Nonlinear Optical Microscopy Imaging Research Articles

Review on Multimodal Nonlinear Optical Microscopy Imaging Technology

Review on Multimodal Nonlinear Optical Microscopy Imaging Technology

RepE: unsupervised representation learning for image enhancement in nonlinear optical microscopy.

We present an unsupervised learning denoising method, RepE (representation and enhancement), designed for nonlinear optical microscopy images, such as second harmonic generation (SHG) and two-photon fluorescence (TPEF). Addressing the challenge of effectively denoising images with various noise types, RepE employs an encoder network to learn noise-free representations and a reconstruction network to generate denoised images. It offers several key advantages, including its ability to (i) operate without restrictive statistic assumptions, (ii) eliminate the need for clean-noisy pairs, and (iii) requires only a few training images. Comparative evaluations on real-world SHG and TPEF images from esophageal cancer tissue slides (ESCC) demonstrate that our method outperforms existing techniques in image quality metrics. The proposed method provides a practical, robust solution for denoising nonlinear optical microscopy images, and it has the potential to be extended to other nonlinear optical microscopy modalities.

A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images.

The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis.

Emerging photonic technologies for cultural heritage studies: the examples of non-linear optical microscopy and photoacoustic imaging

The availability of non-invasive technologies, which can be used separately or in combination for obtaining chemical composition data and structural information of Cultural Heritage (CH) materials, is of prime importance for improving the understanding the environmental or ageing impact on monuments and artefacts and defining optimal strategies for their conservation. This paper overviews and assesses the potential of two emerging photonic technologies, the Non-linear Optical Microscopy (NLOM) and Photoacoustic (PA) imaging modalities, for a variety of diagnostic applications in preservation science. These techniques, which are well-established in biomedical research, during the last few years have been also investigated as non-invasive tools for the in-depth, high-resolution analysis of various CH objects, including paintings, documents and murals. We discuss on the applicability of these diagnostic optical methods to obtain precise stratigraphic information in artefacts, evaluating additionally the presence and the extent of potential morphological or chemical changes in several CH materials due to ageing. Furthermore, we demonstrate that the contrast complementarity of NLOM and PA imaging provides invaluable insights into the structural integrity of an artwork, which can be subsequently utilized for the early and accurate detection of depth degradation effects.

Accurate evaluation of the treatment effects of immunotherapy on subcutaneous ovarian cancer in mice with nonlinear optical imaging and algorithmic analysis.

Immunotherapy and its evaluation have shown great promise for cancer treatment. Here, a mouse subcutaneous transplantable tumor model was applied to testing therapeutic strategies. The mouse model was treated by regulating anti-PD-L1, anti-CTLA-4, cisplatin and their combined therapy. Biochemistry experiments have found that after immunotherapy, mice have more immune responses, which were manifested by an increase in the content of growth factors and the activation of T cells. Meanwhile, multimodal nonlinear optical microscopy imaging combined with algorithms was used to evaluate the treatment's effectiveness. By detecting the metabolism rate and microstructure in tissue, it was proved that combined therapies including immune checkpoint inhibitors do have a better effect on ovarian tumors. Our discovery of valid treatments for mice with ovarian tumor and provides an evaluation tool via nonlinear optics combined with algorithms offers new insights into therapeutic effect.

Multimodal widefield fluorescence imaging with nonlinear optical microscopy workflow for noninvasive oral epithelial neoplasia detection: a preclinical study.

.Significance: Early detection of epithelial cancers and precancers/neoplasia in the presence of benign lesions is challenging due to the lack of robust in vivo imaging and biopsy guidance techniques. Label-free nonlinear optical microscopy (NLOM) has shown promise for optical biopsy through the detection of cellular and extracellular signatures of neoplasia. Although in vivo microscopy techniques continue to be developed, the surface area imaged in microscopy is limited by the field of view. FDA-approved widefield fluorescence (WF) imaging systems that capture autofluorescence signatures of neoplasia provide molecular information at large fields of view, which may complement the cytologic and architectural information provided by NLOM.Aim: A multimodal imaging approach with high-sensitivity WF and high-resolution NLOM was investigated to identify and distinguish image-based features of neoplasia from normal and benign lesions.Approach: In vivo label-free WF imaging and NLOM was performed in preclinical hamster models of oral neoplasia and inflammation. Analyses of WF imaging, NLOM imaging, and dual modality (WF combined with NLOM) were performed.Results: WF imaging showed increased red-to-green autofluorescence ratio in neoplasia compared to inflammation and normal oral mucosa (). In vivo assessment of the mucosal tissue with NLOM revealed subsurface cytologic (nuclear pleomorphism) and architectural (remodeling of extracellular matrix) atypia in histologically confirmed neoplastic tissue, which were not observed in inflammation or normal mucosa. Univariate and multivariate statistical analysis of macroscopic and microscopic image-based features indicated improved performance (94% sensitivity and 97% specificity) of a multiscale approach over WF alone, even in the presence of benign lesions (inflammation), a common confounding factor in diagnostics.Conclusions: A multimodal imaging approach integrating strengths from WF and NLOM may be beneficial in identifying oral neoplasia. Our study could guide future studies on human oral neoplasia to further evaluate merits and limitations of multimodal workflows and inform the development of multiscale clinical imaging systems.

Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

Fractal dimension and directional analysis of elastic and collagen fiber arrangement in unsectioned arterial tissues affected by atherosclerosis and aging.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.

Visualization of Breast Cancer Metabolism Using Multimodal Nonlinear Optical Microscopy of Cellular Lipids and Redox State.

Label-free nonlinear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+) is widely used for high-resolution cellular redox imaging. In this work, we combined three label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: (i) TPEF optical redox ratio (ORR = FAD+/NADH + FAD+), (ii) coherent Raman scattering of cellular lipids, and (iii) second harmonic generation of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial (PME) cells and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17β-estradiol (E2) increased glycolysis in both PME and T47D ER+ breast cancer acini. However, PME cells displayed increased lipid content with no effect on ECM, while T47D cells had decreased lipid storage (P < 0.001) and significant reorganization of collagen. By measuring deuterated lipids synthesized from exogenously administered deuterium-labeled glucose, treatment of T47D cells with E2 increased both lipid synthesis and consumption rates. These results confirm that glucose is a significant source for the cellular synthesis of lipid in glycolytic breast cancer cells, and that the combination of cellular redox and lipid fraction imaging endpoints is a powerful approach with new and complementary information content.Significance: These findings provide unique insight into metabolic processes, revealing correlations between cancer metastasis and cellular redox state, lipid metabolism, and extracellular matrix. Cancer Res; 78(10); 2503-12. ©2018 AACR.

Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications.

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

Nonlinear imaging microscopy for assessing structural and photochemical modifications upon laser removal of dammar varnish on photosensitive substrates.

The nonlinear optical microscopy (NLM) modalities of Multi-Photon Excited Fluorescence (MPEF) and Third Harmonic Generation (THG) have been combined in this work to characterize as a function of depth with micrometric resolution the type and extent of morphological and photochemical modifications that take place upon ultraviolet (UV) pulsed laser removal of a dammar varnish layer applied on a photosensitive substrate. The latter consists on a layer of the synthetic polymer poly-methyl methacrylate doped with a photosensitizer, the aromatic compound 1,4-di[2-(5-phenyloxazolyl)] benzene, that strongly fluoresces upon UV light illumination. A number of laser conditions for partial or total elimination of the varnish coating were explored, namely different wavelengths (266, 248 and 213 nm) and pulse durations, in the nanosecond, picosecond and femtosecond ranges. Changes in the MPEF signals upon laser ablation of the outermost varnish layer successfully signpost photochemical modifications of the varnish or of the photosensitive under-layer, and their dependence with the laser ablation parameters, i.e., wavelength and pulse duration. In turn, THG signals mark the presence of layer boundaries and the reduction by laser ablation of the thickness of the varnish coating. The obtained MPEF and THG data are complemented by morphological observation by optical microscopy and measurements of laser induced fluorescence and micro-Raman spectra of the samples before and after laser ablation at the selected laser irradiation conditions. The results acquired through these non-destructive NLM imaging techniques serve to understand the phenomena that are induced upon laser ablation and to determine the best operating conditions that ensure controlled removal of the varnish with minimal morphological and chemical modifications to the under-layers. This research is of direct application to the UV pulsed laser cleaning of paintings and demonstrates the potential of NLM as a novel assessment tool for non-destructive, on line monitoring of the laser cleaning process.

Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments.

This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material.

Differentiating the extent of cartilage repair in rabbit ears using nonlinear optical microscopy.

Nonlinear optical microscopy (NLOM) was used as a noninvasive and label-free tool to detect and quantify the extent of the cartilage recovery. Two cartilage injury models were established in the outer ears of rabbits that created a different extent of cartilage recovery based on the presence or absence of the perichondrium. High-resolution NLOM images were used to measure cartilage repair, specifically through spectral analysis and image texture. In contrast to a wound lacking a perichondrium, wounds with intact perichondria demonstrated significantly larger TPEF signals from cells and matrix, coarser texture indicating the more deposition of type I collagen. Spectral analysis of cells and matrix can reveal the matrix properties and cell growth. In addition, texture analysis of NLOM images showed significant differences in the distribution of cells and matrix of repaired tissues with or without perichondrium. Specifically, the decay length of autocorrelation coefficient based on TPEF images is 11.2 ± 1.1 in Wound 2 (with perichondrium) and 7.5 ± 2.0 in Wound 1 (without perichondrium), indicating coarser image texture and faster growth of cells in repaired tissues with perichondrium (p < 0.05). Moreover, the decay length of autocorrelation coefficient based on collagen SHG images also showed significant difference between Wound 2 and 1 (16.2 ± 1.2 vs. 12.2 ± 2.1, p < 0.05), indicating coarser image texture and faster deposition of collagen in repaired tissues with perichondrium (Wound 2). These findings suggest that NLOM is an ideal tool for studying cartilage repair, with potential applications in clinical medicine. NLOM can capture macromolecular details and distinguish between different extents of cartilage repair without the need for labelling agents.

Quantitative ultrasound methods for uterine-cervical assessment

The cervix is a remarkable organ. One of its tasks is to remain firm and “closed” (5 mm diameter cervical canal) prior to pregnancy. Shortly after conception the cervix begins to soften through collagen remodeling and increased hydration. As the fetus reaches full-term there is a profound breakdown in the collagen structure. At the end of this process, the cervix is as soft as warm butter and the cervical canal has dilated to about 100 mm diameter. Errors in timing of this process are a cause for preterm birth, which has a cascade of life-threatening consequences. Quantitative ultrasound is well-suited to monitoring these changes. We have demonstrated the ability to accurately assess the elastic properties and acoustic scattering properties (anisotropy in backscatter and attenuation) of the cervix in non-pregnant hysterectomy specimens and in third trimester pregnancy. We’ve shown that acoustic and mechanical properties vary along the length of the cervix. When anisotropy and spatially variability are accounted for, there are clear differences in parameter values with subtle differences in softening. We are corroborating acoustic observations with nonlinear optical microscopy imaging for a reality check on underlying tissue structure. This presentation will provide an overview of this effort.

Multimodal nonlinear optical microscopic imaging provides new insights into acetowhitening mechanisms in live mammalian cells without labeling.

We developed a multimodal nonlinear optical microscopy imaging (e.g., third-harmonic generation (THG) and two-photon excited fluorescence (TPEF)) platform based on a femtosecond laser pumped photonic crystal fiber to investigate the acetowhitening phenomenon induced by acetic acid in live mammalian cells without labeling. After treated by acetic acid with concentrations of higher than 0.2%, THG images show that light scattering is remarkably increased inside the nucleus and cytoplasm in cells. Co-localized TPEF and THG imaging on tryptophan and NADH in cells indicates that the change of scattering property is largely originating from the morphological change of metabolic proteins induced by acetic acids. Further TPEF imaging on NADH and FAD in cells confirms that this change is irreversible when acetic acid concentration is higher than 1.2%. These subcellular-level THG/TPEF imaging results reveal that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

Multimodality imaging of the effects of a novel dentifrice on oral biofilm.

Oral biofilm formation and progression on the surface of the tooth can lead to advanced oral disease such as gingivitis. The purpose of this randomized, controlled, double-blinded study was to evaluate the effects of a novel dental gel on oral plaque biofilm using multimodal imaging techniques. Twenty-five subjects with moderate gingival inflammation (Löe and Silness Gingival Index ≥2) and pocket depths <4 were randomly assigned to brush twice daily for 21 days with the test or the control dental gel. In vivo multimodality in situ imaging was performed over a 3-week period using in vivo Optical Coherence Tomography (OCT) and Non-Linear Optical microscopy (NLOM). Plaque levels, gingival inflammation and gingival bleeding were also charted on days 0, 7, 14, and 21 using standard clinical indices. After 3 weeks, OCT and NLOM images showed a macroscopic break-up of the plaque layer and smaller, fragmented residual deposits in the test group with no apparent changes in the pellicle. Biofilm was also reduced in the control group, but to a lesser degree with regard to thickness, continuity and surface area. Paralleling these imaging results, clinical indices were significantly improved in both groups (P < 0.05) and significantly lower in the test group (P < 0.05). Both dental gels reduced oral biofilm with the test gel showing greater efficacy (P < 0.05) as determined by clinical and imaging parameters.

Automated seeding-based nuclei segmentation in nonlinear optical microscopy

Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.

Characterization of optical properties of ZnO nanoparticles for quantitative imaging of transdermal transport

Widespread applications of ZnO nanoparticles (NP) in sun-blocking cosmetic products have raised safety concerns related to their potential transdermal penetration and resultant cytotoxicity. Nonlinear optical microscopy provides means for high-contrast imaging of ZnO NPs lending in vitro and in vivo assessment of the nanoparticle uptake in skin, provided their nonlinear optical properties are characterized. We report on this characterization using ZnO NP commercial product, Zinclear, mean-sized 21 nm. Two-photon action cross-section of this bandgap material (Ebg = 3.37 eV, λbg = 370 nm) measured by two techniques yielded consistent results of = 6.2 ± 0.8 μGM at 795 nm, and 32 ± 6 μGM at 770 nm per unit ZnO crystal cell, with the quantum efficiency of = (0.9 ± 0.2) %. In order to demonstrate the quantitative imaging, nonlinear optical microscopy images of the excised human skin topically treated with Zinclear were acquired and processed using and values yielding nanoparticle concentration map in skin. Accumulations of Zinclear ZnO nanoparticles were detected only on the skin surface and in skin folds reaching concentrations of 800 NPs per μm3.