DOCSLIB.ORG

Detection of Protozoans Using Molecular Techniques in Routine Clinical Practice

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Detection of Protozoans Using Molecular Techniques in Routine Clinical Practice DETECTION OF PROTOZOANS USING MOLECULAR TECHNIQUES IN ROUTINE CLINICAL PRACTICE C Rune Stensvold Department of Microbiology and Infection Control Statens Serum Institut Copenhagen - Denmark ECCMID 2016 in Amsterdam EW003 - Diagnosis of intestinal protozoan infections: the best way to go 2-hour Educational Workshop ESCMIDSaturday, 9 April 2016eLibrary - 08:45 - 10:45 by author FOCUS POINTS What to test for When to test for it How to test for it Current ‘state-of-the-art’ at the Laboratory of Parasitology at Statens Serum Institut and looking ahead Apology in advance for including a lot of text! I was instructed to submit the presentation for handout production, and so I put priority to including clear text messages rather than cryptic bullet points and illustrations that might be misinterpreted. ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS PCR as as a supplementary tool or as a replacement for detecting parasites, including parasites in human stool samples. - Conventional PCR and Sanger sequencing - Real-time PCR (mono, duplex, multiplex) ESCMID eLibrary by author COMPARISON OF PCR AND MICROSCOPY ESCMIDStensvold & Nielsen, eLibrary J Clin Microbiol. 2012 Feb;50(2):540-1 by author COMPARISON OF PCR AND MICROSCOPY ESCMIDVerweij & Lieshout eLibrary, Parasitology. 2011 Oct;138(12):1492-8 by author TYPICAL SET-UP Automated DNA extraction ESCMIDPCR >> eLibraryMicroscopy by author SOME ADVANTAGES OF DIAGNOSTIC PCR 1. Standardisable 2. Automatable 3. Sensitive 4. PathogenicAutomatiseret/apathogenic species 5. TypingDNA (outbreaks ekstraktion) 6. Eukaryotic diversity ESCMIDPCR >> eLibraryMicroscopy by author PCR – ISSUES AND LIMITATIONS Diagnostic PCR may not be the solution to all diagnostic issues - ”Is PCR too sensitive?” • Maybe. But no contamination issues (as opposed to fungi of clinical relevance, meaning that if the parasite is there, then it’s there) - Should/can Ct-value cut-offs be defined for some parasites? - What influences the diagnostic accuracy of PCR • Correct sampling (and how many samples?) • Test material (fresh, frozen, fixed, time until processing, etc.) • DNA extraction (do we get the DNA out of the parasites? Formalin?) • PCR assay (primer suitability, choice of reagents and PCR conditions) • Competing template (faecal DNA) ESCMID• When should results be confirmed by sequencing? eLibrary by author EMPTY GIARDIA CYSTS PCR + IFA + cyst DAPI + cyst PCR - IFA + cyst DAPI - cyst ESCMID eLibraryCourtesy of Dr Marianne Lebbad by author HELMINTHS VS GIARDIA IN TRAVELLERS (DK) 30,500 samples (10 y) – microscopy data - Helminth prevalence: 0.2% (60 positive samples) - Giardia prevalence: 4.0% ESCMID eLibraryUnpublished data by author SITUATION IN THE NETHERLANDS Helminth prevalence: 0.3% Algorithm: - Standard analysis includes real-time PCR for enteric protozoa - Microscopy for helminths and rare parasites only in cases of • Eosinophilia • Increased IgE levels (> 120 U/L) • Urticaria • Recent history of travel to the (sub)tropics • Cases of adoption/immigration Reduced costs and increased sample turnover. ESCMIDCoppenraet et al., Clin eLibraryMirobiol Infect. 2009 Sep;15(9):869-74. by author SITUATION IN THE NETHERLANDS Helminth prevalence: 0.3% Algorithm: - Standard analysis includes real-time PCR for enteric protozoa - Microscopy for helminths and rare parasites only in cases of • Eosinophilia • Increased IgE levels• NL(> 120 lab: U/L) Reduction of • Urticaria • Recent history of travelmicroscopyto the (sub)tropicsby 90% • Cases of adoption/immigration Reduced costs and increased sample turnover. ESCMIDCoppenraet et al., Clin eLibraryMirobiol Infect. 2009 Sep;15(9):869-74. by author R&D AT SSI Laboratory of Parasitology, Statens Serum Institut: - Reference laboratory for the detection of parasites in Denmark - We detect parasites in all sorts of clinical sample materials, including stool samples - We try and do a bit of research into • The influence of the gut fauna on - Human health and disease - Remaining microbiota ESCMID eLibrary by author CURRENT SITUATION AT SSI Laboratory of Parasitology, Statens Serum Institut offers the following DNA-based methods for detection of intestinal parasites: - Ascaris (real-time PCR) - Blastocystis (real-time PCR) - Blastocystis sp. subtype (conv PCR and Sanger sequencing) - Cryptosporidium spp. and Giardia intestinalis (duplex real-time PCR) - Cyclospora cayetanensis (real-time PCR in development) - Dientamoeba fragilis (real-time PCR) - Entamoeba histolytica and E. dispar (duplex real-time PCR) - Enterocytozoon bieneusi and Encephalitozoon spp. (duplex real-time PCR) - Strongyloides stercoralis (conv PCR; real-time PCR in development) - Taenia spp. (conv PCR and Sanger sequencing) ESCMID- Trichuris spp. (real-time PCR) eLibrary by author CURRENT SITUATION AT SSI Overall line of thinking: - We try to put as much ”on offer” as possible - We try to concentrate on developing the best possible services for parasites that are clinically relevant - We also provide diagnostics for some parasites, the clinical significance of which is subject to debate, e.g. Blastocystis and Dientamoeba fragilis, so as to enable ourselves and others to expand ESCMIDthe knowledge on these organisms eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. ESCMID eLibrary by author WHEN TO TEST FOR PARASITES?? In Denmark, protozoal infection is mainly suspected only in cases of persistent diarrhoea or diarrhoea in - Travellers - Immunocompromised patients - Outbreaks In Halland (Halmstad), Sweden, both Cryptosporidium and Giardia are now included on a routine basis in all cases of diarrhea, also in cases of diarrhoea acquired in Sweden in otherwise healthy individuals. ESCMID eLibrary by author INCIDENCE OF CRYPTOSPORIDIUM IN SWEDEN Incidence per 100,000 inhabitants ESCMIDhttp://www.folkhalsomyndigheten.se/amnesomraden/statistik-och -undersokningar/sjukdomsstatistik/cryptosporidiuminfektion/eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. ESCMID eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed by Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. - In 6–12-year-old children, prevalence is 60%–80% (normal ”flora”) - Eradication of the parasite does not lead to symptom resolution ESCMID eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed by Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. However, novel data from a large cohort study in Denmark suggest that D. fragilis is highly associated with abdominal pain and other non- diarrhoeal intestinal symptoms (unpublished observations). Moreover, the ESCMIDGPs keep testing for it (as a separate eLibraryanalysis). by author QUALITY ASSURANCE Local ring trial (Sweden) Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) – Molecular Parasitology module (/Jaap van Hellemond) ESCMID eLibrary by author LOOKING AHEAD What we have: - DNA-based methods targeting specific parasites (i.e., high sensitivity and specificity but narrow range) - Microscopy, which enables detection of most gut parasites, but which is hampered by low sensitivity and specificity What we want: - A method with the broad range of microscopy and the high sensitivity and specificity of PCR. Solution: - Technology, including suitable primers, that enables amplification of the same gene for all eukaryotic organisms, with ILLUMINA-based sequencing of PCR products and analysis of DNA data using bespoke software (BIONmeta) and well-curated databases. ESCMID eLibrary by author BIONMETA – SHORT DESCRIPTION BIONmeta is a k-mer-based software for species annotation, which can use any of the existing relevant databases for annotation. The most commonly applied databases include RDP, Greengenes, SILVA, and UNITE; however, we also use a locally curated database developed at the SSI. We are going to publish some results from our evaluation studies soon,
Load more

Recommended publications
  • Electron Microscopy and the Investigation of New Infectious Diseases
    Review Electron microscopy and the investigation of new infectious diseases Alan Curry@) Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258 INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology.
    [Show full text]
  • Second Harmonic Imaging Microscopy
    170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison.
    [Show full text]
  • Applications of Microscopy in Bacteriology
    Microscopy Research, 2016, 4, 1-9 Published Online January 2016 in SciRes. http://www.scirp.org/journal/mr http://dx.doi.org/10.4236/mr.2016.41001 Applications of Microscopy in Bacteriology Mini Mishra1, Pratima Chauhan2* 1Centre of Environmental Studies, Department of Botany, University of Allahabad, Allahabad, India 2Department of Physics, University of Allahabad, Allahabad, India Received 28 September 2015; accepted 2 January 2016; published 5 January 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Bacteria are smallest primitive, simple, unicellular, prokaryotic and microscopic organisms. But these organisms cannot be studied with naked eyes because of their minute structure. Therefore in search for the information about the structure and composition of bacterial cells, cell biologist used light microscopes with a numerical aperture of 1.4 and using wavelength of 0.4 µm separa- tion. But there are still certain cellular structures that cannot be seen through naked eyes, and for them electron microscope is used. There are certain improved types of light microscope which can be incorporated to improve their resolving power. Hence microscopy is playing a crucial role in the field of bacteriology. Keywords AFM, SEM, TEM, Microscopy, Bacteriology 1. Introduction To get acquainted with the world of bacteria like small organisms, very effective and advanced technique is re- quired. The size of bacteria ranges between 0.5 - 5.0 micrometer in length; the smallest of them are members of mycoplasma which measures 0.3 micrometers [1].
    [Show full text]
  • The Development of High Performance Liquid
    Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 3-23-2010 The evelopmeD nt of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives Megan N. Bottegal Florida International University, [email protected] DOI: 10.25148/etd.FI10041603 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Recommended Citation Bottegal, Megan N., "The eD velopment of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives" (2010). FIU Electronic Theses and Dissertations. 154. https://digitalcommons.fiu.edu/etd/154 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida THE DEVELOPMENT OF OPTIMIZED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SYSTEMS FOR THE ANALYSIS OF IMPROVISED EXPLOSIVES A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by Megan Nicole Bottegal 2010 To: Dean Kenneth Furton College of Arts and Sciences This dissertation, written by Megan Nicole Bottegal, and entitled The Development of Optimized High Performance Liquid Chromatography Systems for the Anlysis of Improvised Explosives, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. ____________________________________ Jose Almirall ____________________________________ John Berry ____________________________________ William Hearn ____________________________________ Fenfei Leng ____________________________________ DeEtta Mills ____________________________________ Bruce McCord, Major Professor Date of Defense: March 23, 2010 The dissertation of Megan Nicole Bottegal is approved.
    [Show full text]
  • Examples of High Performance Liquid Chromatography (HPLC) Application in Marine Ecology Studies in the Northern Adriatic
    Prejeto / Received: 19.6.2009 Sprejeto / Accepted: 17.12.2009 Examples of high performance liquid chromatography (HPLC) application in marine ecology studies in the northern Adriatic Vesna FLANDER-PUTRLE Marine Biology Station, National Institute of Biology, Fornače 41, SI-6330 Piran; e-mail: [email protected] Abstract. Photosynthetic pigments have proved to be useful biomarkers of the abundance, composition and physiological status of the phytoplankton biomass in the marine environment. Using HPLC pigment analysis, we determined phytoplankton community structure in three different marine environments: in the area of a fish farm, in the area of sewage outlets, and in the mucilaginous aggregates. At the reference site we observed seasonal changes with prevalence of fucoxanthin-containing phytoplankton (i.e. diatoms) during winter/spring and autumn. In the fish farm area the concentration of chlorophyll a degradation products was higher, whereas in the locally enriched environment of sewage outlets we observed only small changes in taxonomic composition and phytoplankton biomass. The impact of season is more expressed than the impact of sewage discharge. With the use of HPLC pigment analysis we determined the development of phytoplankton community in different stages of mucilage aggregates. Phytoplankton biomass was composed primarily of diatoms, and as the aggregates aged, diatoms increased in the relative biomass. Our examples have proven the usefulness and suitability of HPLC pigment analysis in marine ecology studies. Key words: pigments, HPLC, phytoplankton, Adriatic Sea, mucilage, fish farm, sewage outlets Izvleček: PRIMERI UPORABE TEKOČINSKE KROMATOGRAFIJE VISOKE LOČLJIVOSTI (HPLC) PRI ŠTUDIJAH MORSKE EKOLOGIJE SEVERNEGA JADRANA – Fotosintezna barvila so se izkazala kot dobri kazalci abundance, sestave in fiziološkega stanja fitoplanktonske biomase v morskem okolju.
    [Show full text]
  • Microscopy, Optical Spectroscopy, and Macroscopic Techniques Series: Methods in Molecular Biology, Vol
    C. Jones, B. Mulloy, A.H. Thomas Microscopy, Optical Spectroscopy, and Macroscopic Techniques Series: Methods in Molecular Biology, Vol. 22 This is the second of three volumes of Methods in Molecular Biology that deal with Physical Methods of Analysis. The first of these, Spectroscopic Methods and Analyses dealt with NMR spec troscopy, mass spectrometry, and metalloprotein techniques, and the third will cover X-ray crystallographic methods. As with the first volume. Microscopy, Optical Spectroscopy, and Macroscopic Techniques is intended to provide a basic understand ing for the biochemist or biologist who needs to collaborate with spe cialists in applying the techniques of modern physical chemistry to biological macromolecules. The methods treated in this book fall into four groups. Part One covers microscopy, which aims to visualize individual molecules or complexes of several molecules. Electron microscopy is the more familiar of these, while scanning tunneling microscopy is a new and rapidly developing tool. Methods for determining the shapes and sizes of molecules in solution are described in Part Two, which includes chapters on X-ray and neutron scattering, 1994, XI, 251 p. light scattering, and ult- centrifugation. Calorimetry, described in Part Three, provides the means to monitor processes involving thermodynamic changes, whether these are A product of Humana Press intramolecular, such as conformational transition, or the interactions between solutes or between a solute and its sol vent. Part Four is concerned with optical and infrared Printed book spectroscopy and describes applications ranging from the measurement of protein concentration by UV absorbance to the analysis of secondary struc ture using circular Hardcover dichroism and Fourier-transform infrared spec troscopy.
    [Show full text]
  • Proximity Scanning Transmission Electron Microscopy/Spectroscopy
    Proximity Scanning Transmission Electron Microscopy/Spectroscopy Ing-Shouh Hwang . Institute of Physics, Academia Sinica, Nankang, Taipei, Taiwan Email: [email protected] Abstract Here a new microscopic method is proposed to image and characterize very thin samples like few-layer materials, organic molecules, and nanostructures with nanometer or sub-nanometer resolution using electron beams of energies lower than 20 eV. The microscopic technique achieves high resolution through the proximity (or near-field) effect, as in scanning tunneling microscopy (STM), while it also allows detection of transmitted electrons for imaging and spectroscopy, as in scanning transmission electron microscopy (STEM). This proximity transmission electron microscopy (PSTEM) does not require any lens to focus the electron beam. It also allows detailed characterization of the interaction of low-energy electron with materials. PSTEM can operate in a way very similar to scanning tunneling microscopy, which provides high-resolution imaging of geometric and electronic structures of the sample surface. In addition, it allows imaging and characterization of the interior structures of the sample based on the detected transmission electron signals. PSTEM comprises a family of spectroscopies that address the transport and scattering of low-energy electrons in materials. Thus rich information can be extracted from the measurements. PSTEM offers a fundamentally new and powerful way to investigate thin materials. New analysis methods of thin materials and new physics may be uncovered by this method. Scanning tunneling microscopy (STM) [1] has been a powerful technique to study topographic and electronic properties of sample surfaces with atomic resolution. Fig. 1a illustrates a schematic of STM. A metal tip is brought to a conductive sample surface to within ~ 1 nm.
    [Show full text]
  • Introduction to Confocal Laser Scanning Microscopy (LEICA)
    Introduction to Confocal Laser Scanning Microscopy (LEICA) This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics. For questions & registration please contact www.zmb.unizh.ch . Confocal Laser Scanning Microscopy xy yz 100 µm xz 100 µm xy yz xz thick specimens at different depth 3D reconstruction Types of confocal microscopes { { { point confocal slit confocal spinning disc confocal (Nipkov) Best resolution and out-of-focus suppression as well as highest multispectral flexibility is achieved only by the classical single point confocal system ! Fundamental Set-up of Fluorescence Microscopes: confocal vs. widefield Confocal Widefield Fluorescence Fluorescence Microscopy Microscopy Photomultiplier LASER detector Detector pinhole aperture CCD Dichroic mirror Fluorescence Light Source Light source Okular pinhole aperture Fluorescence Filter Cube Objectives Sample Plane Z Focus Confocal laser scanning microscope - set up: The system is composed of a a regular florescence microscope and the confocal part, including scan head, laser optics, computer. Comparison: Widefield - Confocal Y X Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer. Only a small volume can be visualized by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling.
    [Show full text]
  • Label-Free Multiphoton Microscopy: Much More Than Fancy Images
    International Journal of Molecular Sciences Review Label-Free Multiphoton Microscopy: Much More than Fancy Images Giulia Borile 1,2,*,†, Deborah Sandrin 2,3,†, Andrea Filippi 2, Kurt I. Anderson 4 and Filippo Romanato 1,2,3 1 Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy; fi[email protected] 2 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy; [email protected] (D.S.); andrea.fi[email protected] (A.F.) 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, 35128 Padua, Italy 4 Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London NW1 1AT, UK; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment.
    [Show full text]
  • Scanning Tunneling Microscopy and Spectroscopy
    WORKSHOP Nanoscience on the Tip Scanning Tunneling Microscopy and Spectroscopy Table of Contents 1. Motivation ......................................................................................................................................... 1 2. Scanning Tunneling Microscopy....................................................................................................... 1 3. Tunneling Spectroscopy .................................................................................................................... 5 4. Layered Structure of HOPG .............................................................................................................. 7 5. References ......................................................................................................................................... 8 Recommended Reading ......................................................................................................................... 8 1. Motivation With the development of quantum mechanics in the early 20th century, mankind’s perception of nature was stretched to a great degree leading to new axioms, and the recognition of the particle-wave dualism. It was found that particles with small masses such electrons could interchangeably be described as waves or as corpuscular objects. With the wave character of matter, particles exhibit a probability of existence at places, where they can classically not exist. One of these phenomena is the tunnel effect, which describes the ability of an electron to tunnel through
    [Show full text]
  • All-Digital Histopathology by Infrared-Optical Hybrid Microscopy
    All-digital histopathology by infrared-optical hybrid microscopy Martin Schnella, Shachi Mittala,b, Kianoush Falahkheirkhaha,c, Anirudh Mittala,b, Kevin Yeha,b, Seth Kenkela,d, Andre Kajdacsy-Ballae, P. Scott Carneyf, and Rohit Bhargavaa,b,c,d,g,h,i,1 aBeckman Institute for Advanced Science and Technology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; bDepartment of Bioengineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; cDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbana– Champaign, Urbana, IL 61801; dDepartment of Mechanical Science and Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; eDepartment of Pathology, University of Illinois at Chicago, Chicago, IL 60612; fThe Institute of Optics, University of Rochester, Rochester, NY 14620; gCancer Center at Illinois, University of Illinois at Urbana–Champaign, Urbana, IL 61801; hDepartment of Electrical and Computer Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and iDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Christian Huck, University of Innsbruck, Innsbruck, Austria, and accepted by Editorial Board Member John A. Rogers December 20, 2019 (received for review July 19, 2019) Optical microscopy for biomedical samples requires expertise in fidelity imaging in both point-scanning (15) and wide-field (16–19) staining to visualize structure and composition. Midinfrared (mid-IR) modalities, IR pixel sizes are ∼100-fold larger than those easily spectroscopic imaging offers label-free molecular recording and achieved in visible microscopy, and data acquisition speed is still virtual staining by probing fundamental vibrational modes of three to four orders of magnitude slower than in visible imaging molecular components.
    [Show full text]
  • Lab 4 Pre-Lab Name___Microbial Diversity & Microscopy Protozoa 1) Using Campbell, Draw a Rough S
    Lab 4 Pre-Lab Name______________________________ Microbial Diversity & Microscopy Protozoa 1) Using Campbell, draw a rough sketch and briefly describe the following organisms: Amoeba Paramecium Vorticella Spirogyra Diatoms 2) Using the information in your text book, draw a phylogenetic tree that includes the following organisms. You do not need to include information about when the last common ancestor existed. Do include the names of the kingdoms and phyla. Please draw it on the back of this sheet. humans fish c-ferns (they are regular ferns) Paramecium Volvox Vorticella 3) Which two organisms from question 2 are the most closely-related? Microbial Diversity & Microscopy Objectives: To become familiar with the proper use of a compound microscope in order to comparatively study and measure cells. To learn how to make a wet mount of a living culture and observe with a microscope. To observe eukaryotic unicellular organisms (protozoa) and their subcellular components. To look at some organisms on the border between plants and animals and to contrast them with organisms of other domains. Introduction: All living things are made up of cells, but as life varies greatly so do the cells that make it up. Prokaryotes (Archaea and Bacteria) are organisms in a group which lack true nuclei and contain few organelles. Eukaryotes (Fungi, Plants and Animals) on the other hand have true nuclei, cytoplasm, and a plasma membrane surrounding their cells and contain a variety of other organelles. They also differ in that some are unicellular organisms and others are multicellular. Whether eukaryotic or prokaryotic, a general term for any life form needing magnification in order to be seen is “microbe” and many (but not all) of these are single-celled organisms rather than multicellular.
    [Show full text]