High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.     

Tissue profiling by mass spectrometry: a review of methodology and applications

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. For example, protein profiles from tumors may allow accurate prediction of tumor behavior, diagnosis, and prognosis and uncover etiologies underlying idiopathic diseases. MALDI MS, in conjunction with laser capture microdissection, is able to produce protein expression profiles from a relatively small number of cells from specific regions of heterogeneous tissue architectures. Imaging mass spectrometry enables the investigator to assess the spatial distribution of proteins, drugs, and their metabolites in intact tissues. This article provides an overview of several tissue profiling and imaging applications performed by MALDI MS, including sample preparation, matrix selection and application, histological staining prior to MALDI analysis, tissue profiling, imaging, and data analysis. Several applications represent direct translation of this technology to clinically relevant problems.     

In situ metabolomic mass spectrometry imaging: recent advances and difficulties

MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.     

High Definition Infrared Spectroscopic Imaging for Lymph Node Histopathology

Chemical imaging is a rapidly emerging field in which molecular information within samples can be used to predict biological function and recognize disease without the use of stains or manual identification. In Fourier transform infrared (FT-IR) spectroscopic imaging, molecular absorption contrast provides a large signal relative to noise. Due to the long mid-IR wavelengths and sub-optimal instrument design, however, pixel sizes have historically been much larger than cells. This limits both the accuracy of the technique in identifying small regions, as well as the ability to visualize single cells. Here we obtain data with micron-sized sampling using a tabletop FT-IR instrument, and demonstrate that the high-definition (HD) data lead to accurate identification of multiple cells in lymph nodes that was not previously possible. Highly accurate recognition of eight distinct classes - naïve and memory B cells, T cells, erythrocytes, connective tissue, fibrovascular network, smooth muscle, and light and dark zone activated B cells was achieved in healthy, reactive, and malignant lymph node biopsies using a random forest classifier. The results demonstrate that cells currently identifiable only through immunohistochemical stains and cumbersome manual recognition of optical microscopy images can now be distinguished to a similar level through a single IR spectroscopic image from a lymph node biopsy.     

Multimodality imaging in vivo for preclinical assessment of tumor-targeted doxorubicin nanoparticles

This study presents a new multimodal imaging approach that includes high-frequency ultrasound, fluorescence intensity, confocal, and spectral imaging to improve the preclinical evaluation of new therapeutics in vivo. Here we use this approach to assess in vivo the therapeutic efficacy of the novel chemotherapy construct, HerDox during and after treatment. HerDox is comprised of doxorubicin non-covalently assembled in a viral-like particle targeted to HER2+ tumor cells, causing tumor cell death at over 10-fold lower dose compared to the untargeted drug, while sparing the heart. Whereas our initial proof-of-principle studies on HerDox used tumor growth/shrinkage rates as a measure of therapeutic efficacy, here we show that multimodal imaging deployed during and after treatment can supplement traditional modes of tumor monitoring to further characterize the particle in tissues of treated mice. Specifically, we show here that tumor cell apoptosis elicited by HerDox can be monitored in vivo during treatment using high frequency ultrasound imaging, while in situ confocal imaging of excised tumors shows that HerDox indeed penetrated tumor tissue and can be detected at the subcellular level, including in the nucleus, via Dox fluorescence. In addition, ratiometric spectral imaging of the same tumor tissue enables quantitative discrimination of HerDox fluorescence from autofluorescence in situ. In contrast to standard approaches of preclinical assessment, this new method provides multiple/complementary information that may shorten the time required for initial evaluation of in vivo efficacy, thus potentially reducing the time and cost for translating new drug molecules into the clinic.     

FTIR spectro-imaging of collagens for characterization and grading of gliomas

Collagens are a family of at least 30 protein types organized as networks. They constitute the main support material of cells under the form of extracellular matrix as well as for membranes in vessels, organs, and tissue compartments. Collagen network abnormalities are at the origin of many diseases, including myopathies and fibroses. The characterization of collagens remains an analytical challenge due to the insolubility of these molecules and the difficulty encountered in isolating given types without altering their structure or in maintaining network organization, which is critical to diagnosing related pathologies. We have proposed using a vibrational spectroscopy based imaging technique, namely Fourier-transform infrared (FTIR) imaging, for a spatially-resolved analysis of secondary structure of different collagen types in complex samples, and more specifically for characterizing gliomas. With newly developed spectral data treatments and chemometrics using secondary structure parameters of collagen proteins, FTIR imaging is now able to distinguish between several types. On this basis, gliomas have been investigated as specific collagen-rich tissues developing in a non-collagenous environment, providing high specificity to this FTIR imaging utilization. Here, we review the recent advances in this imaging approach for understanding glioma development, with FTIR imaging now being proposed as a molecular histopathology tool for clinicians.     

Adaptive filtering for enhancement of the osteocyte cell network in 3D microtomography images

The osteocyte cell network in bone tissue is thought to orchestrate tissue adaptation and remodeling, thus holding responsibility for tissue quality. Previously, this structure has been studied mainly in 2D and its architecture and functions are not fully elucidated. The assessment of the osteocyte system is prerequisite for deeper understanding of bone remodeling and for advances in management of bone diseases. Our goal is to enable 3D isotropic imaging of bone at cellular level and to develop algorithms for quantitative image analysis of the cell network. We recently demonstrated accurate 3D imaging of this cell structure with synchrotron radiation tomography at submicrometric scale. Due to the limited spatial resolution of the imaging system and the constraints in terms of radiation dose, the images suffer from low signal to noise ratio and the detection of the cell dendrites is challenging. Here we detail a method for enhancement of the osteocyte network in human bone from 3D microtomography images. The approach combines Hessian-based 3D line enhancement and bilateral filtering. Our method enables extraction of the interconnected cells from noisy images, preserving the integrity of the cells and of their slender dendrites. Qualitative and quantitative results are presented.     

Diffusion MRI: a new strategy for assessment of cancer therapeutic efficacy

The use of anatomical imaging in clinical oncology practice traditionally relies on comparison of patient scans acquired before and following completion of therapeutic intervention. Therapeutic success is typically determined from inspection of gross anatomical images to assess changes in tumor size. Imaging could provide significant additional insight into therapeutic impact if a specific parameter or combination of parameters could be identified which reflect tissue changes at the cellular or physiologic level. This would provide an early indicator or treatment response/outcome in an individual patient before completion of therapy. Moreover, response of a tumor to therapeutic intervention may be heterogeneous. The use of imaging could assist in delineating therapeutic-induced spatial heterogeneity within a tumor mass by providing information related to specific regions that are resistant or responsive to treatment. Largely untapped potential resides in exploratory methods such as diffusion MRI, which is a nonvolumetric intravoxel measure of tumor response based upon water molecular mobility. Alterations in water mobility reflect changes in tissue structure at the cellular level. While the clinical utility of diffusion MRI for oncologic practice is still under active investigation, this overview on the use of diffusion MRI for the evaluation of brain tumors will serve to introduce how this approach may be applied in the future for the management of patients with solid tumors.     

Photoacoustic imaging in cancer detection, diagnosis, and treatment guidance

Imaging modalities play an important role in the clinical management of cancer, including screening, diagnosis, treatment planning and therapy monitoring. Owing to increased research efforts during the past two decades, photoacoustic imaging (a non-ionizing, noninvasive technique capable of visualizing optical absorption properties of tissue at reasonable depth, with the spatial resolution of ultrasound) has emerged. Ultrasound-guided photoacoustics is noted for its ability to provide in vivo morphological and functional information about the tumor within the surrounding tissue. With the recent advent of targeted contrast agents, photoacoustics is now also capable of in vivo molecular imaging, thus facilitating further molecular and cellular characterization of cancer. This review examines the role of photoacoustics and photoacoustic-augmented imaging techniques in comprehensive cancer detection, diagnosis and treatment guidance.     

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging,Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.For this purpose, we injected 10⁵ MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg¹. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula¹. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified^2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions^5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles⁷.In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.     

Molecular MALDI imaging: an emerging technology for neuroscience studies

Mass spectrometry (MS) has become an essential tool for the detection, identification, and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of the nervous system. Generally, the application of these powerful techniques requires the destruction of the specimen under study, but recent technological advances have made it possible to apply the matrix-assisted laser desorption/ionization (MALDI) MS technique directly to tissue sections. The major advantage of direct MALDI analysis is that it enables the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps, which have the potential for introducing artifacts. With automation and the ability to display complex spectral data using imaging software, it is now possible to create multiple 2D maps of selected biomolecules in register with tissue sections, a method now known as MALDI Imaging, or MSI (for Mass Spectrometry Imaging). This creates, for example, an opportunity to correlate functional states, determined a priori with live recording or imaging, with the corresponding molecular maps obtained at the time the tissue is frozen and analyzed with MSI. We review the increasing application of MALDI Imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both vertebrates and invertebrates, focusing in particular on recent studies of neurodegenerative diseases and early efforts to implement assays of neuronal development.     

3-D Visualization and Quantitation of Microvessels in Transparent Human Colorectal Carcinoma

Microscopic analysis of tumor vasculature plays an important role in understanding the progression and malignancy of colorectal carcinoma. However, due to the geometry of blood vessels and their connections, standard microtome-based histology is limited in providing the spatial information of the vascular network with a 3-dimensional (3-D) continuum. To facilitate 3-D tissue analysis, we prepared transparent human colorectal biopsies by optical clearing for in-depth confocal microscopy with CD34 immunohistochemistry. Full-depth colons were obtained from colectomies performed for colorectal carcinoma. Specimens were prepared away from (control) and at the tumor site. Taking advantage of the transparent specimens, we acquired anatomic information up to 200 μm in depth for qualitative and quantitative analyses of the vasculature. Examples are given to illustrate: (1) the association between the tumor microstructure and vasculature in space, including the perivascular cuffs of tumor outgrowth, and (2) the difference between the 2-D and 3-D quantitation of microvessels. We also demonstrate that the optically cleared mucosa can be retrieved after 3-D microscopy to perform the standard microtome-based histology (H&E staining and immunohistochemistry) for systematic integration of the two tissue imaging methods. Overall, we established a new tumor histological approach to integrate 3-D imaging, illustration, and quantitation of human colonic microvessels in normal and cancerous specimens. This approach has significant promise to work with the standard histology to better characterize the tumor microenvironment in colorectal carcinoma.     

A realistic utilization of nanotechnology in molecular imaging and targeted radiotherapy of solid tumors

Precise dose delivery to malignant tissue in radiotherapy is of paramount importance for treatment efficacy while minimizing morbidity of surrounding normal tissues. Current conventional imaging techniques, such as magnetic resonance imaging (MRI) and computerized tomography (CT), are used to define the three-dimensional shape and volume of the tumor for radiation therapy. In many cases, these radiographic imaging (RI) techniques are ambiguous or provide limited information with regard to tumor margins and histopathology. Molecular imaging (MI) modalities, such as positron emission tomography (PET) and single photon-emission computed-tomography (SPECT) that can characterize tumor tissue, are rapidly becoming routine in radiation therapy. However, their inherent low spatial resolution impedes tumor delineation for the purposes of radiation treatment planning. This review will focus on applications of nanotechnology to synergize imaging modalities in order to accurately highlight, as well as subsequently target, tumor cells. Furthermore, using such nano-agents for imaging, simultaneous coupling of novel therapeutics including radiosensitizers can be delivered specifically to the tumor to maximize tumor cell killing while sparing normal tissue.     

A Novel In Vivo Vascular Imaging Approach for Hierarchical Quantification of Vasculature Using Contrast Enhanced Micro-Computed Tomography

The vasculature of body tissues is continuously subject to remodeling processes originating at the micro-vascular level. The formation of new blood vessels (angiogenesis) is essential for a number of physiological and pathophysiological processes such as tissue regeneration, tumor development and the integration of artificial tissues. There are currently no time-lapsed in vivo imaging techniques providing information on the vascular network at the capillary level in a non-destructive, three-dimensional and high-resolution fashion. This paper presents a novel imaging framework based on contrast enhanced micro-computed tomography (micro-CT) for hierarchical in vivo quantification of blood vessels in mice, ranging from largest to smallest structures. The framework combines for the first time a standard morphometric approach with densitometric analysis. Validation tests showed that the method is precise and robust. Furthermore, the framework is sensitive in detecting different perfusion levels after the implementation of a murine ischemia-reperfusion model. Correlation with both histological data and micro-CT analysis of vascular corrosion casts confirmed accuracy of the method. The newly developed time-lapsed imaging approach shows high potential for in vivo monitoring of a number of different physiological and pathological conditions in angiogenesis and vascular development.     

Nuclear morphology measurements with angle-resolved low coherence interferometry for application to cell biology and early cancer detection

The study of intact, living cells using non-invasive optical spectroscopic methods offers the opportunity to assess cellular structure and organization in a way that is not possible with commonly used cell biology imaging techniques. We have developed a novel spectroscopic technique for diagnosing disease at the cellular level based on using low-coherence interferometry (LCI) to detect the angular distribution of scattered light. Angle-resolved LCI (a/LCI) combines the ability of LCI to isolate scattering from sub-surface tissue layers with the ability of light scattering spectroscopy to obtain structural information on sub-wavelength scales. In application to examining cellular structure, a/LCI enables quantitative measurements of changes in the size and texture of cell nuclei. These quantitative measurements are characteristic of different pathological states. The capabilities of a/LCI were demonstrated using a clinical system that can be applied in endoscopic surveillance of esophageal tissue, producing high sensitivity and specificity for detecting dysplastic tissues in vivo. Experiments with in vitro cell samples also show the utility of a/LCI in observing structural changes due to environmental stimuli as well as detecting apoptosis due to chemotherapeutic agents.     

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally‐ and temporally‐resolved fluorescence under one‐ and two‐photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically‐derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi‐quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally‐resolved fluorescence lifetime parameters, two‐photon fluorescence imaging and second harmonic generation imaging.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Promise of new imaging technologies for assessing ovarian function

Advancements in imaging technologies over the last two decades have ushered a quiet revolution in research approaches to the study of ovarian structure and function. The most significant changes in our understanding of the ovary have resulted from the use of ultrasonography which has enabled sequential analyses in live animals. Computer-assisted image analysis and mathematical modeling of the dynamic changes within the ovary has permitted exciting new avenues of research with readily quantifiable endpoints. Spectral, color-flow and power Doppler imaging now facilitate physiologic interpretations of vascular dynamics over time. Similarly, magnetic resonance imaging (MRI) is emerging as a research tool in ovarian imaging. New technologies, such as three-dimensional ultrasonography and MRI, ultrasound-based biomicroscopy and synchrotron-based techniques each have the potential to enhance our real-time picture of ovarian function to the near-cellular level. Collectively, information available in ultrasonography, MRI, computer-assisted image analysis and mathematical modeling heralds a new era in our understanding of the basic processes of female and male reproduction.