imaging time is 25 min 
during anesthesia of the mouse. Scale bars, 50 μ m.
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4Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
fibroblasts and immune cells. Moxifloxacin fluorescence-based MP imaging of isolated immune cells suggested 
that while neutrophils/monocytes, macrophages, eosinophils, and T cells could be visualized by moxifloxacin in 
the dermis (Supplementary Fig. 5), the identification of specific cells in the skin may be confirmed by usage of 
transgenic mice23 or immunofluorescent labeling. Furthermore, we conducted time-lapse MP imaging, in which 
we observed dynamic movement of leukocytes within the dermis (Fig. 2d and Supplementary Video 5). Weak AF 
signal necessitates the extension of pixel duration time or the increase of incident laser power, which has been an 
obstacle for AF-based video-rate imaging and has also raised concerns of inducing thrombosis by two-photon 
excitation in in vivo skin samples. Therefore, we have conducted high-speed imaging on moxifloxacin-labeled 
and unlabeled mouse hind limb skin in vivo (Supplementary Fig. 6). Excitation laser powers of 31.2 mW and 
51 mW were used for moxifloxacin-labeled and unlabeled dermis, respectively. Although AF-based skin imaging 
allowed for the monitoring of the dynamic cellular structures, extended imaging appeared to induce thrombosis 
due to high excitation power, as seen by the aggregation of endogenous fluorophores within the blood vessels in 
the dermis (Supplementary Video 6). In contrast, high-speed imaging of moxifloxacin-labeled dermis showed 
no signs of thrombosis and visualized more of the cellular microenvironment at 1.63 times reduced laser power 
(Supplementary Video 7).
Lastly, we briefly investigated labeling of freshly excised mouse small intestine and rat bladder by moxiflox-
acin. Moxifloxacin, as expected, effectively visualized cells in these tissues with a laser power in the range of 
8 mW to 14 mW depending on the depth. In the moxifloxacin-labeled small intestine (jejunum), abundant cells 
within the villus (Fig. 3a and Supplementary Video 8), as well as enterocytes in the epithelial layer, can be clearly 
observed. Particularly, capillary structures were visible in the villus. Meanwhile in the rat bladder, the structurally 
diverse cell layers of the urothelium, lamina propria, and submucosa were clearly visualized with high contrast 
based on moxifloxacin fluorescence (Fig. 3b and Supplementary Video 9) suggesting the possible enablement of 
this approach as a real-time diagnostic tool for MP clinical endoscopy24–26. Additionally, endothelial cells outlin-
ing the capillaries in the lamina propria were observed.
Discussion
In this study, we demonstrated a novel MP tissue imaging method by using moxifloxacin, an FDA-approved 
compound, as a contrast agent for tissues such as cornea, skin, small intestine, and bladder. Moxifloxacin 
fluorescence-based MP imaging, even with 2 to 4 times reduced laser power than AF-based imaging, enabled 
effective and comparable visualization of various features of cells and their microenvironment, thereby allowing 
wide-field and high-speed imaging.
Optimized imaging parameters with short staining time and fast imaging speed are important for in vivo clin-
ical imaging. Staining time may be dependent on the kinds of tissues and purpose of tissue studies. Staining time 
of less than approximately 20 minutes was enough for MP imaging of most tissues in vivo. On the other hand, 
imaging speeds should be at minimum 5–10 frames/s or preferably performed at video rates (> 20 frames/s). 
Figure 3. MPM images of freshly extracted mouse small intestine and rat bladder after topical treatment of 
moxifloxacin. (a) 3D rendered image of villus in small intestine (jejunum) and zoomed MPM images consisting 
of goblet cells (yellow arrowhead) and enterocytes (blue arrowhead) in epithelium, inner immune cells (white 
arrowhead), and capillaries (red arrowhead). Scale bars, 25 μ m. (b) MPM images of rat bladder composed of 
structurally diverse layers such as the superficial urothelium (z = 2 μ m), dense intermediate cell layer (z = 10 μ m), 
lamina propria (z = 32 μ m) with capillary endothelial cells (green arrow), and submucosa (z = 100 μ m) consisting 
of muscle layer (black asterisk). Scale bars, 50 μ m.
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5Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
Faster imaging speed would be possible by adapting high-speed imaging methods. As such, other imaging param-
eters also have more room for improvement and, notwithstanding, experimental conditions for various human 
tissue imaging should be optimized in the future.
Limitations are still present in imaging human skin in vivo due to thicker and more folded characteristics 
compared to mouse skin. With an MPM imaging depth of less than 200 μ m in the mouse skin, our current imag-
ing depth will be an issue in the human skin. More careful optimization of imaging parameters is necessary 
for human skin imaging. Longer excitation wavelengths could be used to achieve an improved imaging depth, 
although it may reduce the advantage of moxifloxacin-based signal enhancement. In addition, usage of immer-
sion materials for refractive index matching may help to reduce aberration and scattering of excitation light on 
the skin surface.
Because moxifloxacin is an antibiotic preventing DNA replication of both Gram-positive and Gram-negative 
bacteria, treatment of moxifloxacin for MP imaging may affect microflora. Therefore, moxifloxacin-based MPM 
may not be applicable to some research settings where undisturbed microflora is important. Notwithstanding, 
the negligible influence of moxifloxacin treatment on the investigation of cellular morphology in tissue strongly 
suggests the potential application of moxifloxacin as a nonspecific cell-labeling agent for MP clinical imaging.
Meanwhile, two-photon fluorescence lifetime imaging (FLIM) of tissue AF is a method used to detect metabolic 
changes of cells in tissue because fluorescence lifetime is highly sensitive to changes in cellular environment27–29. 
FLIM has been used in measuring oxidative stress30 and detecting cancer cells. On the other hand, moxifloxa-
cin used as a cell-labeling agent for signal enhancement in MP imaging may allow for high-speed imaging or 
imaging with relatively lower excitation power. However, moxifloxacin-based MP imaging does not provide any 
further information about cell functionality. Therefore, the combination of moxifloxacin-based MP imaging and 
AF-based metabolic MP imaging with fluorescence lifetime30–34 will be useful. Because moxifloxacin fluorescence 
is stronger than AF, measurement of weak AF and fluorescence lifetime under the presence of moxifloxacin flu-
orescence will be difficult. However, since moxifloxacin concentration within cells will decrease over time, AF 
intensity and lifetime measurements before or a few hours after treatment will be possible.
Overall, we believe that moxifloxacin, an FDA-approved compound, is a promising contrast agent for MP 
clinical imaging due to sufficient intrinsic fluorescence and high intracellular concentration within tissue.
Methods
Moxifloxacin. Moxifloxacin ophthalmic solution Vigamox (Alcon Laboratories, Fort Worth, US), was used 
to label various cell lines and cells in several tissues. This solution contains 0.5% (5 mg/mL) moxifloxacin hydro-
chloride (12.4 mM, pH 6.8) as the active ingredient. The moxifloxacin ophthalmic solution batch was always kept 
in its box to prevent possible photobleaching.
Multiphoton microscopy and data processing. Multiphoton microscopy (MPM) with a Ti-Sapphire 
laser (Chameleon Vision II, Coherent) at 140 fs pulse width and 80 MHz pulse repetition rate was used (TCS SP5 
II, Leica, Germany). 3D MPM imaging was performed with either of two 20× objective lenses (HCX APO L 20× , 
1.0NA water immersion, Leica, Germany; XLUMPLFLN-20XW, 1.0NA, water immersion, Olympus, Japan) or a 
25× objective lens (HCX IRAPO L 25× , 0.95NA, water immersion, Leica, Germany). Laser power was measured 
by a power meter (S310C, Thorlabs) and was accounted for beam clipping at the objective lens back pupil and 
transmittance across the objective lens. MPM images, composed of 512 × 512 pixels, were acquired and processed 
by LAS AF Lite (Leica, Germany) and MATLAB.
Cell preparation and imaging. Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial 
Institute (RPMI) medium, fetal bovine serum (FBS), penicillin-streptomycin (PS), phosphate buffered saline 
(PBS), and trypsin-EDTA were purchased from Hyclone. HT29 human colon carcinoma and CCDTr841 human 
normal colon cells were obtained from Korean Cell Line Bank. HT29 cells were maintained in RPMI while 
CCDTr841 cells were maintained in DMEM, both supplemented with 10% (v/v) FBS and 1% (w/v) PS at 37 °C 
in a humidified atmosphere of 5% CO2 in air. Cells were passaged when they reached approximately 80% con-
fluence. Cells were seeded onto cell culture dish at a density of 1 × 105 cells and incubated overnight at 37 °C 
under 5% CO2. Cells were treated with 10 μ M of moxifloxacin for 1 hr before MP imaging. A 20× objective lens 
(XLUMPLFLN-20XW× , 1.0NA, water immersion, Olympus, Japan) was used for MPM. Prepared individual 
cell lines in culture dish were excited at 780 nm with an MP laser power of 2.52 mW. The emission light from 
moxifloxacin-treated cells was collected by 2 channels (blue: 300–430 nm; green: 430–680 nm). 3D volumetric 
scanning of cell lines in culture dish was performed at 0.08 frames/s and a stepwise increment of 1 μ m in the 
z direction. MPM images of moxifloxacin-treated cell lines were processed as maximum intensity projection 
(MIP), and quantified as fluorescence intensities by using MIP images.
Animal preparation and ethical conduct. Animal models were bred at the animal facility of POSTECH 
Biotech Center under specific pathogen-free (SPF) condition and had access to food and water ad libitum. All 
animal experimental procedures were conducted in accordance with institutional guidelines and regulations and 
approved by the Institutional Animal Care & Use Committee at POSTECH (2014-0073).
Rat cornea imaging. Rat (Sprague-Dawley, male) was anesthetized via face mask administering a gas mix-
ture of 1.5%/v isoflurane (Terrell™ , Piramal) and medical grade oxygen, and was then sacrificed by cervical dis-
location. An eye ball was extracted and mounted onto a custom-made acrylic eyeball holder. Images of cornea 
were acquired by MPM and reflectance confocal microscopy (RCM) based on topical treatment of moxifloxacin 
(12.4 mM) for 10 min of staining time. A 20× objective lens (HCX APO L 20× , 1.0NA water immersion, Leica, 
Germany) was used for both MPM and RCM. Excitation lasers were tuned to 780 nm and 633 nm for MPM 
and RCM, respectively. Laser power was approximately 12.15 mW at the ocular tissue for both MPM and RCM. 
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6Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
Emission light of moxifloxacin fluorescence-based MP imaging was collected by a single channel (green: 455–
680 nm). Following, that of corneal cells from RCM was also taken into a single channel (green: 618–648 nm). 
3D volumetric scanning of rat cornea was performed at 0.78 frames/s and a stepwise increment of 2 μ m in the z 
direction.
Preparation for in vivo mouse hind limb skin imaging. Mice (SKH1-HrHr, 6 weeks, female; 
BALB/c, 7 weeks, female) were anesthetized via face mask administering a gas mixture of 1.5%/vol 
isoflurane (Terrell™ , Piramal) and medical grade oxygen. The mice were placed on a custom-made hind limb 
holder, which also functioned as a heating plate secured to a motorized x-y translational stage. Prior to imaging, 
fur was shaved when necessary, after which, mild tape stripping of the skin was performed approximately 10 
times to remove the stratum corneum for efficient delivery of moxifloxacin.
Mouse hind limb skin imaging. Excitation laser for AF-based imaging was tuned to 780 nm with a laser 
power held constant at 5 mW and 19 mW, for individual experiments, throughout the skin. Emission light was 
collected into 2 channels (blue: 300–430 nm; green: 430–680 nm). Following, moxifloxacin (12.4 mM) was top-
ically applied to the tape stripped area and was left to diffuse for 30 min. Moxifloxacin was excited with 780 nm 
with a constant power of 5 mW throughout the skin. The emission light of moxifloxacin-based MP imaging was 
collected into the 2 aforementioned channels. 3D volumetric scanning was performed at 0.08 frames/s and step-
wise increment of 2 μ m in the z direction. For fluorescent labeling of cells and cell nuclei, 100 μ L of a mixture of 
5% (v/v) Hoechst 33342 (b2261, Sigma-Aldrich, Saint Louis, US) dissolved in moxifloxacin solution was treated 
for 30 min. To identify vascular structures, we intravenously injected 100 μ L of 10 mg/mL tetramethylrhodamine 
(TAMRA, D-7139, ThermoFisher Scientific, Waltham, US) in PBS. Emission peaks of counterstained skin were 
furthest separated at 800 nm excitation, which allowed for maximum contrast with a laser power in the range of 
8 mW to 21 mW depending on the depth. This emission light was collected into 4 channels (blue: 430–450 nm; 
green: 467–499 nm; yellow: 520–525 nm; red: 565–605 nm). 3D volumetric scanning of counterstained skin was 
performed at 0.25 frames/s with 2 lines averaging and a stepwise increment of 1.5 μ m in the z direction.
Time-lapse imaging of hind limb skin. To monitor the dynamic movement of cellular structures in 
moxifloxacin-labeled dermis, excitation laser was tuned to 780 nm for moxifloxacin fluorescence with a laser 
power of approximately 18.8 mW. The emission light of moxifloxacin fluorescence-based imaging was collected 
into 2 channels (blue: 300–490 nm; green: 490–680 nm). Imaging speed was 0.08 frames/s and total elapsed imag-
ing time was 25 min during anesthesia of the mouse.
Antibodies. All fluorescence-conjugated antibodies used for flow cytometric analysis were purchased from 
BD Biosciences and eBioscience. T cells were labeled with antibodies against TCRβ (H57-597) after FcR blocking 
with anti-CD16/CD32 antibodies (2.4G2). For eosinophil (MHC II−CCR3+) labeling, cells were stained with 
CD193 (83103). Neutrophils/monocytes (MHC II−CCR3−Gr1+CD11b+) were stained with Gr1 (RB6-8C5) and 
CD11b (M1/70). Macrophages (MHC II+F4/80+CD11b+) were stained with MHC II (M5/114.15.2) and F4/80 
(BM8).
Isolation of immune cells and imaging. For isolation of spleen macrophages, spleens were minced and 
digested in enzyme media (RPMI 1640 media containing 400 U/mL of collagenase D, 10 μ g/mL DNase I, 3% 
FCS, 20 mM HEPES, 100 U/mL penicillin, 100 μ g/mL streptomycin, 1 mM sodium pyruvate, and 1 mM NEAA) 
for 45 min at 37 °C. EDTA (final concentration of 10 mM) was added to the cell suspension and the cells were 
incubated for an additional 5 min at 37 °C. After filtering through a 100 μ m cell strainer, the cells were spun 
in 17.5% Accudenz solution (Accurate Chemical & Scientific Co.) to enrich antigen presenting cells. Immune 
cells were purified by MoFlo Astrios cell sorter (Beckman Coulter). These isolated cells settled to the bottom of 
the Eppendorf tube and were labeled with moxifloxacin (12.4 mM) for 2 hr at 4 °C. Vortexed cell groups within 
moxifloxacin were moved onto well slide glass and sealed with a coverslip. A 20× objective lens (HCX APO L 
20× , 1.0NA water immersion, Leica, Germany) was used for MPM. The excitation laser was tuned to 780 nm 
for moxifloxacin with a constant laser power of 3.6 mW. The emission light was collected into 4 channels (green: 
430–480 nm; green: 500–550 nm; red: 565–605 nm; red: 625–675 nm). 3D volumetric scanning of isolated cells 
was performed at 0.78 frames/s with 2 lines averaging and a stepwise increment of 1 μ m in the z direction. 
Representative MPM images of moxifloxacin-treated sorted cells were presented as MIP images (Supplementary 
Fig. 5). Intracellular and extracellular moxifloxacin concentrations were analyzed by boundary selection of single 
cells.
Mouse small intestine imaging. To investigate the applicability of moxifloxacin for gut staining, fresh 
intact small intestine was surgically excised from wildtype mouse (BALB/c, 6 weeks, female) and was longitu-
dinally cut to expose the villi of small intestine. The small intestine (jejunum) was labeled with moxifloxacin 
(12.4 mM) for 20 min and washed with PBS for 5 min. A prepared mouse small intestine, flattened with the villus 
side exposed, was mounted on glass slide and then covered with a coverslip. A 20× objective lens (HCX APO L 
20× , 1.0NA, water immersion, Leica, Germany) was used for MPM. The excitation laser was tuned to 780 nm 
for moxifloxacin with a laser power in the range of 9.72 mW to 14.26 mW depending on the depth. The emission 
light was collected into 4 channels (green: 430–480 nm; green: 500–550 nm; red: 565–605 nm; red: 625–675 nm). 
3D volumetric scanning was performed at 0.78 frames/s with 2 lines averaging and a stepwise increment of 2 μ m 
in the z direction (from top of the villus to serosa).
Rat bladder imaging. Rat (Sprague-Dawley, male) was anesthetized via face mask administering a gas 
mixture of 1.5%/v isoflurane (Terrell™ , Piramal) and medical grade oxygen, and was then sacrificed by cervical 
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dislocation. The preparation of a fresh intact bladder tissue was the same as that of mouse small intestine. A 20× 
objective lens (XLUMPLFLN-20XW, 1.0NA, water immersion, Olympus, Japan) was used for MPM. The excita-
tion laser was tuned to 780 nm for moxifloxacin excitation with a laser power in the range of 8.2 mW to 14.05 mW 
depending on the depth. The emission light was collected into 2 channels (green: 300–560 nm; red: 565–680 nm). 
3D volumetric scanning was performed was the same as that of mouse small intestine (from urothelium to 
submucosa).
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Acknowledgements
This research was supported in part by the Bio and Medical Technology Development Program (No. 2011-
0019633), the Engineering Research Center grant (No. 2011-0030075) of the National Research Foundation 
(NRF) funded by the Korean government (MEST), BK21 Plus (No. 10Z20130012243) funded by the Ministry of 
Education of Korea, Institute for Basic Science grant (No. IBS-R005-S1-2015-a00), NIH-5-O41-EB015871-27, 
DP3-DK101024 01, 1-U01-NS090438-01, 1-R01-EY017656-0,6A1, 1-R01-HL121386-01A1, the Biosym IRG of 
Singapore-MIT Alliance Research and Technology Center, the Koch Institute for Integrative Cancer Research 
Bridge Initiative, the Hamamatsu Inc., and the Samsung GRO program.
www.nature.com/scientificreports/
8Scientific RepoRts | 6:27142 | DOI: 10.1038/srep27142
Author Contributions
T.W. performed overall MP imaging experiments, processed data, and wrote the manuscript. W.H.J. and S.L. 
performed mouse hind limb skin imaging. C.J.Y. wrote the manuscript with T.W., checked cellular labeling of 
mouse hind limb skin using moxifloxacin and Hoechst 33342, and intravenously injected TAMRA with V.-H.L 
J.H.L. performed mouse and rat cornea imaging with S.L. B.K. helped with time-lapse imaging of mouse hind 
limb skin with W.H.J. S.H. and S.K. prepared cultured cell lines and cell-labeling protocol for cell lines. C.-P.H. 
and M.H.J. performed cell sorting from mice and helped with intracellular signal analysis. Y.Y. performed rat 
bladder imaging. G.L. performed data quantification with T.W. S.B. and G.-O.A. helped with discussion on 
immune cells in the skin. J.L. and Y.S.G. helped with discussion on cellular interactions and dynamics in the skin. 
E.C. provided various in vivo imaging protocols using animal models. S.M. and P.T.C.S. guided the experiments 
and revised the manuscript. M.J.K. prepared moxifloxacin solution and helped with corneal imaging. K.H.K. 
directed the overall project, analyzed experimental results and reviewed the manuscript with input from other 
authors.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Wang, T. et al. Moxifloxacin: Clinically compatible contrast agent for multiphoton 
imaging. Sci. Rep. 6, 27142; doi: 10.1038/srep27142 (2016).
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