Unstained Samples Research Articles

G66(P) To Determine the Accuracy of Phase Contrast Microscopy in Predicting Urine Culture Results

AimsTo determine the accuracy of phase contrast microscopy in predicting urine culture results.Design and methodsA prospective study comparing the results of phase contrast microscopy interpretation of urine samples with that...

Use of touch imprint cytology as a simple method to enrich tumor cells for molecular analysis

A relative excess of nonneoplastic cells in frozen carcinoma samples is often a cause of false-negative results in molecular assays. Given the greater cohesiveness of epithelial tumor cells compared with nonneoplastic epithelium and mesenchymal stroma, the authors hypothesized that tumor procurement by touch imprinting would provide a simple, cost-effective method of obtaining enriched neoplastic cells compared with frozen whole-tumor samples. Eleven adenocarcinomas with known KRAS gene mutations were tested. Two sets of 8 touch imprint (TP) slides and 1 frozen whole-tumor sample (FS), both with a corresponding hematoxylin and eosin-stained slide, were obtained from each tumor. DNA from unstained TP and FS samples was tested for KRAS exon 2 mutations by Sanger sequencing. The percentage of carcinoma cells was determined by light microscopy of hematoxylin and eosin-stained slides. The fold increase in the mutant-enriched DNA in TP versus FS samples was determined by calculating the height ratio between the mutant and wild-type peaks on the sequencing electropherogram. Using light microscopy, TP demonstrated a 1.1-fold to 3.5-fold (mean, 1.8-fold) enrichment in neoplastic cells compared with the FS. The mutant-to-wild-type peak height ratio was 1.4-fold to 7.1-fold (mean, 3.1-fold) higher in TP compared with the corresponding FS samples. The average amount of extracted DNA ranged from 145 ng to 7.9 μg per TP slide. The procurement of carcinoma samples by TP is rapid, simple, and inexpensive; consistently provides a tumor-enriched sample; is an excellent source of high-quality tumor DNA; and could compensate for the relatively low sensitivity of direct sequencing. Cancer (Cancer Cytopathol) 2013;121:354-360. © 2013 American Cancer Society.

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

BackgroundIn standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals. MethodsSingle-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations. ResultsWe show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images. ConclusionOur results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage. General significanceThis technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.

Enhancement in statistical and image analysis forin situµSXRF studies of elemental distribution and co-localization, usingDioscorea balcanica

Synchrotron-based X-ray microfluorescence (µSXRF) is an analytical method suitable for in situ investigation of the distribution of micronutrient and macronutrient elements in several-micrometres-thick unstained biological samples, e.g. single cells and tissues. Elements are mapped and quantified at sub-p.p.m. concentrations. In this study the quantity, distribution and grouping/co-localization of various elements have been identified in straight and twisted internodes of the stems of the monocotyledonous climber D. balcanica Košanin. Three different statistical methods were employed to analyse the macronutrient and micronutrient distributions and co-localization. Macronutrient elements (K, P, Ca, Cl) are distributed homogeneously in both straight and twisted internodes. Micronutrient elements are mostly grouped in the vasculature and in the sclerenchyma cell layer. In addition, co-localization of micronutrient elements is much more prominent in twisted than in straight internodes. These image analyses and statistical methods provided very similar outcomes and could be applied to various types of biological samples imaged byµSXRF.

Chemically specific imaging and in‐situ chemical analysis of articular cartilage with stimulated Raman scattering

Stimulated Raman scattering (SRS) has been applied to unstained samples of articular cartilage enabling the investigation of living cells within fresh tissue. Hyperspectral SRS measurements over the CH vibrational region showed variations in protein and lipid content within the cells, pericellular matrix and interterritorial matrix. Changes in the cells and pericellular matrix were investigated as a function of depth into the cartilage. Lipid was detected in the pericellular matrix of superficial zone chondrocytes. The spectral profile of lipid droplets within the chondrocytes indicated that they contained predominantly unsaturated lipids. The mineral content has been imaged by using the PO₄³⁻ vibration at 959 cm⁻¹ and the CO₃²⁻ vibration at 1070 cm⁻¹. Both changes in cells and mineralization are known to be important factors in the progression of osteoarthritis. SRS enables these to be visualized in fresh unstained tissue and consequently should benefit osteoarthiritis research.

Elemental bioimaging of haematoxylin and eosin-stained tissues by laser ablation ICP-MS

Haematoxylin and eosin are frequently used histological stains (“H & E-stains”) for the visualization of tissue structures, which is particularly important for the diagnosis of various diseases including cancer. These stains contain aluminum and bromine in their chemical structures, thus providing access for their visualization by means of ICP-MS imaging. Different tissues including appendix, lymph nodes, Fallopian tube and esophageal tumor were investigated. By means of this novel approach, orthogonal data in comparison to optical micrographs, commonly used by the pathologist, were obtained. Hence, different entities of the parallel or serial tissue sections can now be allocated to the manifold set of elemental information acquired by LA-ICP-MS. In surgically removed and H & E-stained human tissues, the images of aluminum and bromine with resolution down to 10 μm were generated. In addition, elemental distribution maps of carbon, aluminum, bromine and platinum in unstained and stained human esophageal tumor sections after Cisplatin therapy were generated, showing a further expanding the capabilities of this technique. As both stained and unstained samples showed a similar platinum distribution, integrity of the platinum distribution after the staining process could be verified. In addition, no significant background of aluminum or bromine in unstained samples was observed, showing the successful reduction of polyatomic, isobaric interferences by utilization of a collision/reaction cell.

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

SummaryWe developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.

Confocal Raman Microscopy. Thomas Dieing, Olaf Hollricher, and Jan Toporski (Eds.)

Confocal Raman Microscopy. Thomas Dieing, Olaf Hollricher, and Jan Toporski (Eds.). Springer, New York, 2011, 289 pages. ISBN 978-3642125218Raman microscopy is the combination of Raman spectroscopy with optical light microscopy and was first presented in the mid-1970s. It combines the advantages of vibrational Raman spectroscopy as a noninvasive technique providing a wealth of chemical information on the properties of molecules and solids with the imaging capabilities and small sample volume requirements of an optical microscope. “Seeing is believing”: Raman microscopy can visualize the inherent chemical inhomogeneity of a sample without the need of external labels, information that cannot be obtained from simply looking at an unstained sample in the standard optical microscope. During the last decade, confocal Raman microscopy has gone through a rapid development with respect to instrumentation and software as well as the diversity of applications.

Phase-gradient microscopy in thick tissue with oblique back-illumination.

Phase contrast techniques, such as differential interference contrast (DIC) microscopy, are widely used to provide morphological images of unstained biological samples. The trans-illumination geometry required for these techniques restricts their application to thin samples. We introduce oblique back-illumination microscopy (OBM), a method of collecting en face phase gradient images of thick scattering samples, enabling near video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

BackgroundNonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.Methodology/Principal FindingsWe obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.Conclusions/SignificanceNLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

Vibrational Spectroscopic Imaging and Multiphoton Microscopy of Spinal Cord Injury

Spinal cord injury triggers a series of complex biochemical alterations of nervous tissue. Up to now, such cellular events could not be studied without conventional tissue staining. The development of optical, label-free imaging techniques could provide powerful monitoring tools with the potential to be applied in vivo. In this work, we assess the ability of vibrational spectroscopy to generate contrast at molecular level between normal and altered regions in a rat model of spinal cord injury. Using tissue sections, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy and spontaneous Raman spectroscopy are able to identify the lesion, the surrounding scar, and unharmed normal tissue, delivering insight into the biochemical events induced by the injury and allowing mapping of tissue degeneration. The FT-IR and Raman spectroscopic imaging provides the basis for fast multimodal nonlinear optical microscopy (coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence, and second harmonic generation). The latter proves to be a fast tool for imaging of the lesion on unstained tissue samples, based on the alteration in lipid content, extracellular matrix composition, and microglia/macrophages distribution pattern. The results establish these technologies in the field of regeneration in central nervous system, with the long-term goal to extend them to intravital use, where fast and nonharmful imaging is required.

Evaluating Different Fixation Protocols for Spectral Cytopathology, Part 2: Cultured Cells

Spectral cytopathology (SCP) is a robust and reproducible diagnostic technique that employs infrared spectroscopy and multivariate statistical methods, such as principal component analysis to interrogate unstained cellular samples and discriminate changes on the biochemical level. In the past decade, SCP has taken considerable strides in its application for disease diagnosis. Cultured cell lines have proven to be useful model systems to provide detailed biological information to this field; however, the effects of sample fixation and storage of cultured cells are still not entirely understood in SCP. Conventional cytopathology utilizes fixation and staining methods that have been established and widely accepted for nearly a century and are focused on maintaining the morphology of a cell. Conversely, SCP practices must implement fixation protocols that preserve the sample's biochemical composition and maintain its spectral integrity so not to introduce spectral changes that may mask variance significant to disease. It is not only necessary to evaluate the effects on fixed exfoliated cells but also fixed cultured cells because although they are similar systems, they exhibit distinct differences. We report efforts to study the effects of fixation methodologies commonly used in traditional cytopathology and SCP including both fixed and unfixed routines applied to cultured HeLa cells, an adherent cervical cancer cell line. Data suggest parallel results to findings in Part 1 of this series for exfoliated cells, where the exposure time in fixative and duration of sample storage via desiccation contribute to minor spectral changes only. The results presented here reinforce observations from Part 1 indicating that changes induced by disease are much greater than changes observed as a result of alternate fixation methodologies. Principal component analysis of HeLa cells fixed via the same conditions and protocols as exfoliated cells (Part 1) yield nearly identical results. More importantly, the overall conclusion is that it is necessary that all samples subjected to comparative analysis should be prepared identically because although changes are minute, they are present.

Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

3PS011 走査電子顕微鏡を用いた非染色生物試料の高コントラスト・低ダメージ観察方法(日本生物物理学会第50回年会(2012年度))

3PS011 走査電子顕微鏡を用いた非染色生物試料の高コントラスト・低ダメージ観察方法(日本生物物理学会第50回年会(2012年度))

Quantitative phase restoration by direct inversion using the optical transfer function

Quantitative phase recovery of phase objects is achieved by a direct inversion using the defocused weak object transfer function. The presented method is noniterative and is based on partially coherent principles. It also takes into account the optical properties of the system and gives the phase of the object directly. The proposed method is especially suitable for application to weak phase objects, such as live and unstained biological samples but, surprisingly, has also been shown to work with comparatively strong phase objects.

Energy-filtered transmission electron microscopy of biological samples on highly transparent carbon nanomembranes

Ultrathin carbon nanomembranes (CNM) comprising crosslinked biphenyl precursors have been tested as support films for energy-filtered transmission electron microscopy (EFTEM) of biological specimens. Due to their high transparency CNM are ideal substrates for electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) of stained and unstained biological samples. Virtually background-free elemental maps of tobacco mosaic virus (TMV) and ferritin have been obtained from samples supported by ∼1nm thin CNM. Furthermore, we have tested conductive carbon nanomembranes (cCNM) comprising nanocrystalline graphene, obtained by thermal treatment of CNM, as supports for cryoEM of ice-embedded biological samples. We imaged ice-embedded TMV on cCNM and compared the results with images of ice-embedded TMV on conventional carbon film (CC), thus analyzing the gain in contrast for TMV on cCNM in a quantitative manner. In addition we have developed a method for the preparation of vitrified specimens, suspended over the holes of a conventional holey carbon film, while backed by ultrathin cCNM.

Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis

Contemporary brain tumor research focuses on two challenges: First, tumor typing and grading by analyzing excised tissue is of utmost importance for choosing a therapy. Second, for prognostication the tumor has to be removed as completely as possible. Nowadays, histopathology of excised tissue using haematoxylin-eosine staining is the gold standard for the definitive diagnosis of surgical pathology specimens. However, it is neither applicable in vivo, nor does it allow for precise tumor typing in those cases when only nonrepresentative specimens are procured. Infrared and Raman spectroscopy allow for very precise cancer analysis due to their molecular specificity, while nonlinear microscopy is a suitable tool for rapid imaging of large tissue sections. Here, unstained samples from the brain of a domestic pig have been investigated by a multimodal nonlinear imaging approach combining coherent anti-Stokes Raman scattering, second harmonic generation, and two photon excited fluorescence microscopy. Furthermore, a brain tumor specimen was additionally analyzed by linear Raman and Fourier transform infrared imaging for a detailed assessment of the tissue types that is required for classification and to validate the multimodal imaging approach. Hence label-free vibrational microspectroscopic imaging is a promising tool for fast and precise in vivo diagnostics of brain tumors.

Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Amyloid β proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by dark-field and transmission electron microscopy in a preparation of seeded amyloid β protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when mass-per-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.

The use of cotton blue stain to improve the efficiency of picking and identifying chironomid head capsules

Cotton blue was added to sediment samples at least 2 h before chironomid head capsules were picked under a binocular microscope and mounted on slides for identification. The use of stain greatly increased the visibility of chironomid head capsules during picking and enhanced the contrast of various parts of the head capsules (pores, ventromental plates, striations on ventromental plates), which could aid identification. In the seven samples studied, there was no significant difference between the percentages of taxa found in stained and unstained samples. The number of taxa were also similar in stained and unstained samples. This method allowed samples to be picked faster.

High-resolution x-ray observation of unstained samples by a newly developedSGXM

Analytical methods with nanometre-scale resolution are indispensable in various scientificfields, including biology, chemistry and nanotechnology. One suitable tool, the soft x-raymicroscope, provides high spatial resolution of unstained specimens. Recently, we developedan x-ray microscope called a scanning electron generation x-ray microscope (SGXM). Thissystem had attached additional x-ray detection equipment to the scanning electronmicroscope (SEM). Here, we describe a new sample stage containing an improved x-rayemission film that enables the acquisition of high-resolution x-ray images of unstainedsamples. The method proposed in this study, which uses the new SGXM, enables us toacquire x-ray images by using an intact, conventional SEM without additional x-raydetection equipment. Moreover, experiments demonstrate that the new SGXM achieves aspatial resolution of 11 nm. Therefore, our system can be easily used for acquiringhigh-resolution images of a variety of samples across a broad range of scientific fields.