Bumju Kim

Quantum Dots in an Amphiphilic Polyethyleneimine Derivative Platform for Cellular Labeling, Targeting, Gene Delivery, and Ratiometric Oxygen Sensing.

Amphiphilic polyethyleneimine derivatives (amPEIs) were synthesized and used to encapsulate dozens of quantum dots (QDs). The QD-amPEI composite was ∼100 nm in hydrodynamic diameter and had the slightly positive outer surface that suited well for cellular internalization. The QD-amPEI showed very efficient QD cellular labeling with the labeled cell fluorescence intensity more than 10 times higher than conventional techniques such as Lipofectamine-assisted QD delivery. QD-amPEI was optimal for maximal intracellular QD delivery by the large QD payload and the rapid endocytosis kinetics. QD-amPEI platform technology was demonstrated for gene delivery, cell-specific labeling, and ratiometric oxygen sensing. Our QD-amPEI platform has two partitions: positive outer surface and hydrophobic inside pocket. The outer positive surface was further exploited for gene delivery and targeting. Co-delivery of QDs and GFP silencing RNAs was successfully demonstrated by assembling siRNAs to the outer surfaces, which showed the transfection efficiency an order of magnitude higher than conventional gene transfections. Hyaluronic acids were tethered onto the QD-amPEI for cell-specific targeted labeling which showed the specific-to-nonspecific signal ratio over 100. The inside hydrophobic compartment was further applied for cohosting oxygen sensing phosphorescence Ru dyes along with QDs. The QD-Ru-amPEI oxygen probe showed accurate and reversible oxygen sensing capability by the ratiometric photoluminescence signals, which was successfully applied to cellular and spheroid models.

In vivo wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography of human oral cavity with a forward-viewing probe.

We report multimodal imaging of human oral cavity in vivo based on simultaneous wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography (PS-OCT) with a forward-viewing imaging probe. Wide-field reflectance/fluorescence imaging and PS-OCT were to provide both morphological and fluorescence information on the surface, and structural and birefringent information below the surface respectively. The forward-viewing probe was designed to access the oral cavity through the mouth with dimensions of approximately 10 mm in diameter and 180 mm in length. The probe had field of view (FOV) of approximately 5.5 mm in diameter, and adjustable depth of field (DOF) from 2 mm to 10 mm by controlling numerical aperture (NA) in the detection path. This adjustable DOF was to accommodate both requirements for image-based guiding with high DOF and high-resolution, high-sensitivity imaging with low DOF. This multimodal imaging system was characterized by using a tissue phantom and a mouse model in vivo, and was applied to human oral cavity. Information of surface morphology and vasculature, and under-surface layered structure and birefringence of the oral cavity tissues was obtained. These results showed feasibility of this multimodal imaging system as a tool for studying oral cavity lesions in clinical applications.

In vivo visualization of skin inflammation by optical coherence tomography and two-photon microscopy

Inflammation is a non-specific immune response to injury intended to protect biological tissue from harmful stimuli such as pathogens, irritants, and damaged cells. In vivo optical tissue imaging has been used to provide spatial and dynamic characteristics of inflammation within the tissue. In this paper, we report in vivo visualization of inflammation in the skin at both cellular and physiological levels by using a combination of label-free two-photon microscopy (TPM) and optical coherence tomography (OCT). Skin inflammation was induced by topically applying lipopolysaccharide (LPS) on the mouse ear. Temporal OCT imaging visualized tissue swelling, vasodilation, and increased capillary density 30 min and 1 hour after application. TPM imaging showed immune cell migration within the inflamed skin. Combined OCT and TPM was applied to obtain complementary information from each modality in the same region of interest. The information provided by each modality were consistent with previous reports about the characteristics of inflammation. Therefore, the combination of OCT and TPM holds potential for studying inflammation of the skin.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

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.

Dark-field polarization-sensitive optical coherence tomography

Polarization-sensitive optical coherence tomography (PS-OCT) is a functional OCT providing both structural and birefringent information of the sample, and it has been applied to the studies of various organs having polarization properties. Fiber-based PS-OCT is sensitive to specular reflection from the sample surface, because signal saturation due to the strong specular reflection can make the polarization measurement difficult. We developed a dark-field PS-OCT which can avoid the specular reflection problem. Dark-field PS-OCT was implemented by adapting a hybrid method of Bessel-beam illumination and Gaussian-beam detection, and a PS-OCT method based on passive delay unit (PDU). The new system was characterized in comparison with the conventional Gaussian-beam based method in both polarization components and various samples including the human skin. Dark-field PS-OCT performed as good as the conventional PS-OCT without the specular reflection artifact. Dark-field PS-OCT may be useful in practical situations where the specular reflection is unavoidable.

Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin.

Both polarization sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are 3D optical imaging methods providing information related to collagen in the skin. PS-OCT provides birefringence information which is due to the collagen composition of the skin. SHG microscopy visualizes collagen fibers in the skin based on their SHG property. These two modalities have been applied to the same skin pathologies associated with collagen changes, but their relationship has not been examined. In this study, we tried to find the relationship by imaging the same skin samples with both modalities. Various parts of the normal rat skin and burn damaged skin were imaged ex vivo, and their images were analyzed both qualitatively and quantitatively. PS-OCT images were analyzed to obtain tissue birefringence. SHG images were analyzed to obtain collagen orientation indices by applying 2D Fourier transform. The skin samples having higher birefringence values had higher collagen orientation indices, and a linear correlation was found between them. Burn damaged skin showed decreases in both parameters compared to the control skins. This relationship between the bulk and microscopic properties of skin may be useful for further skin studies.

Combined two-photon microscopy and angiographic optical coherence tomography

Abstract. A combined two-photon microscopy (TPM) and angiographic optical coherence tomography (OCT) is developed, which can provide molecular, cellular, structural, and vascular information of tissue specimens in vivo. This combined system is implemented by adding an OCT vasculature visualization method to the previous combined TPM and OCT, and then is applied to in vivo tissue imaging. Two animal models, a mouse brain cranial window model and a mouse ear cancer model, are used. Both molecular, cellular information at local regions of tissues, and structural, vascular information at relatively larger regions are visualized in the same sections. In vivo tissue microenvironments are better elucidated by the combined TPM and angiographic OCT.

Dermoscopy guided dark-field multi-functional optical coherence tomography

Dermoscopy is a skin surface microscopic technique allowing specular reflection free observation of the skin, and has been used to examine pigmented skin lesions. However, dermoscopy has limitations in providing depth information due to lack of 3D resolution. In order to overcome the limitations, we developed dermoscopy guided multi-functional optical coherence tomography (MF-OCT) providing both high-contrast superficial information and depth-resolved structural, birefringent, and vascular information of the skin simultaneously. Dermoscopy and MF-OCT were combined by using a dichroic mirror, and dark-field configuration was adapted for MF-OCT to reduce specular reflection. After characterization, dermoscopy guided MF-OCT was applied to several human skin lesions such as the scar, port-wine stain (PWS) as well as the normal skin for demonstration. Various features of the scar and PWS were elucidated by both dermoscopy and MF-OCT. Dermoscopy guided MF-OCT may be useful for evaluation and treatment monitoring of skin lesions in clinical applications.

NO-dependent attenuation of TPA-induced immunoinflammatory skin changes in Balb/c mice by pindolol, heptaminol or ATRA, but not by verapamil

Recently a mouse skin carcinogenesis study reported that a β-blocker carvedilol displayed antitumor-properties via antihyperplastic effects. However, the antihyperplastic mechanism is unclear as the β-blocker is characterized with multiple pleiotropic effects including stimulation of endothelial NO release and verapamil-like calcium channel blocking activity. To investigate the nature and the origin of the antihyperplastic effects, we tested topical pretreatment with pindolol, heptaminol, ATRA or verapamil against Balb/c mouse ear skin hyperplasia that was induced by TPA. We found that pindolol, heptaminol or ATRA, but not verapamil, inhibited the TPA-induced immunoinflammatory skin changes in an NO-dependent manner, which included epidermal hyperplasia, skin edema and fibrosis. Furthermore, we also observed NO-dependent alleviation of the TPA-induced NK cell depletion in the ear tissues by heptaminol pretreatment. Together our results suggest that stimulation of NO generation from constitutive synthases may be primarily responsible for the reported antihyperplastic and NK cell-preserving effects of the β-blockers, and that similar effects may be observed in other immunity normalizing compounds that also promote endothelial NO synthesis.

Three-photon tissue imaging using moxifloxacin

Moxifloxacin is an antibiotic used in clinics and has recently been used as a clinically compatible cell-labeling agent for two-photon (2P) imaging. Although 2P imaging with moxifloxacin labeling visualized cells inside tissues using enhanced fluorescence, the imaging depth was quite limited because of the relatively short excitation wavelength (<800 nm) used. In this study, the feasibility of three-photon (3P) excitation of moxifloxacin using a longer excitation wavelength and moxifloxacin-based 3P imaging were tested to increase the imaging depth. Moxifloxacin fluorescence via 3P excitation was detected at a >1000 nm excitation wavelength. After obtaining the excitation and emission spectra of moxifloxacin, moxifloxacin-based 3P imaging was applied to ex vivo mouse bladder and ex vivo mouse small intestine tissues and compared with moxifloxacin-based 2P imaging by switching the excitation wavelength of a Ti:sapphire oscillator between near 1030 and 780 nm. Both moxifloxacin-based 2P and 3P imaging visualized cellular structures in the tissues via moxifloxacin labeling, but the image contrast was better with 3P imaging than with 2P imaging at the same imaging depths. The imaging speed and imaging depth of moxifloxacin-based 3P imaging using a Ti:sapphire oscillator were limited by insufficient excitation power. Therefore, we constructed a new system for moxifloxacin-based 3P imaging using a high-energy Yb fiber laser at 1030 nm and used it for in vivo deep tissue imaging of a mouse small intestine. Moxifloxacin-based 3P imaging could be useful for clinical applications with enhanced imaging depth.