Functionalizing Collagen with Vessel-Penetrating Two-Photon Phosphorescence Probes: A New In Vivo Strategy to Map Oxygen Concentration in Tumor Microenvironment and Tissue Ischemia.

The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio-probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI ) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI -diimine complex already exhibits excellent emission yield (34%, λem   = 583 nm) and large two-photon absorption cross-sections (σ2   = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen-bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post-extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial-empowered phosphorescence sensing and imaging.

In vivo dual-mode full-field optical coherence tomography for differentiation of types of melanocytic nevi.

SIGNIFICANCE: Melanocytic nevi represent the most common dermal melanocytic lesions in humans. Nevus is typically diagnosed clinically with the naked eye or with dermoscopy. However, it is essential to identify the type of nevus by invasive biopsy for histopathological examination. The use of noninvasive imaging tools can be used to evaluate the types of nevi to reduce unnecessary excisions of benign entities. AIM: To evaluate the feasibility of using en face and cross-sectional full-field optical coherence tomography (FF-OCT) in differentiation of melanocytic nevi that can facilitate the reduction of unnecessary excisions of benign entities. APPROACH: Dual-mode Mirau-type FF-OCT for cross-sectional imaging (B-scan) and en face imaging were used to distinguish the types of nevi. RESULTS: Although the B-scan reveals the distribution of melanosomes, users can set a specific depth of the en face image to explore the morphology of surrounding skin cells instantly. According to the locations of nevus nests, the different types of nevi, including junction nevus and compound nevus, can be identified using this dual-mode FF-OCT system. CONCLUSIONS: Combining B-scan and en face imaging in vivo FF-OCT enables the examination and navigation of skin tissues in real time and in three dimensions.

Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy.

Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of −0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

Infrared-active quadruple contrast FePt nanoparticles for multiple scale molecular imaging.

A single nanomaterial with multiple imaging contrasts and functions is highly desired for multiscale theragnosis. Herein, we demonstrate single 1-1.9 µm infrared-active FePt alloy nanoparticles (FePt NPs) offering unprecedented four-contrast-in-one molecular imaging - computed tomography (CT), magnetic resonance imaging (MRI), photoacoustic (PA) imaging, and high-order multiphoton luminescence (HOMPL) microscopy. The PA response of FePt NPs outperforms that of infrared-active gold nanorods by 3- to 5.6-fold under identical excitation fluence and particle concentrations. HOMPL (680 nm) of an isolated FePt NP renders spatial full-width-at-half-maximum values of 432 nm and 300 nm beyond the optical diffraction limit for 1230-nm and 920-nm excitation, respectively. The in vivo targeting function was successfully visualized using HOMPL, PA imaging, CT, and MRI, thereby validating FePt as a single nanomaterial system covering up to four types (Optical/PA/CT/MRI) of molecular imaging contrast, ranging from the microscopic level to whole-body scale investigation.

Imaging Endogenous Bilirubins with Two-Photon Fluorescence of Bilirubin Dimers.

On the basis of an infrared femtosecond Cr:forsterite laser, we developed a semiquantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly differentiated hepatocellular carcinoma (HCC) tissues on average showed 3.7 times lower concentration of bilirubins than the corresponding nontumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than that of nontumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of nontumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.

Differential diagnosis of nonmelanoma pigmented skin lesions based on harmonic generation microscopy.

In vivo harmonic generation microscopy (HGM) has been applied successfully in healthy human skin and can achieve a submicron resolution, similar to histopathologic examination, even at a penetration depth up to 270 µm. This study aims to investigate the clinical applicability of HGM imaging for differential diagnosis of nonmelanoma pigmented skin lesions. A total of 42 pigmented skin tumors, including pigmented basal cell carcinoma, melanocytic nevus, and seborrheic keratosis were evaluated by HGM ex vivo or in vivo. Based on the standard histopathologic characteristics, we established the corresponding HGM imaging criteria for each pigmented tumor. Diagnostic performance of HGM for differentiating nonmelanoma pigmented skin tumors was evaluated through the observers' direct general assessment (overall evaluation) or the presence of two imaging criteria with the highest sensitivity and specificity (major criteria evaluation). Our results show that, based on the direct general assessment, the sensitivity is 92% [95% confidence interval (CI): 67 to 97%] and the specificity is 96% (95% CI: 83 to 99%); by major criteria evaluation, 94% sensitivity (95% CI: 70 to 99%) and 100% specificity (95% CI: 87 to 100%) are achieved. Our study indicates that HGM serves as a promising histopathological examination tool for noninvasive differential diagnostics of nonmelanoma pigmented skin tumors.

Automatic cell segmentation and nuclear-to-cytoplasmic ratio analysis for third harmonic generated microscopy medical images.

Traditional biopsy procedures require invasive tissue removal from a living subject, followed by time-consuming and complicated processes, so noninvasive in vivo virtual biopsy, which possesses the ability to obtain exhaustive tissue images without removing tissues, is highly desired. Some sets of in vivo virtual biopsy images provided by healthy volunteers were processed by the proposed cell segmentation approach, which is based on the watershed-based approach and the concept of convergence index filter for automatic cell segmentation. Experimental results suggest that the proposed algorithm not only reveals high accuracy for cell segmentation but also has dramatic potential for noninvasive analysis of cell nuclear-to-cytoplasmic ratio (NC ratio), which is important in identifying or detecting early symptoms of diseases with abnormal NC ratios, such as skin cancers during clinical diagnosis via medical imaging analysis.

Applying tattoo dye as a third-harmonic generation contrast agent for in vivo optical virtual biopsy of human skin.

Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.

Characterization of oral squamous cell carcinoma based on higher-harmonic generation microscopy.

In vivo higher-harmonic generation microscopy (HGM) performed on healthy human oral mucosa not only provides images with a <500 nm lateral resolution at a 280 µm penetration depth, but also leaves no photodamages in the tissues. These advantages suggest that HGM could serve as an ideal virtual biopsy tool for in vivo, in situ, and immediate histopathological diagnosis of oral cancer. However, translation of such mechanism for clinical cancer diagnosis requires evidence based algorithm capable to differentiate cancerous tissues from normal. It is thus critical to investigate if the endogenous contrast provided by the HGM would be high enough to differentiate cancerous versus normal tissues in human oral mucosa. In this report, ex vivo HGM study was performed on the cancerous mucosa from 10 patients with oral squamous cell carcinoma. Compared with histology, HGM revealed histopathological features including the cytological abnormalities, loss of differentiation, interruption of basement membrane, and irregular epithelial stratification in all 10 specimens. In addition, distinct patterns of collagen fibers and increased distribution area of actin filaments in tumor cells were noted. These results indicate HGM holds great potential for the optical biopsy screening of oral cancer lesions.

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy.

Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 µm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.

Applying harmonic optical microscopy for spatial alignment of atrial collagen fibers.

BACKGROUND: Atrial fibrosis creates a vulnerable tissue for atrial fibrillation (AF), but the spatial disarray of collagen fibers underlying atrial fibrosis is not fully elucidated. OBJECTIVE: This study hypothesizes that harmonics optical microscopy can illuminate the spatial mal-alignment of collagen fibers in AF via a layer-by-layer approach. PATIENTS AND METHODS: Atrial tissues taken from patients who underwent open-heart surgery were examined by harmonics optical microscopy. Using the two-dimensional Fourier transformation method, a spectral-energy description of image texture was constituted and its entropy was used to quantify the mal-alignment of collagen fibers. The amount of collagen fiber was derived from its area ratio to total atrial tissue in each image. Serum C-terminal pro-collagen pro-peptide (CICP), pro-matrix metalloproteinase-1 (pro-MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were also evaluated. RESULTS: 46 patients were evaluated, including 20 with normal sinus rhythm and 26 with AF. The entropy of spectral-energy distribution of collagen alignment was significantly higher in AF than that in sinus rhythm (3.97 ± 0.33 vs. 2.80 ± 0.18, p<0.005). This difference was more significant in the permanent AF group. The amount of collagen was also significantly higher in AF patients (0.39 ± 0.13 vs. 0.18 ± 0.06, p<0.005) but serum markers of cardiac fibrosis were not significantly different between the two groups. CONCLUSIONS: Harmonics optical microscopy can quantify the spatial mal-alignment of collagen fibers in AF. The entropy of spectral-energy distribution of collagen alignment is a potential tool for research in atrial remodeling.