Design and Validation of a Custom-Made Hyperspectral Microscope Imaging System for Biomedical Applications.

Hyperspectral microscope imaging (HMI) is an emerging modality that integrates spatial information collected by standard laboratory microscopy and the spectral-based contrast obtained by hyperspectral imaging and may be instrumental in establishing novel quantitative diagnostic methodologies, particularly in histopathology. Further expansion of HMI capabilities hinges upon the modularity and versatility of systems and their proper standardization. In this report, we describe the design, calibration, characterization, and validation of the custom-made laboratory HMI system based on a Zeiss Axiotron fully motorized microscope and a custom-developed Czerny-Turner-type monochromator. For these important steps, we rely on a previously designed calibration protocol. Validation of the system demonstrates a performance comparable to classic spectrometry laboratory systems. We further demonstrate validation against a laboratory hyperspectral imaging system for macroscopic samples, enabling future comparison of spectral imaging results across length scales. An example of the utility of our custom-made HMI system on a standard hematoxylin and eosin-stained histology slide is also shown.

Design and Validation of a Custom-Made Laboratory Hyperspectral Imaging System for Biomedical Applications Using a Broadband LED Light Source.

Hyperspectral imaging (HSI) is a promising optical modality that is already being used in numerous applications. Further expansion of the capabilities of HSI depends on the modularity and versatility of the systems, which would, inter alia, incorporate profilometry, fluorescence imaging, and Raman spectroscopy while following a rigorous calibration and verification protocols, thus offering new insights into the studied samples as well as verifiable, quantitative measurement results applicable to the development of quantitative metrics. Considering these objectives, we developed a custom-made laboratory HSI system geared toward biomedical applications. In this report, we describe the design, along with calibration, characterization, and verification protocols needed to establish such systems, with the overall goal of standardization. As an additional novelty, our HSI system uses a custom-built broadband LED-based light source for reflectance imaging, which is particularly important for biomedical applications due to the elimination of sample heating. Three examples illustrating the utility and advantages of the integrated system in biomedical applications are shown. Our attempt presents both the development of a custom-based laboratory HSI system with novel LED light source as well as a framework which may improve technological standards in HSI system design.

Curvature and height corrections of hyperspectral images using built-in 3D laser profilometry.

Optical imaging systems use a light source that illuminates a sample and a photodetector that detects light reflected from or transmitted through the sample. The sample surface curvature, surface-to-camera distance, and illumination-source-to-surface distance significantly affect the measured signal, resulting in image artifacts. To correct the images, a three-dimensional (3D) profilometry system was used to obtain 3D surface information. The 3D information enables image correction using Lambert cosine law and height correction. In this study, the feasibility of the correction method for push-broom hyperspectral imaging of three different objects is presented. Results show a significant reduction of image artifacts, making further image analysis more accurate and robust. The presented 3D profilometry method is applicable to all push-broom imaging systems and the described correction procedure can be applied to all spectral or monochromatic imaging systems.

Temperature Depth Profiles Induced in Human Skin In Vivo Using Pulsed 975 nm Irradiation.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the temperature depth profiles induced in human skin in vivo by using a pulsed 975 nm diode laser (with 5 ms pulse duration) and compare them with those induced by the more common 532 nm (KTP) and 1,064 nm (Nd:YAG) lasers. Quantitative assessment of the energy deposition characteristics in human skin at 975 nm should help design of safe and effective treatment protocols when using such lasers. STUDY DESIGN/MATERIALS AND METHODS: Temperature depth profiles induced in the human skin by the three lasers were determined using pulsed photothermal radiometry (PPTR). This technique involves time-resolved measurement of mid-infrared emission from the irradiated test site and reconstruction of the laser-induced temperature profiles using an earlier developed optimization algorithm. Measurements were performed on volar sides of the forearms in seven volunteers with healthy skin. At irradiation spot diameters of 3-4 mm, the radiant exposures were 0.24, 0.36, and 5.7 J/cm2 for the 975, 532, and 1,064 nm lasers, respectively. RESULTS: Upon normalization to the same radiant exposure of 1 J/cm 2 , the assessed maximum temperature rise in the epidermis averaged 0.8 °C for the 975 nm laser, 7.4 °C for the 532 nm, and 0.6 °C for the 1,064 nm laser. The characteristic subsurface depth to which 50% of the absorbed laser energy was deposited was on average 0.31 mm at 975 nm irradiation, and slightly deeper at 1,064 nm, and 0.15 mm at 532 nm. The experimentally obtained relations were reproduced in a dedicated numerical simulation. CONCLUSIONS: The assessed energy deposition characteristics show that the pulsed 975 nm diode laser is very suitable for controlled heating of the upper dermis as required, for example, for nonablative skin rejuvenation. The risks of nonselective overheating of the epidermis and subcutis are significantly reduced in comparison with irradiation at 532 and 1,064 nm, respectively. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Effects of phantom microstructure on their optical properties.

Significance: Developing stable, robust, and affordable tissue-mimicking phantoms is a prerequisite for any new clinical application within biomedical optics. To this end, a thorough understanding of the phantom structure and optical properties is paramount. Aim: We characterized the structural and optical properties of PlatSil SiliGlass phantoms using experimental and numerical approaches to examine the effects of phantom microstructure on their overall optical properties. Approach: We employed scanning electron microscope (SEM), hyperspectral imaging (HSI), and spectroscopy in combination with Mie theory modeling and inverse Monte Carlo to investigate the relationship between phantom constituent and overall phantom optical properties. Results: SEM revealed that microspheres had a broad range of sizes with average (13.47±5.98) µm and were also aggregated, which may affect overall optical properties and warrants careful preparation to minimize these effects. Spectroscopy was used to measure pigment and SiliGlass absorption coefficient in the VIS-NIR range. Size distribution was used to calculate scattering coefficients and observe the impact of phantom microstructure on scattering properties. The results were surmised in an inverse problem solution that enabled absolute determination of component volume fractions that agree with values obtained during preparation and explained experimentally observed spectral features. HSI microscopy revealed pronounced single-scattering effects that agree with single-scattering events. Conclusions: We show that knowledge of phantom microstructure enables absolute measurements of phantom constitution without prior calibration. Further, we show a connection across different length scales where knowledge of precise phantom component constitution can help understand macroscopically observable optical properties.

Hyperspectral imaging of 4T1 mammary carcinomas grown in dorsal skinfold window chambers: spectral renormalization and fluorescence modeling.

Significance: Hyperspectral imaging (HSI) of murine tumor models grown in dorsal skinfold window chambers (DSWCs) offers invaluable insight into the tumor microenvironment. However, light loss in a glass coverslip is often overlooked, and particular tissue characteristics are improperly modeled, leading to errors in tissue properties extracted from hyperspectral images. Aim: We highlight the significance of spectral renormalization in HSI of DSWC models and demonstrate the benefit of incorporating enhanced green fluorescent protein (EGFP) excitation and emission in the skin tissue model for tumors expressing genes to produce EGFP. Approach: We employed an HSI system for intravital imaging of mice with 4T1 mammary carcinoma in a DSWC over 14 days. We performed spectral renormalization of hyperspectral images based on the measured reflectance spectra of glass coverslips and utilized an inverse adding-doubling (IAD) algorithm with a two-layer murine skin model, to extract tissue parameters, such as total hemoglobin concentration and tissue oxygenation ( StO 2 ). The model was upgraded to consider EGFP fluorescence excitation and emission. Moreover, we conducted additional experiments involving tissue phantoms, human forearm skin imaging, and numerical simulations. Results: Hyperspectral image renormalization and the addition of EGFP fluorescence in the murine skin model reduced the mean absolute percentage errors (MAPEs) of fitted and measured spectra by up to 10% in tissue phantoms, 0.55% to 1.5% in the human forearm experiment and numerical simulations, and up to 0.7% in 4T1 tumors. Similarly, the MAPEs for tissue parameters extracted by IAD were reduced by up to 3% in human forearms and numerical simulations. For some parameters, statistically significant differences ( p < 0.05 ) were observed in 4T1 tumors. Ultimately, we have shown that fluorescence emission could be helpful for 4T1 tumor segmentation. Conclusions: The results contribute to improving intravital monitoring of DWSC models using HSI and pave the way for more accurate and precise quantitative imaging.

Estimating quantitative physiological and morphological tissue parameters of murine tumor models using hyperspectral imaging and optical profilometry.

Understanding tumors and their microenvironment are essential for successful and accurate disease diagnosis. Tissue physiology and morphology are altered in tumors compared to healthy tissues, and there is a need to monitor tumors and their surrounding tissues, including blood vessels, non-invasively. This preliminary study utilizes a multimodal optical imaging system combining hyperspectral imaging (HSI) and three-dimensional (3D) optical profilometry (OP) to capture hyperspectral images and surface shapes of subcutaneously grown murine tumor models. Hyperspectral images are corrected with 3D OP data and analyzed using the inverse-adding doubling (IAD) method to extract tissue properties such as melanin volume fraction and oxygenation. Blood vessels are segmented using the B-COSFIRE algorithm from oxygenation maps. From 3D OP data, tumor volumes are calculated and compared to manual measurements using a vernier caliper. Results show that tumors can be distinguished from healthy tissue based on most extracted tissue parameters ( p < 0.05 ). Furthermore, blood oxygenation is 50% higher within the blood vessels than in the surrounding tissue, and tumor volumes calculated using 3D OP agree within 26% with manual measurements using a vernier caliper. Results suggest that combining HSI and OP could provide relevant quantitative information about tumors and improve the disease diagnosis.

Hyperspectral evaluation of vasculature in induced peritonitis mouse models.

Imaging of blood vessel structure in combination with functional information about blood oxygenation can be important in characterizing many different health conditions in which the growth of new vessels contributes to the overall condition. In this paper, we present a method for extracting comprehensive maps of the vasculature from hyperspectral images that include tissue and vascular oxygenation. We also show results from a preclinical study of peritonitis in mice. First, we analyze hyperspectral images using Beer-Lambert exponential attenuation law to obtain maps of hemoglobin species throughout the sample. We then use an automatic segmentation algorithm to extract blood vessels from the hemoglobin map and combine them into a vascular structure-oxygenation map. We apply this methodology to a series of hyperspectral images of the abdominal wall of mice with and without induced peritonitis. Peritonitis is an inflammation of peritoneum that leads, if untreated, to complications such as peritoneal sclerosis and even death. Characteristic inflammatory response can also be accompanied by changes in vasculature, such as neoangiogenesis. We demonstrate a potential application of the proposed segmentation and processing method by introducing an abnormal tissue fraction metric that quantifies the amount of tissue that deviates from the average values of healthy controls. It is shown that the proposed metric successfully discriminates between healthy control subjects and model subjects with induced peritonitis and has a high statistical significance.

Molecular and Cellular Markers in Chlorhexidine-Induced Peritoneal Fibrosis in Mice.

Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering.

Effect of curvature correction on parameters extracted from hyperspectral images.

SIGNIFICANCE: Hyperspectral imaging (HSI) has emerged as a promising optical technique. Besides optical properties of a sample, other sample physical properties also affect the recorded images. They are significantly affected by the sample curvature and sample surface to camera distance. A correction method to reduce the artifacts is necessary to reliably extract sample properties. AIM: Our aim is to correct hyperspectral images using the three-dimensional (3D) surface data and assess how the correction affects the extracted sample properties. APPROACH: We propose the combination of HSI and 3D profilometry to correct the images using the Lambert cosine law. The feasibility of the correction method is presented first on hemispherical tissue phantoms and next on human hands before, during, and after the vascular occlusion test (VOT). RESULTS: Seven different phantoms with known optical properties were created and imaged with a hyperspectral system. The correction method worked up to 60 deg inclination angle, whereas for uncorrected images the maximum angles were 20 deg. Imaging hands before, during, and after VOT shows good agreement between the expected and extracted skin physiological parameters. CONCLUSIONS: The correction method was successfully applied on the images of tissue phantoms of known optical properties and geometry and VOT. The proposed method could be applied to any reflectance optical imaging technique and should be used whenever the sample parameters need to be extracted from a curved surface sample.

Thermophoretic tweezers for single nanoparticle manipulation.

We present the trapping and manipulation of a single nano-object in an aqueous medium by optically induced temporally varying temperature gradients. By real-time object tracking and control of the position of the heating laser focus, we can precisely employ thermophoretic drift to oppose the random diffusive motion. As a result, a nano-object is confined in a micrometer-sized trap. Numerical modeling gives a quantitative prediction of the effect. Traps can be dynamically created and relocated, which we demonstrate by the controlled independent manipulation of two nanoparticles.

Hyperspectral evaluation of peritoneal fibrosis in mouse models.

Analysis of morphological changes of the peritoneal membrane is an essential part of animal studies when investigating molecular mechanisms involved in the development of peritoneal fibrosis or testing the effects of potential therapeutic agents. Current methods, such as histology and immunohistochemistry, require time consuming sample processing and analysis and result in limited spatial information. In this paper we present a new method to evaluate structural and chemical changes in an animal model of peritoneal fibrosis that is based on hyperspectral imaging and a model of light transport. The method is able to distinguish between healthy and diseased subjects based on morphological as well as physiological parameters such as blood and scattering parameters. Furthermore, it enables evaluation of changes, such as degree of inflammation and fibrosis, that are closely related to histological findings.

Erratum: Optical properties of PlatSil SiliGlass tissue-mimicking phantoms: erratum.

[This corrects the article on p. 3753 in vol. 11.].

Optical properties of PlatSil SiliGlass tissue-mimicking phantoms.

In this work, we revise the preparation procedure and conduct an in depth characterization of optical properties for the recently proposed silicone-based tissue-mimicking optical phantoms in the spectral range from 475 to 925 nm. The optical properties are characterized in terms of refractive index and its temperature dependence, absorption and reduced scattering coefficients and scattering phase function related quantifiers. The scattering phase function and related quantifiers of the optical phantoms are first assessed within the framework of the Mie theory by using the measured refractive index of SiliGlass and size distribution of the hollow silica spherical particles that serve as scatterers. A set of purely absorbing optical phantoms in cuvettes is used to evaluate the linearity of the absorption coefficient with respect to the concentration of black pigment that serves as the absorber. Finally, the optical properties in terms of the absorption and reduced scattering coefficients and the subdiffusive scattering phase function quantifier γ are estimated for a subset of phantoms from spatially resolved reflectance using deep learning aided inverse models. To this end, an optical fiber probe with six linearly arranged optical fibers is used to collect the backscattered light at small and large distances from the source fiber. The underlying light propagation modeling is based on the stochastic Monte Carlo method that accounts for all the details of the optical fiber probe.