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Occasionally, tissue samples cannot be processed completely and are stored under varying conditions for extended periods. This is particularly beneficial in interinstitutional studies where a given research setting may lack the expertise or infrastructure for sample processing, imaging and data analysis. Currently, there is limited literature available on the controlled storage of biological tissues in primary fixatives for fluorescence and electron microscopy. In this contribution, we mimicked various tissue storage scenarios by taking different fixation conditions, storage temperatures and storage durations into account. Rat liver tissue was used for its well-known diversity of cellular ultrastructure and microscopy analysis. Fluorescent labelling of actin, DNA and lipids were employed in conjunction with high-resolution electron microscopy imaging. Herein, we tested three different fixative solutions (1.5% glutaraldehyde, 0.4% glutaraldehyde and 4% formaldehyde and 4% formaldehyde) and stored samples for 1–28 days at room temperature and refrigerator temperature. We found that liver tissue can be stored for up to 2 weeks in a 0.4% glutaraldehyde + 4% formaldehyde fixative solution, while still enabling reliable fluorescent labelling and ultrastructural studies. Ultrastructural integrity was eminent up to 1 month using either glutaraldehyde or formaldehyde fixation protocols. When liver tissue is fixed with a mixture of 0.4% glutaraldehyde and 4% formaldehyde and stored at 4 °C, it retains its capacity for electron microscopy analysis for several years, but loses its capacity for reliable fluorescent labelling studies. In conclusion, we demonstrated that liver tissue can be stored for extended periods enabling profound structure–function analysis across length scales. [ABSTRACT FROM AUTHOR]

The success of machine learning (ML) models in object or pattern recognition naturally leads to ML being employed in the classification of the microstructure of steel surfaces. Light optical microscopy (LOM) is the traditional imaging process in this field. However, the increasing use of ML to extract or relate more aspects of the aforementioned materials and the limitations of LOM motivated us to provide an improvement to the established image acquisition process. In essence, we perform style transfer from LOM to scanning electron microscopy (SEM) combined with “intelligent” upscaling. This is achieved by employing an ML model trained on a multimodal dataset to generate an SEM-like image from the corresponding LOM image. This transformation, in our opinion, which is corroborated by a detailed analysis of the source, target and prediction, successfully pushes the limits of LOM in the case of steel surfaces. The expected consequence is the improvement of the precise characterization of advanced multiphase steels’ structure based on these transformed LOM images.

The success of machine learning (ML) models in object or pattern recognition naturally leads to ML being employed in the classification of the microstructure of steel surfaces. Light optical microscopy (LOM) is the traditional imaging process in this field. However, the increasing use of ML to extract or relate more aspects of the aforementioned materials and the limitations of LOM motivated us to provide an improvement to the established image acquisition process. In essence, we perform style transfer from LOM to scanning electron microscopy (SEM) combined with "intelligent" upscaling. This is achieved by employing an ML model trained on a multimodal dataset to generate an SEM-like image from the corresponding LOM image. This transformation, in our opinion, which is corroborated by a detailed analysis of the source, target and prediction, successfully pushes the limits of LOM in the case of steel surfaces. The expected consequence is the improvement of the precise characterization of advanced multiphase steels' structure based on these transformed LOM images. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fibers, axonopathy, and inflammatory responses leading to consequent sensory-motor and cognitive deficits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which offers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals. [ABSTRACT FROM AUTHOR]

The combination of multiple imaging modalities in a single microscopy system can enable new insights into biological processes. In this work, we describe the construction and rigorous characterization of a custom microscope with multimodal imaging in a single, cost-effective system. Our design utilizes advances in LED technology, robotics, and open-source software, along with existing optical components and precision optomechanical parts to offer a modular and versatile design. This microscope is operated using software written in Arduino and Python and has the ability to run multi-day automated imaging experiments when placed inside of a cell culture incubator. Additionally, we provide and demonstrate methods to validate images taken in brightfield and darkfield, along with validation and optimization for differential phase contrast (DPC) quantitative phase imaging.

Background and Objectives: Oral cancer is a global health challenge. The disease can be successfully treated if detected early, but the survival rate drops significantly for late stage cases. There is a growing interest in a shift from the current standard of invasive and time-consuming tissue sampling and histological examination, towards non-invasive brush biopsies and cytological examination, facilitating continued risk group monitoring. For cost effective and accurate cytological analysis there is a great need for reliable computer-assisted data-driven approaches. However, infeasibility of accurate cell-level annotation hinders model performance, and limits evaluation and interpretation of the results. This study aims to improve AI-based oral cancer detection by introducing additional information through multimodal imaging and deep multimodal information fusion., Methods: We combine brightfield and fluorescence whole slide microscopy imaging to analyze Papanicolaou-stained liquid-based cytology slides of brush biopsies collected from both healthy and cancer patients. Given the challenge of detailed cytological annotations, we utilize a weakly supervised deep learning approach only relying on patient-level labels. We evaluate various multimodal information fusion strategies, including early, late, and three recent intermediate fusion methods., Results: Our experiments demonstrate that: (i) there is substantial diagnostic information to gain from fluorescence imaging of Papanicolaou-stained cytological samples, (ii) multimodal information fusion improves classification performance and cancer detection accuracy, compared to single-modality approaches. Intermediate fusion emerges as the leading method among the studied approaches. Specifically, the Co-Attention Fusion Network (CAFNet) model achieves impressive results, with an F1 score of 83.34% and an accuracy of 91.79% at cell level, surpassing human performance on the task. Additional tests highlight the importance of accurate image registration to maximize the benefits of the multimodal analysis., Conclusion: This study advances the field of cytopathology by integrating deep learning methods, multimodal imaging and information fusion to enhance non-invasive early detection of oral cancer. Our approach not only improves diagnostic accuracy, but also allows an efficient, yet uncomplicated, clinical workflow. The developed pipeline has potential applications in other cytological analysis settings. We provide a validated open-source analysis framework and share a unique multimodal oral cancer dataset to support further research and innovation., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Occasionally, tissue samples cannot be processed completely and are stored under varying conditions for extended periods. This is particularly beneficial in interinstitutional studies where a given research setting may lack the expertise or infrastructure for sample processing, imaging and data analysis. Currently, there is limited literature available on the controlled storage of biological tissues in primary fixatives for fluorescence and electron microscopy. In this contribution, we mimicked various tissue storage scenarios by taking different fixation conditions, storage temperatures and storage durations into account. Rat liver tissue was used for its well-known diversity of cellular ultrastructure and microscopy analysis. Fluorescent labelling of actin, DNA and lipids were employed in conjunction with high-resolution electron microscopy imaging. Herein, we tested three different fixative solutions (1.5% glutaraldehyde, 0.4% glutaraldehyde and 4% formaldehyde and 4% formaldehyde) and stored samples for 1-28 days at room temperature and refrigerator temperature. We found that liver tissue can be stored for up to 2 weeks in a 0.4% glutaraldehyde + 4% formaldehyde fixative solution, while still enabling reliable fluorescent labelling and ultrastructural studies. Ultrastructural integrity was eminent up to 1 month using either glutaraldehyde or formaldehyde fixation protocols. When liver tissue is fixed with a mixture of 0.4% glutaraldehyde and 4% formaldehyde and stored at 4 °C, it retains its capacity for electron microscopy analysis for several years, but loses its capacity for reliable fluorescent labelling studies. In conclusion, we demonstrated that liver tissue can be stored for extended periods enabling profound structure-function analysis across length scales., Competing Interests: Declarations. Conflict of Interest: There is no conflict of interest associated with any of the senior authors or other co-authors who contributed their efforts in this manuscript., (© 2024. The Author(s).)

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering-two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughputmetabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders ofmagnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRSFISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail. [ABSTRACT FROM AUTHOR]

The Prospective Instruments MPX-series is a turnkey compact multimodal microscope enabling advanced multiphoton imaging without requiring a laboratory or optical bench. One of the key design features is the ultrafast fiber laser engine integrated into the free moving scan head, which is lightweight, rugged, and allows the ultimate imaging flexibility. A modular design concept allows the user to explore a wide variety of biological applications and measurements, without compromising performance, in any indoor working space. In contrast, standard setups are non-transportable, utility-demanding, and the complexity can be complicated, therefore degrading the value and adding significant initial, short- and long-term expenses. Every clinical and biological researcher should have access to high-quality reliable optical microscopy modalities without being hardware experts. In this article we outline the demand and design of the MPX-microscope and present multiphoton imaging results from experiments in various configurations. There are no existing instruments on the market that are portable and combine easily switchable modes in one composite industry-ready device for life science, clinical, and pharmacological research. [ABSTRACT FROM AUTHOR]

Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second-harmonic generation (SHG) and third-harmonic generation images from ex vivo human skin and showed that a sub-40 fs, 1060-nm Yb-fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber-based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low-barrier-to-access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.

Integrated multimodal cross-sectional or volumetric imaging techniques give us fruitful information to understand the behavior or status of target objects such as biological samples. Most of the reported systems for this purpose are either time consuming due to scanning or use additional reference beams such as in interferometry. Therefore, fast, simple, highly efficient, and powerful multimodal imaging systems that can perform cross-sectional imaging with simple algorithms are worth to be investigated. In this paper, a multimodal technique for cross-sectional quantitative phase and fluorescence imaging with computational microscopy is presented. We combine cross-sectional fluorescence and quantitative phase imaging by using the transport of intensity equation (TIE) and numerical wave propagation. The amplitude and phase of the fluorescence light wave with partially spatial coherence are obtained from three defocused intensity patterns. The proposed hybrid imaging system is simple, compact, and non-iterative. We present experimental results of microbeads and fluorescent protein-labeled living cells of the moss Physcomitrella patens to demonstrate the performance of the proposed imaging system. [ABSTRACT FROM AUTHOR]

Fullerenes are candidates for theranostic applications because of their high photodynamic activity and intrinsic multimodal imaging contrast. However, fullerenes suffer from low solubility in aqueous media, poor biocompatibility, cell toxicity, and a tendency to aggregate. C70@lysozyme is introduced herein as a novel bioconjugate that is harmless to a cellular environment, yet is also photoactive and has excellent optical and optoacoustic contrast for tracking cellular uptake and intracellular localization. The formation, water‐solubility, photoactivity, and unperturbed structure of C70@lysozyme are confirmed using UV‐visible and 2D 1H, 15N NMR spectroscopy. The excellent imaging contrast of C70@lysozyme in optoacoustic and third harmonic generation microscopy is exploited to monitor its uptake in HeLa cells and lysosomal trafficking. Last, the photoactivity of C70@lysozyme and its ability to initiate cell death by means of singlet oxygen (1O2) production upon exposure to low levels of white light irradiation is demonstrated. This study introduces C70@lysozyme and other fullerene‐protein conjugates as potential candidates for theranostic applications. [ABSTRACT FROM AUTHOR]

Abstract Although liver transport routes have been extensively studied in rodents, live imaging under in situ and in vivo conditions of large volumes is still proven to be difficult. In this study, we took advantage of the optical transparency of zebrafish and their small size to explore their usefulness for correlative imaging studies and liver transport experimentations. First, we assessed the micro-architecture of the zebrafish liver and compared its fine structure to the rodent and humans' literature. Next, we investigated the transport routes and cellular distribution of albumin using combined and correlative microscopy approaches. These methods permitted us to track the injected proteins at different time points through the process of liver uptake and clearance of albumin. We demonstrate strong structural and functional resemblance between the zebrafish liver and its rodents and humans' counterparts. In as short as 5 min post-injection, albumin rapidly accumulated within the LSECs. Furthermore, albumin entered the space of Disse where it initially accumulated then subsequently was taken up by the hepatocytes. We propose the zebrafish as a viable alternative experimental model for hepatic transport studies, allowing swift multimodal imaging and direct quantification on the hepatic distribution of supramolecular complexes of interest. [ABSTRACT FROM AUTHOR]

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

The combination of multiple imaging modalities in a single microscopy system can enable new insights into biological processes. In this work, we describe the construction and rigorous characterization of a custom microscope with multimodal imaging in a single, cost-effective system. Our design utilizes advances in LED technology, robotics, and open-source software, along with existing optical components and precision optomechanical parts to offer a modular and versatile design. This microscope is operated using software written in Arduino and Python and has the ability to run multi-day automated imaging experiments when placed inside of a cell culture incubator. Additionally, we provide and demonstrate methods to validate images taken in brightfield and darkfield, along with validation and optimization for differential phase contrast (DPC) quantitative phase imaging., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Fluorescent labels are well suited as tracers for cancer drug monitoring. Identifying cellular target regions of these drugs with a high resolution is important to assess the working principle of a drug. We investigate the applications of label-free nonresonant four-wave mixing (NR-FWM) microscopy in biological imaging in combination with fluorescence imaging of fluorescently labeled cancer drugs. Results from human A431 tumor cells with stained nuclei and incubated with IRdye 800CW labeled cancer drug cetuximab targeting epidermal growth factor receptor at the cell membrane show that NR-FWM is well suited for cellular imaging. A comparison of vibrationally nonresonant FWM imaging with vibrational resonant coherent anti-Stokes Raman scattering signals revealed nearly identical qualitative information in cellular imaging. NR-FWM is also suitable for tumor tissue imaging in combination with fluorescence imaging of IRdye 800CW labeled, human epidermal growth factor 2 targeting cancer drug pertuzumab and provides additional information over transmission microscopy. [ABSTRACT FROM AUTHOR]

Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark- or bright-field illumination. Thus, the particular visual information associated with interference and phase contrast, dark- and bright-field illumination is joined in real-time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark-field illumination. In particular, haloing and shade-off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators. [ABSTRACT FROM AUTHOR]

We have developed a multimodal optical platform for label-free imaging on the basis of nonlinear optical microscopy (NLO) for applications in biology and biomedical science. Three application areas have been chosen as examples: regenerative medicine, drug delivery monitoring and cancer diagnosis. Obtained data showed the potential of NLO microscopy for the following: (1) investigating the stem cell differentiation states prior their use in regenerative medicine and tissue repair, (2) tracking drug molecules in living cells and (3) detecting cancer by imaging biopsies ex vivo. The results of this study illustrate the potential to translate NLO microscopy from ex vivo to in vivo applications. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Intracellular imaging is a key tool in the investigation of host-pathogen interactions. Advances in this area are particularly sought to understand the effect of viral infection processes on the host cell and its metabolic functions including those cases where host cell lipid metabolism is modulated as a result of infection. We demonstrate the use of combined coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence microscopies to image fibroblast cells infected by cytomegalovirus. CARS is used to image the host cell membrane, lipid droplets and morphology of the nucleus. Cell nuclei are found to expand during infection, approximately doubling in area. Some cells also show accumulations of lipid droplets at the nuclear periphery. Using a genetically modified virus strain expressing the green fluorescent protein also enables two-photon imaging of the same cells to reveal the location, nature and extent of viral protein expression. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Objectives: To increase the chance for a cure, cancer must be detected as early as possible. This can be achieved with cytopathological diagnostic methods. For a further increase of the diagnostic accuracy of these methods we introduced the multimodal cell analysis, viz, cells on the slide have to be relocalized to enable successive analysis of identical cells in different stains. For practical reasons the relocalization step must be automated.Methods: For a fully automatic acquisition of successive cell images we use a passive autofocus that is adaptive to the material, i.e., to the cells, followed by a comparison of the scenes, i.e., the cell constellation, of two such obtained images from different stains. In case that no sub-scene match can be found the search is extended to the surrounding area. A set of 1556 scenes from seven specimens have been subject to our algorithm. The automatically relocalized and acquired images from a second stain have been manually compared to the images from a first stain.Results: An overall relocalization rate of 85.4% is achieved. 14.3% of the images could not be relocalized and are lost for the following diagnostic process, while the critical case of erroneously matched images was observed in only 0.3% of cases.Conclusions: We could show that it is possible to automatically acquire images of successive stains of identical cells on cytopathological specimens. The method presented achieves acceptable relocalization rates. Wrong image acquisitions are very rare and can mostly be ascribed to images with single cells, i.e., without scene information. [ABSTRACT FROM AUTHOR]

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering-two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

Rapid emergence of multimodal imaging in scanning probe, electron, and optical microscopies has brought forth the challenge of understanding the information contained in these complex data sets, targeting the intrinsic correlations between different channels, and further exploring the underpinning causal physical mechanisms. Here, we develop such an analysis framework for Piezoresponse Force Microscopy. We argue that under certain conditions, we can bootstrap experimental observations with the prior knowledge of materials structure to get information on certain nonobserved properties, and demonstrate linear causal analysis for PFM observables. We further demonstrate that the strength of individual causal links between complex descriptors can be ascertained using the deep kernel learning (DKL) model. In this DKL analysis, we use the prior information on domain structure within the image to predict the physical properties. This analysis demonstrates the correlative relationships between morphology, piezoresponse, elastic property, etc., at nanoscale. The prediction of morphology and other physical parameters illustrates a mutual interaction between surface condition and physical properties in ferroelectric materials. This analysis is universal and can be extended to explore the correlative relationships of other multichannel data sets, and allow for high-fidelity reconstruction of underpinning functionalities and physical mechanisms.

Abstract A correlative approach employing optical microscopy and scanning electron microscopy is proposed for the quantification of phases and pores in iron ore pellets. Combining mosaic images covering the full cross-sections of pellets, obtained with both techniques, it is possible to improve the discrimination of pores, quartz and silicates. First, the images must be carefully registered to show the exact same regions. The procedure for registration, discrimination and quantification was developed with Fiji open source software. A qualitative and quantitative analysis confirmed the advantages of the correlative method as compared to the individual techniques.

The healing process is often significantly impaired under conditions of chronic or large area wounds, which are often treated clinically using autologous split-thickness skin grafts. However, in many cases, harvesting of donor tissue presents a serious problem such as in the case of very large area burns. In response to this, engineered biomaterials have emerged that attempt to mimic the natural skin environment or deliver a suitable therapy to assist in the healing process. In this study, a custom-built multimodal optical microscope capable of noninvasive structural and functional imaging is used to investigate both the engineered tissue microenvironment and the in vivo wound healing process. Investigation of various engineered scaffolds show the strong relationship among the microenvironment of the scaffold, the organization of the cells within the scaffold, and the delivery pattern of these cells onto the healing wound. Through noninvasive tracking of these processes and parameters, multimodal optical microscopy provides an important tool in the assessment of engineered scaffolds both in vitro and in vivo.

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

In this paper, we demonstrate high-resolution, multimodal in vivo imaging of human skin using optical coherence (OCM) and multiphoton microscopy (MPM). These two modalities are integrated into a single instrument to enable simultaneous acquisition and coregistration. The system design and the OCM image processing architecture enable sufficient performance of both modalities for in vivo imaging of human skin. Examples of multimodal in vivo imaging are presented as well as time lapse imaging of blood flow in single capillary loops. By making use of multiple intrinsic contrast mechanisms this integrated technique improves the ability to noninvasively visualize living tissue. Integrated OCM and MPM has potential applications for in vivo diagnosis of various pathological skin conditions, such as skin cancer, as well as potential pharmaceutical and cosmetic research applications.