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Significance: The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and postprocessing methods to differentiate cortical cells, such as neurons and small blood vessels. Aim: We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination. Approach: We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography. Results: The phase contrast implementation is successful to retrieve deep images in the cortex up to 800 µm using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network. Conclusions: Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved fromthe dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design. [ABSTRACT FROM AUTHOR]

Significance: Confocal microscopy is an indispensable tool for biologists to observe samples and is useful for fluorescence imaging of living cells with high spatial resolution. Recently, phase information induced by the sample has been attracting attention because of its applicability such as the measurability of physical parameters and wavefront compensation. However, commercially available confocal microscopy has no phase imaging function., Aim: We reborn an off-the-shelf confocal microscope as a phase measurement microscope. This is a milestone in changing the perspective of researchers in this field. We would meet the demand of biologists if only they had measured the phase with their handheld microscopes., Approach: We proposed phase imaging based on the transport of intensity equation (TIE) in commercially available confocal microscopy. The proposed method requires no modification using a bright field imaging module of a commercially available confocal microscope., Results: The feasibility of the proposed method is confirmed by evaluating the phase difference of a microlens array and living cells of the moss Physcomitrium patens and living mammalian cultured cells. In addition, multi-modal imaging of fluorescence and phase information is demonstrated., Conclusions: TIE-based quantitative phase imaging (QPI) using commercially available confocal microscopy is proposed. We evaluated the feasibility of the proposed method by measuring the microlens array, plant, and mammalian cultured cells. The experimental result indicates that QPI can be realized in commercially available confocal microscopy using the TIE technique. This method will be useful for measuring dry mass, viscosity, and temperature of cells and for correcting phase fluctuation to cancel aberration and scattering caused by an object in the future., (© 2024 The Authors.)

Accurate identification of epidermal cells on reflectance confocal microscopy (RCM) images is important in the study of epidermal architecture and topology of both healthy and diseased skin. However, analysis of these images is currently done manually and therefore time-consuming and subject to human error and inter-expert interpretation. It is also hindered by low image quality due to noise and heterogeneity. We aimed to design an automated pipeline for the analysis of the epidermal structure from RCM images. Two attempts have been made at automatically localizing epidermal cells, called keratinocytes, on RCM images: the first is based on a rotationally symmetric error function mask, and the second on cell morphological features. Here, we propose a dual-task network to automatically identify keratinocytes on RCM images. Each task consists of a cycle generative adversarial network. The first task aims to translate real RCM images into binary images, thus learning the noise and texture model of RCM images, whereas the second task maps Gabor-filtered RCM images into binary images, learning the epidermal structure visible on RCM images. The combination of the two tasks allows one task to constrict the solution space of the other, thus improving overall results. We refine our cell identification by applying the pre-trained StarDist algorithm to detect star-convex shapes, thus closing any incomplete membranes and separating neighboring cells. The results are evaluated both on simulated data and manually annotated real RCM data. Accuracy is measured using recall and precision metrics, which is summarized as the F1 -score. We demonstrate that the proposed fully unsupervised method successfully identifies keratinocytes on RCM images of the epidermis, with an accuracy on par with experts' cell identification, is not constrained by limited available annotated data, and can be extended to images acquired using various imaging techniques without retraining. [ABSTRACT FROM AUTHOR]

Significance: Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures., Aim: We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy., Approach: In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels., Results: Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image's cell distribution., Conclusion: This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies., (© 2024 The Authors.)

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acoustooptic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTLs) play a key role in adoptive cell therapy (ACT) by destroying tumor cells. Although some mechanisms of CTLs killing tumor cells have already been revealed, the precise dynamic information of CTLs' interaction with tumor cells is still not known. Here, we used confocal microscopy to visualize the whole process of how CTLs kill tumor cells in vitro. According to imaging data, CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or more tumor cells, and the average time for CTLs destroying one or more tumor cells in vitro is dozens of minutes only. Our study displayed the temporal events of CTLs' interaction with tumor cells at the beginning up to the point of killing them. Furthermore, the imaging data presented strong cytotoxicity of CTLs toward the specific tumor cells. These results could help us to well understand the mechanism of CTLs' elimination of tumor cells and improve the efficacy of ACT in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectancebased handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15 frames/s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1 µm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device. [ABSTRACT FROM AUTHOR]

An optical technique called line-field confocal optical coherence tomography (LC-OCT) is introduced for high-resolution, noninvasive imaging of human skin in vivo. LC-OCT combines the principles of time-domain optical coherence tomography and confocal microscopy with line illumination and detection using a broadband laser and a line-scan camera. LC-OCT measures the echo-time delay and amplitude of light backscattered from cutaneous microstructures through low-coherence interferometry associated with confocal spatial filtering. Multiple A-scans are acquired simultaneously while dynamically adjusting the focus. The resulting crosssectional B-scan image is produced in real time at 10 frame/s. With an isotropic spatial resolution of ~1 µm, the LC-OCT images reveal a comprehensive structural mapping of skin at the cellular level down to a depth of ~500 µm. LC-OCT has been applied to the imaging of various skin lesions, in vivo, including carcinomas and melanomas. LC-OCT images are found to strongly correlate with conventional histopathological images. The use of LC-OCT as an adjunct tool in medical practice could significantly improve clinical diagnostic accuracy while reducing the number of biopsies of benign lesions. [ABSTRACT FROM AUTHOR]

Constructing models of cells' realistic internal and external morphology is vital for correlation between light scattering and morphology of the scattering structure. The image stack obtained from fluorescent confocal microscopy is at present used to construct the cell's three-dimensional (3-D) morphology. However, due to the poor labeling quality and unavoidable optical noise present in the image stacks, 3-D morphologies are difficult to construct and are an impediment to the statistical analyses of cell structures. We propose a method called the "area and shape constraint method (ASCM)" for constructing 3-D morphology. Blurred 3-D morphologies constructed by common methods from image stacks considered as defective and which are commonly discarded are well restored by the ASCM. Seventy-four clinical blood samples and a series of standard fluorescent spheres are selected to evaluate the validity and precision of our proposed ASCM. Both the qualitative and quantitative results obtained by ASCM indicate the good performance of the method in constructing the cell's 3-D morphology. [ABSTRACT FROM AUTHOR]

We present a fiber-optic dual-confocal laser caliper method for noncontact high-precision sensing and measuring thickness and refractive index of intraocular lens (IOL) implants. The principle of the method is based on sensing and measuring the confocal intensity response of the laser beam reflection from the opposite object surfaces, which provides the advanced feature of having no limitations on the object shape, thickness, and transparency. Using single-mode optical fibers and a 658-nm laser source, the thickness measurement accuracy was assessed to be as high as 5 μm. In addition, refractive index of a transparent object with thickness smaller than the working distance of the focusing lenses can be measured. The thickness and refractive index of a planoconvex IOL were measured with a high accuracy. [ABSTRACT FROM AUTHOR]

Rare-earth-doped nanocomposites have appealing optical properties for use as biomedical contrast agents, but few systems exist for imaging these materials. We describe the design and characterization of (i) a preclinical system for whole animal in vivo imaging and (ii) an integrated optical coherence tomography/confocal microscopy system for high-resolution imaging of ex vivo tissues. We demonstrate these systems by administering erbiumdoped nanocomposites to a murine model of metastatic breast cancer. Short-wave infrared emissions were detected in vivo and in whole organ imaging ex vivo. Visible upconversion emissions and tissue autofluorescence were imaged in biopsy specimens, alongside optical coherence tomography imaging of tissue microstructure. We anticipate that this work will provide guidance for researchers seeking to image these nanomaterials across a wide range of biological models. [ABSTRACT FROM AUTHOR]

Delivery and spatial localization of upconversion luminescent microparticles [Y2O3:Yb, Er] (mean size ~1.6 µm) and quantum dots (QDs) (CuInS2/ZnS nanoparticles coated with polyethylene glycol-based amphiphilic polymer, mean size ~20 nm) inside rat skin was studied in vivo using a multimodal optical imaging approach. The particles were embedded into the skin dermis to the depth from 300 to 500 µm through microchannels performed by fractional laser microablation. Low-frequency ultrasound was applied to enhance penetration of the particles into the skin. Visualization of the particles was revealed using a combination of luminescent spectroscopy, optical coherence tomography, confocal microscopy, and histochemical analysis. Optical clearing was used to enhance the image contrast of the luminescent signal from the particles. It was demonstrated that the penetration depth of particles depends on their size, resulting in a different detection time interval (days) of the luminescent signal from microparticles and QDs inside the rat skin in vivo. We show that luminescent signal from the upconversion microparticles and QDs was detected after the particle delivery into the rat skin in vivo during eighth and fourth days, respectively. We hypothesize that the upconversion microparticles have created a long-time depot localized in the laser-created channels, as the QDs spread over the surrounding tissues. [ABSTRACT FROM AUTHOR]

Plastic embedding is widely applied in light microscopy analyses. Previous studies have shown that embedding agents and related techniques can greatly affect the quality of biological tissue embedding and fluorescent imaging. Specifically, it is difficult to preserve endogenous fluorescence using currently available acidic commercial embedding resins and related embedding techniques directly. Here, we developed a neutral embedding resin that improved the green fluorescent protein (GFP), yellow fluorescent protein (YFP), and DsRed fluorescent intensity without adjusting the pH value of monomers or reactivating fluorescence in lye. The embedding resin had a high degree of polymerization, and its fluorescence preservation ratios for GFP, YFP, and DsRed were 126.5%, 155.8%, and 218.4%, respectively. [ABSTRACT FROM AUTHOR]

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells. [ABSTRACT FROM AUTHOR]

Autofluorescence photobleaching describes the decrease of fluorescence intensity of endogenous fluorophores in biological tissue upon light irradiation. The origin of autofluorescence photobleaching is not fully understood. In the skin, the spatial distribution of various endogenous fluorophores varies within the skin layers. Most endogenous fluorophores are excited in the ultraviolet and short visible wavelength range, and only a few, such as porphyrins (red) and melanin (near-infrared), are excited at longer wavelengths. The excitation wavelength- and depth-dependent irradiation of skin will therefore excite different fluorophores, which will likely influence the photobleaching characteristics. The autofluorescence photobleaching of porcine ear skin has been measured ex vivo using 325, 473, 633, and 785 nm excitation at different skin depths from the surface to the dermis at 150 µm. Confocal Raman microscopes were used to achieve sufficient spatial resolution of the measurements. The autofluorescence area under the curve was measured for 21 consecutive acquisitions of 15 s. In all cases, the photobleaching follows a two-exponential decay function approximated by nonlinear regression. The results show that photobleaching can be applied to improve the signal-to-noise ratio in Raman spectroscopy for all of the applied excitation wavelengths and skin depths. [ABSTRACT FROM AUTHOR]

The separation zone between enamel and dentin [dentin-enamel junction (DEJ)] with different properties in biomechanical composition has an important role in preventing crack propagation from enamel to dentin. The understanding of the chemical structure (inorganic and organic components), physical properties, and chemical composition of the human DEJ could benefit biomimetic materials in dentistry. Spatial distribution of calcium phosphate crystallinity and the collagen crosslinks near DEJ were studied using confocal Raman microscopy and calculated by different methods. To obtain collagen crosslinking, the ratio of two peaks 1660 cm-1 over 1690 cm-1 (amide I bands) is calculated. For crystallinity, the inverse full-width at half maximum of phosphate peak at 960 cm-1, and the ratio of two Raman peaks of phosphate at 960/950 cm-1 is provided. In conclusion, the study of chemical and physical properties of DEJ provides many benefits in the biomaterial field to improve the synthesis of dentalmaterials in respect to the natural properties of human teeth. Confocal Raman microscopy as a powerful tool provides the molecular structure to identify the changes along DEJ and can be expanded for other mineralized tissues. [ABSTRACT FROM AUTHOR]

Laser ablation offers a procedure for precise, fast, and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided approach offers cellular-level histopathology-like feedback directly on the patient, which may then guide and help improve the efficacy of the ablation procedure. Following an ex vivo benchtop study (reported in our earlier papers), we performed an initial study on 44 BCCs on 21 patients in vivo, using a pulsed erbium:ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). In 10 lesions on six patients, the RCM imagingguided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally,34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, with immediate confirmation for clearance of tumor (no histopathology), followed by longer-term monitoring, currently in progress, with follow-up imaging (again, no histopathology) at 3, 6, and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy of ablation in the wound, both immediately postablation and subsequently during recovery.The efficacy results appear to be promising, with observed clearance in 19 cases of 22 cases with follow-ups ranging from 6 to 21 months. An additional 12 cases with 1 to 3 months of follow-ups has shown clearance of tumor but a longer follow-up time is required to establish conclusive results. Further instrumentation development will be necessary to cover larger areas with a more automatically controlled instrument for more uniform, faster, and deeper imaging of margins. [ABSTRACT FROM AUTHOR]

We present a hand-held implementation and preliminary evaluation of a combined optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) probe for detecting and delineating the margins of basal cell carcinomas (BCCs) in human skin in vivo. A standard OCT approach (spectrometer-based) with a central wavelength of 1310 nm and 0.11 numerical aperture (NA) was combined with a standard RCM approach (830-nm wavelength and 0.9 NA) into a common path hand-held probe. Cross-sectional OCT images and enface RCM images are simultaneously displayed, allowing for three-dimensional microscopic assessment of tumor morphology in real time. Depending on the subtype and depth of the BCC tumor and surrounding skin conditions, OCT and RCM imaging are able to complement each other, the strengths of each helping overcome the limitations of the other. Four representative cases are summarized, out of the 15 investigated in a preliminary pilot study, demonstrating how OCT and RCM imaging may be synergistically combined to more accurately detect BCCs and more completely delineate margins. Our preliminary results highlight the potential benefits of combining the two technologies within a single probe to potentially guide diagnosis as well as treatment of BCCs. [ABSTRACT FROM AUTHOR]

This article proposes a multispectral system that uses the analysis of the spatial distribution of color and spectral features to improve the detection of skin cancer lesions, specifically melanomas and basal cell carcinomas. The system consists of a digital camera and light-emitting diodes of eight different wavelengths (414 to 995 nm). The parameters based on spectral features of the lesions such as reflectance and color, as well as others empirically computed using reflectance values, were calculated pixel-by-pixel from the images obtained. Statistical descriptors were calculated for every segmented lesion [mean ( x), standard deviation (σ), minimum, and maximum]; descriptors based on the first-order statistics of the histogram [entropy (Ep), energy (En), and third central moment (μ3)] were also obtained. The study analyzed 429 pigmented and nonpigmented lesions: 290 nevi and 139 malignant (95 melanomas and 44 basal cell carcinomas), which were split into training and validation sets. Fifteen parameters were found to provide the best sensitivity (87.2% melanomas and 100% basal cell carcinomas) and specificity (54.5%). The results suggest that the extraction of textural information can contribute to the diagnosis of melanomas and basal cell carcinomas as a supporting tool to dermoscopy and confocal microscopy. [ABSTRACT FROM AUTHOR]

Optical coherence tomography (OCT) is capable of imaging microstructures within translucid samples. A time-domain version of the OCT technology is employed here due to its compatibility with the dynamic focus (DF) procedure. DF means moving the confocal gate in synchronism with the depth scanning via the coherence gate. A DF-OCT setup was implemented for imaging samples at 1300 nm. Its confocal gate of 180 μm allows the achievement of good and similar transversal resolution along its much larger axial range. Images of a phantom, human skin, teeth, and larynx with and without DF are demonstrated. [ABSTRACT FROM AUTHOR]

A reflectance confocal endomicroscope with double-clad fiber coupler and electrically tunable focus lens is applied to imaging of the oral mucosa. The instrument is designed to be lightweight and robust for clinical use. The tunable lens allows axial scanning through >250 µm in the epithelium when the probe tip is placed in contact with tissue. Images are acquired at 6.6 frames per second with a field of view diameter up to 850 µm. In vivo imaging of a wide range of normal sites in the oral cavity demonstrates the accessibility of the handheld probe. In vivo imaging of clinical lesions diagnosed as inflammation and dysplasia illustrates the ability of reflectance confocal endomicroscopy to image cellular changes associated with pathology. [ABSTRACT FROM AUTHOR]

Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750 x 750 µm². We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2 x 2 cm² of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant. [ABSTRACT FROM AUTHOR]

Breast conserving surgery allows complete tumor resection while maintaining acceptable cosmesis for patients. Safe and rapid intraoperative margin assessment during the procedure is important to establish the completeness of tumor excision and minimizes the need for reoperation. Confocal laser endomicroscopy has demonstrated promise for real-time intraoperative margin assessment using acriflavine staining, but it is not approved for routine in-human use. We describe a custom high-speed line-scan confocal laser endomicroscopy (LS-CLE) system at 660 nm that enables high-resolution histomorphological imaging of breast tissue stained with methylene-blue, an alternative fluorescent stain for localizing sentinel nodes during breast surgery. Preliminary imaging results on freshly excised human breast tissue specimens are presented, demonstrating the potential of methylene-blue aided rapid LS-CLE to determine the oncological status of surgical margins in-vivo. [ABSTRACT FROM AUTHOR]

Reflectance confocal microscopy (RCM) allows for real-time in vivo visualization of the skin at the cellular level. The study of RCM images provides information on the structural properties of the epidermis. These may change in each layer of the epidermis, depending on the subject's age and the presence of certain dermatological conditions. Studying RCM images requires manual identification of cells to derive these properties, which is time consuming and subject to human error, highlighting the need for an automated cell identification method. We aim to design an automated pipeline for the analysis of the structure of the epidermis from RCM images of the Stratum granulosum and Stratum spinosum. We identified the region of interest containing the epidermal cells and the individual cells in the segmented tissue area using tubeness filters to highlight membranes. We used prior biological knowledge on cell size to process the resulting detected cells, removing cells that were too small and reapplying the used filters locally on detected regions that were too big to be considered a single cell. The proposed full image analysis pipeline (FIAP) was compared with machine learning-based approaches (cell cutter, different U-Net configurations, and loss functions). All methods were evaluated both on simulated data (four images) and on manually annotated RCM data (seven images). Accuracy was measured using recall and precision metrics. Both accuracy metrics were higher in the proposed FIAP for both real (precision = 0.720 ± 0.068, recall = 0.850 ± 0.11) and synthetic images (precision = 0.835 ± 0.067, recall = 0.925 ± 0.012). The tested machine learning methods failed to identify and segment keratinocytes on RCM images with a satisfactory accuracy. We showed that automatic cell segmentation can be achieved using a pipeline based on membrane detection, with an accuracy that matches expert manual cell identification. To our knowledge, this is the first method based on membrane detection to study healthy skin using RCM images evaluated against manually identified cell positions. [ABSTRACT FROM AUTHOR]

Current diagnostic capabilities and limitations of fluorescence endomicroscopy in the cervix are assessed by qualitative and quantitative image analysis. Four cervical tissue types are investigated: normal columnar epithelium, normal and precancerous squamous epithelium, and stromal tissue. This study focuses on the perceived variability within and the subtle differences between the four tissue groups in the context of endomicroscopic in vivo pathology. Conclusions are drawn on the general ability to distinguish and diagnose tissue types, on the need for imaging depth control to enhance differentiation, and on the possible risks for diagnostic misinterpretations. [ABSTRACT FROM AUTHOR]

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. [ABSTRACT FROM AUTHOR]

Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration. [ABSTRACT FROM AUTHOR]

In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, selective plane illumination microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single-photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (~10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly. [ABSTRACT FROM AUTHOR]

In the confocal Raman spectra of skin dermis, the band area in the spectral region of proline and hydroxyproline varies according to the age and health condition of the volunteers, classified as healthy young women, healthy elderly women, and diabetic elderly women. Another observation refers to the intensity variation and negative Raman shift of the amide I band. To understand these effects, we adopted a model system using the DFT/B3LYP:3-21G procedure, considering the amino acid chain formed by glycine, hydroxyproline, proline, and alanine, which interacts with two and six water molecules. Through these systems, polarizability variations were analyzed to correlate its values with the observed Raman intensities of the three groups of volunteers and to assign the vibrational spectra of the skin dermis. As a way to correlate other experimental trends, we propose a model of chemical reaction of water interchange between the bonding amino acids, in which water molecules are attached with glucose by hydrogen bonds. The theoretical results are in accordance with the observed experimental trends. [ABSTRACT FROM AUTHOR]

We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams. [ABSTRACT FROM AUTHOR]

Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related "fingerprints" for accurate and label-free classification of the two prostate cell types. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other. [ABSTRACT FROM AUTHOR]

Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation. [ABSTRACT FROM AUTHOR]

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT's basic principles, major implementations, imaging contrasts, and recent applications. [ABSTRACT FROM AUTHOR]

The increased ability of TMX-202 (derivative of imiquimod) to penetrate the intact stratum corneum (SC) and the follicular orifices of porcine ear skin was shown ex vivo using confocal Raman microscopy and laser scanning microscopy. Moreover, to assess whether TMX-202 is able to reach the immune cells, Langerhans cells extracted from pretreated human skin were investigated ex vivo using confocal Raman microscopy combined with multivariate statistical methods. Tracking the Raman peak of dimethyl sulfoxide centered at 690 cm-1, the absorption of TMX-202 containing formulation by Langerhans cells was shown. To answer the question whether the TMX-202 active ingredient is able to reach Langerhans cells, the attraction of immune cells to TMX-202 containing formulation treated skin was measured in the in vivo rodent model Mastomys coucha. The results show that TMX-202 active ingredient is able to reach Langerhans cells after penetrating through the intact skin and subsequently attract immune cells. Both the intercellular/transcellular as well as the follicular pathways allow the penetration through the intact barrier of the SC. [ABSTRACT FROM AUTHOR]

Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micromagnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development. [ABSTRACT FROM AUTHOR]

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions. [ABSTRACT FROM AUTHOR]

Reflectance-mode confocal microscopy (RCM) enables in vivo assessment of the human skin. Impact of overweight on both human skin microcirculation and histomorphology has not been investigated in vivo. The purpose of this study is to evaluate both microcirculation and histomorphology in vivo in overweight. In 10 normotensive overweight nondiabetic individuals (OW-group, BMI 29.1 ± 2.7 kg/m²) and 10 age- and sex-matched healthy lean controls (CO-group, BMI 20.4 ± 1.9 kg/m²) the following parameters were evaluated using RCM: dermal blood cell flow (DBCF), density of dermal capillaries (DDC), epidermal thickness (ET), and epidermal cell size (ECS). DBCF was counted at 63.11 & #177; 4.14 cells/ min in OW-group and at 51.06 ± 3.84 cells/ min in COgroup (P < 0.05). DDC was reduced in OW-group (4.91 ± 0.39 capillaries/mm2) compared to the controls (6.02 ± 0.64 capillaries/mm², P < 0.05). Histometric evaluation of ET reveals thickening in OW-group compared to the CO-group (54.79 ± 4.25 µm versus 44.03 ± 3.11 µm, P < 0.05). ECS differed significantly (P < 0.05) in OW-group (821.3 ± 42.02 µm²) compared to the controls (772.6 ± 34.79 µm²). Inverse correlation of dermal capillary density and overweight point to reduced total tissue perfusion while positive related blood cell flow reveals vasodilatation. Increase of both ET and cell size indicates remodeling of cutaneous histomorphology, maybe as an early stage of adiposity-related skin condition. [ABSTRACT FROM AUTHOR]

We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ~1 µm, OCT imaging delineates BCC depth spreading (margins) with resolution of ~7 µm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ~600 µm, and agreement with histology was within about ±50 µm. [ABSTRACT FROM AUTHOR]

Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering. [ABSTRACT FROM AUTHOR]

A digital adaptive optics line-scanning confocal imaging (DAOLCI) system is proposed by applying digital holographic adaptive optics to a digital form of line-scanning confocal imaging system. In DAOLCI, each line scan is recorded by a digital hologram, which allows access to the complex optical field from one slice of the sample through digital holography. This complex optical field contains both the information of one slice of the sample and the optical aberration of the system, thus allowing us to compensate for the effect of the optical aberration, which can be sensed by a complex guide star hologram. After numerical aberration compensation, the corrected optical fields of a sequence of line scans are stitched into the final corrected confocal image. In DAOLCI, a numerical slit is applied to realize the confocality at the sensor end. The width of this slit can be adjusted to control the image contrast and speckle noise for scattering samples. DAOLCI dispenses with the hardware pieces, such as Shack-Hartmann wavefront sensor and deformable mirror, and the closedloop feedbacks adopted in the conventional adaptive optics confocal imaging system, thus reducing the optomechanical complexity and cost. Numerical simulations and proof-of-principle experiments are presented that demonstrate the feasibility of this idea. [ABSTRACT FROM AUTHOR]

Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er; Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enablesmicroscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for realtime in vivo imaging in the clinic. [ABSTRACT FROM AUTHOR]

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development. [ABSTRACT FROM AUTHOR]

Intrinsic optical signal (IOS) imaging promises to be a noninvasive method for high-resolution examination of retinal physiology, which can advance the study and diagnosis of eye diseases. While specialized optical instruments are desirable for functional IOS imaging of retinal physiology, in depth understanding of multiple IOS sources in the complex retinal neural network is essential for optimizing instrument designs. We provide a brief overview of IOS studies and relationships in rod outer segment suspensions, isolated retinas, and intact eyes. Recent developments of line-scan confocal and functional optical coherence tomography (OCT) instruments have allowed in vivo IOS mapping of photoreceptor physiology. Further improvements of the line-scan confocal and functional OCT systems may provide a feasible solution to pursue functional IOS mapping of human photoreceptors. Some interesting IOSs have already been detected in inner retinal layers, but better development of the IOS instruments and software algorithms is required to achieve optimal physiological assessment of inner retinal neurons. [ABSTRACT FROM AUTHOR]

Reflectance confocal microscopy is successfully used in infant skin research. Infant skin structure, function, and composition are undergoing a maturation process. We aimed to uncover how the epidermal architecture and cellular topology change with time. Images were collected from three age groups of healthy infants between one and four years of age and adults. Cell centers were manually identified on the images at the stratum granulosum (SG) and stratum spinosum (SS) levels. Voronoi diagrams were used to calculate geometrical and topological parameters. Infant cell density is higher than that of adults and decreases with age. Projected cell area, cell perimeter, and average distance to the nearest neighbors increase with age but do so distinctly between the two layers. Structural entropy is different between the two strata, but remains constant with time. For all ages and layers, the distribution of the number of nearest neighbors is typical of a cooperator network architecture. The topological analysis provides evidence of the maturation process in infant skin. The differences between infant and adult are more pronounced in the SG than SS, while cell cooperation is evident in all cases of healthy skin examined. [ABSTRACT FROM AUTHOR]

We synthesized and characterized gold nanostars and their silica-coated derivatives with 7- to 50-nm shell thicknesses as contrast agents for optical imaging. The scattering and absorption coefficients of the nanoparticles (NPs) were estimated by means of collimated transmittance and diffuse reflectance/transmittance analyses. The contrasting properties of the nanostructures were studied in optical coherence tomography glass capillary imaging. The silica-coated nanostars with the thickest shell have higher scattering ability in comparison with bare nanostars. Viability assays confirmed weak in vitro toxicity of nanostructures at up to ~200-µg/mL concentrations. We showed real-time visualization of nanostars in both agarose and cultured cells by analyzing the backscattering signal using a conventional laser confocal microscope. The signal intensity detected from the silica-coated NPs was almost 1.5 times higher in comparison with bare nanostars. To the best of our knowledge, this is the first time that conventional laser confocal microscopy was applied in combined scattering and transmitted light modes to detect the backscattered signal of gold nanostars, which is useful for direct monitoring of the uptake, translocation, and accumulation of NPs in living cells. [ABSTRACT FROM AUTHOR]

Mohs surgery for the removal of nonmelanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is time consuming and labor intensive. Real-time intraoperative reflectance confocal microscopy (RCM), combined with video mosaicking, may enable rapid detection of residual tumor directly in the surgical wounds on patients. We report our initial experience on 25 patients, using aluminum chloride for nuclear contrast. Imaging was performed in quadrants in the wound to simulate the Mohs surgeons examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin and detectable in residual NMSC tumors. The presence of residual tumor and normal skin features could be detected in the peripheral and deep dermal margins. Intraoperative RCM imaging may enable detection of residual tumor directly on patients during Mohs surgery, and may serve as an adjunct for frozen pathology. Ultimately, for routine clinical utility, a stronger tumor-to-dermis contrast may be necessary, and also a smaller microscope with an automated approach for imaging in the entire wound in a rapid and controlled manner. [ABSTRACT FROM AUTHOR]

We present an implementation of spectrally encoded slit confocal microscopy. The method employs a rapid wavelength-swept laser as the light source and illuminates a specimen with a line focus that scans through the specimen as the wavelength sweeps. The reflected light from the specimen is imaged with a stationary line scan camera, in which the finite pixel height serves as a slit aperture. This scanner-free operation enables a simple and cost-effective implementation in a small form factor, while allowing for the three-dimensional imaging of biological samples. [ABSTRACT FROM AUTHOR]

We present a swept source optical coherence tomography (OCT) system at 1060 nm equipped with a wavefront sensor at 830 nm and a deformable mirror in a closed-loop adaptive optics (AO) system. Due to the AO correction, the confocal profile of the interface optics becomes narrower than the OCT axial range, restricting the part of the B-scan (cross section) with good contrast. By actuating on the deformable mirror, the depth of the focus is changed and the system is used to demonstrate Gabor filtering in order to produce B-scan OCT images with enhanced sensitivity throughout the axial range from a Drosophila larvae. The focus adjustment is achieved by manipulating the curvature of the deformable mirror between two user-defined limits. Particularities of controlling the focus for Gabor filtering using the deformable mirror are presented. [ABSTRACT FROM AUTHOR]