A comparative Raman and CARS imaging study of colon tissue

An experimental evaluation of the information content of two complimentary techniques, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy, is presented. CARS is a nonlinear variant of Raman spectroscopy that enables rapid acquisition of images within seconds in combination with laser scanning microscopes. CARS images were recorded from thin colon tissue sections at 2850, 1660, 1450 and 1000 cm^–1 and compared with Raman images. Raman images were obtained from univariate and multivariate (k‐means clustering) methods, whereas all CARS images represent univariate results. Variances within tissue sections could be visualized in chemical maps of CARS and Raman images. However, identification of tissue types and characterization of variances between different tissue sections were only possible by analysis of cluster mean spectra, obtained from k‐means cluster analysis. This first comparison establishes the foundation for further development of the CARS technology to assess tissue. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

A preliminary clinical trial using state‐of‐the‐art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three‐dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub‐cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm³ at a frame rate of 0.14 Hz (512 × 512 pixels). State‐of‐the‐art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ～8 μm and ～20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm³ at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre‐screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub‐cellular level information of the respective regions (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The use of Fourier‐transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.)

In the present study, Fourier‐transform infrared spectroscopy (FTIR) is investigated as a method to measure connective tissue components that are important for the quality of Atlantic cod filets (Gadus morhua L.). The Atlantic cod used in this study originated from a feeding trial, which found that fish fed a high starch diet contained relative more collagen type I, while fish fed a low starch (LS) diet contained relative more glycosaminoglycans (GAGs) in the connective tissue. FTIR spectra of pure commercial collagen type I and GAGs were acquired to identify spectral markers and compare them with FTIR spectra and images from connective tissue. Using principal component analysis, high and LS diets were separated due to collagen type I in the spectral region 1800 to 800 cm^?1. The spatial distribution of collagen type I and GAGs were further investigated by FTIR imaging in combination with immunohistochemistry. Pixel‐wise correlation images were calculated between preprocessed connective tissue images and preprocessed pure components spectra of collagen type I and GAGs, respectively. For collagen, the FTIR images reveal a collagen distribution that closely resembles the collagen distribution as imaged by immunohistochemistry. For GAGs, the concentration is very low. Still, the FTIR images detect the most GAGs rich regions.      

Comparison of pulsed photothermal radiometry,optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non‐invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three‐dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 μm–1.7 mm and 120–910 μm respectively) but poor axial resolution (75 and 120 μm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14–450 μm) but highest axial resolution (14 μm). The Monte Carlo simulation and three‐dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (<$>><$>400 μm), while OCT is better to measure thin melanoma lesions (<$><<$>400 μm). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Development and first in‐human use of a Raman spectroscopy guidance system integrated with a brain biopsy needle

Navigation‐guided brain biopsies are the standard of care for diagnosis of several brain pathologies. However, imprecise targeting and tissue heterogeneity often hinder obtaining high‐quality tissue samples, resulting in poor diagnostic yield. We report the development and first clinical testing of a navigation‐guided fiberoptic Raman probe that allows surgeons to interrogate brain tissue in situ at the tip of the biopsy needle prior to tissue removal. The 900 μm diameter probe can detect high spectral quality Raman signals in both the fingerprint and high wavenumber spectral regions with minimal disruption to the neurosurgical workflow. The probe was tested in three brain tumor patients, and the acquired spectra in both normal brain and tumor tissue demonstrated the expected spectral features, indicating the quality of the data. As a proof‐of‐concept, we also demonstrate the consistency of the acquired Raman signal with different systems and experimental settings. Additional clinical development is planned to further evaluate the performance of the system and develop a statistical model for real‐time tissue classification during the biopsy procedure.      

Imaging of plant cell walls by confocal Raman microscopy

Raman imaging of plant cell walls represents a nondestructive technique that can provide insights into chemical composition in context with structure at the micrometer level (<0.5 μm). The major steps of the experimental procedure are described: sample preparation (embedding and microcutting), setting the mapping parameters, and finally the calculation of chemical images on the basis of the acquired Raman spectra. Every Raman image is based on thousands of spectra, each being a spatially resolved molecular 'fingerprint' of the cell wall. Multiple components are analyzed within the native cell walls, and insights into polymer composition as well as the orientation of the cellulose microfibrils can be gained. The most labor-intensive step of this process is often the sample preparation, as the imaging approach requires a flat surface of the plant tissue with intact cell walls. After finishing the map (acquisition time is ～10 min to 10 h, depending on the size of the region of interest and scanning parameters), many possibilities exist for the analysis of spectral data and image generation.     

A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture

The role of microcalcifications (μCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44), using a very limited tissue sample, that μCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-μm resolution and undecalcified histology with special emphasis on calcified particles <50 μm in diameter. Our results reveal the presence of thousands of μCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 μCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these μCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the μCalc and surrounding tissue, μCalc size, and clustering. We found that local tissue stress could be increased fivefold when μCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 μm) if no μCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous μCalcs < 15 μm in the caps, not visible at 6.7-μm resolution, and that our failure to find any nonruptured caps between 30 and 66 μm is a strong indication that many of these caps contained μCalcs.     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

One- and Two-Dimensional Electron Paramagnetic Resonance Imaging in Skin

EPR imaging with modulated field gradients provides the possibility for obtaining an EPR spectrum in a selected volume We demonstrate the feasibility of X-band (9.5GHz) electron paramagnetic resonance (EPR) imaging in skin biopsies of hairless mice. One- (ID) and two-dimensional (2D) EPR images of the persistent free radical di-tertiary-butyl-nitroxide are measured. At a microwave frequency of 9.5 GHz (X-band), 2D images are obtained in skin biopsies with an actual point distinction resolution of 25 μm. In a biological model system. 2D images are measured at L-band frequency (2.0 GHz) with a pixel resolution of 61 μm. and a theoretical spatial resolution of 12.5 μm. In combination with the spin labeling and spin trapping technique. EPR imaging is the most direct approach to analyzing spatial distribution of physico-chemical properties in skin, such as membrane fluidity and polarity. as well as detection of free radicals.     

Ex vivo three-dimensional elemental imaging of mouse brain tissue block by laser-induced breakdown spectroscopy

Measurement and reconstruction of an elemental image of large brain tissue will be beneficial to the diagnosis of neurological brain diseases. Herein, laser-induced breakdown spectroscopy (LIBS) is introduced for three dimensional (3D) elemental analysis of paraffin-embedded mouse brain tissue blocks. It is used for the first time towards the mapping of mouse brain block samples. A micro-LIBS prototype is developed for brain elemental imaging and a layer-by-layer approach is used to reconstruct the 3D distribution of Ca, Mg, Na, Cu, and P in the brain tissue. Images are captured with 50 μm lateral resolution and 300 μm depth resolution. The images show that the reclamation area of the cortex surface is enriched with Ca and Mg. In contrast, the Cu distribution is circular and is found primarily in the entirety of the cerebral cortex for the paraffin-embedded brain samples. Elemental imaging results suggest that the highest P intensity is found in the cerebellum nearby the middle sagittal plane in the left-brain paraffin block. These preliminary results indicate that LIBS is a potentially powerful tool for elemental bioimaging of the whole brain and may further improve the understanding of complex brain mechanisms.     

Histological classification of atherosclerotic arteries using high-speed confocal Raman microscopy with machine learning

Confocal Raman microscopy is a useful tool to observe composition and constitution of label-free samples at high spatial resolution. However, accurate characterization of microstructure of tissue and its application in diagnostic imaging are challenging due to weak Raman scattering signal and complex chemical composition of tissue. We have developed a method to improve imaging speed, diffraction efficiency, and spectral resolution of confocal Raman microscopy. In addition to the novel imaging technique, the machine learning method enables confocal Raman microscopy to visualize accurate histology of tissue sections. Here, we have demonstrated the performance of the proposed method by measuring histological classification of atherosclerotic arteries and compared the histological confocal Raman images with the conventional staining method. Our new confocal Raman microscopy enables us to comprehend the structure and biochemical composition of tissue and diagnose the buildup of atherosclerotic plaques in the arterial wall without labeling.     

A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy

ABSTRACT

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100? plastic resin. Thin sections, 1?2.5 μm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.     

Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells

Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.     

Multi-scale molecular photoacoustic tomography of gene expression

Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ～1.0 mm and ～0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 μm and an axial resolution of 15 μm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.     

Studies on the Minimum Reproducible Unit of Staphylococcal L-Forms

The minimum size of a reproducible unit of staphylococcal L-forms was determined by filtration and electron microscopic methods. Ultrathin sections of an induced strain of Staphylococcal L-forms (STA-EMT-1) in liquid medium revealed several types of structures, all of which were bound by a single membrane and most of which possessed ribosome-like granules. Many of the small granules were less than 0.3 μm and were attached to the membrane of the large bodies. Using a serial filtration method, it was observed that viable L-forms were still detected in 0.22 μm filtrate, but the viable cell count of L-forms decreased in number with the decrease in pore size of membrane filters. A fractionation technique, using L-forms filtered through a membrane filter with a 0.45 μm pore size, revealed that there were three classes of small bodies but only the first class with ribosome-like granules over approximately 0.2 μm in diameter seems to be able to reproduce.     

Lensfree fluorescent on-chip imaging of transgenic Caenorhabditis elegans over an ultra-wide field-of-view

We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2-8 cm(2) with a spatial resolution of ～10 μm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2-8 cm(2), without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ～10 μm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research.     

Investigation on the potential of Mueller matrix imaging for digital staining

Digital staining based on Mueller matrix measurements and their derivatives was investigated. Mueller matrix imaging was performed at the microscopic level on gastric tissue sections. Full Mueller matrices (4 × 4) were reconstructed using recorded images, followed by the extraction of polarization parameters. The most effective parameters and their combinations were extracted from Mueller matrix elements, principal component scores and polarization parameters respectively to classify samples into three categories – i.e. cancer, dysplasia and intestinal metaplasia/normal glands for various regions of interest sizes. It was observed that two‐step classification yielded higher classification accuracy than the traditional one‐step classification and that pixel classification based on Mueller matrix elements yielded higher accuracy than that based on polarization parameters and derived principal components. Moreover, Mueller matrix images with a lower spatial resolution generated higher classification accuracy but those with a higher spatial resolution revealed more morphological details.ns.

The original stained image (top) and the digital staining image (bottom).     

Visualization of lateral water transport pathways in soybean by a time of flight-secondary ion mass spectrometry cryo-system

Water movement between cells in a plant body is the basic phenomenon of plant solute transport; however, it has not been well documented due to limitations in observational techniques. This paper reports a visualization technique to observe water movement among plant cells in different tissues using a time of flight-secondary ion mass spectrometry (Tof-SIMS) cryo-system. The specific purpose of this study is to examine the route of water supply from xylem to stem tissues. The maximum resolution of Tof-SIMS imaging was 1.8 μm (defined as the three pixel step length), which allowed detection of water movement at the cellular level. Deuterium-labelled water was found in xylem vessels in the stem 2.5 min after the uptake of labelled water by soybean plants. The water moved from the xylem to the phloem, cambium, and cortex tissues within 30-60 min after water absorption. Deuterium ion counts in the phloem complex were slightly higher than those in the cortex and cambium tissue seen in enlarged images of stem cell tissue during high transpiration. However, deuterium ion counts in the phloem were lower than those in the cambium at night with no evaporative demand. These results indicate that the stem tissues do not receive water directly from the xylem, but rather from the phloem, during high evaporative demand. In contrast, xylem water would be directly supplied to the growing sink during the night without evaporative demand.     

Nuclear magnetic resonance microscopy ofAncistrocladus heyneanus

Summary The tropical lianaAncistrocladus heyneanus, which is known for its biologically active naphthylisoquinoline alkaloids, has been studied by nuclear magnetic resonance (NMR) microscopy for the first time. The spatial resolution of the cross-sectional NMR images was of the order of 20 m. Quantitative NMR relaxation time images of the root and the shoot show great contrast between different tissue regions. In addition, we observed the regional distribution of chemical compounds inAncistrocladus heyneanus by chemical-shift NMR microscopy. The NMR imaging results were compared with light and fluorescence microscopic images and reveal the excellent tissue characterization using NMR technology.Abbreviations NMR nuclear magnetic resonance - CSI chemical-shift magnetic resonance imaging - FOV field of view - TE echo time - TR repetition time     

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 μm×50 μm×2.5 μm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.