ANAT2241 The Virtual Microscope

From Embryology
ANAT2241 This practical support page content is not part of the virtual science practical class and provides additional information for student self-directed learning purposes. 2018 - Moodle ANAT2241 | Virtual slides

General Objective

An introduction to the use of the virtual microscope.

Specific Objective and Learning Activities

To learn the correct use of virtual microscopy using computers and to employ various virtual histological databases for enhancing learning as well as for revision purposes.


In Histology, you are expected to study the features of histological preparations as virtual images, which were scanned from real stained sections, which were mounted on glass slides and listed in the Learning Activities. In these classes you may encounter structures and terminology not defined in lectures. You will need to read about these structures in your textbooks.

Learning Activity

Read sections in your course manual on General Hints on Staining Procedures and Routine Staining Protocols.

The Virtual Microscope

Introduction

Histological sections, which are slices of tissue usually from 5 - 8µm thick (see Dimensions below), can be examined efficiently as follows:

  1. Use the Slide Catalogue in your course handbook to determine:
    1. The animal from which the section is taken.
    2. The stains used (thereby defining the colours of the major tissue components). Note that stains are not examinable.
  2. Low power sketches or notes made may help you to remember the main histological features of a section, e.g., which major tissue components are present.
  3. Note the 2-D shapes in the section and the major tissue components present and try to determine the approximate 3-D shape of the whole organ from which the section was taken. Is the section cut randomly through the organ? Is there an obvious lumen in the section?

Abbreviations

  • XS - cross section
  • TS - transverse section
  • LS - longitudinal section
  • LM - light microscope or light micrograph
  • EM - electron microscope, or electron micrograph
  • 2-D = 2-dimensional
  • 3-D = 3-dimensional

Dimensions

1mm = 103 micrometres (µm) = 106 nanometres (nm)

A micrometre is often called a "micron" (µm); 1µm = 10-6m

Resolving Powers

  • Unaided eye - approx. 0.1 mm = 100µm
  • Light microscope - approx. 0.1 µm = 100nm
  • Electron microscope - approx. 1 nm

What are Virtual Slides?

High magnification scanned digital images of tissue sections on glass slides acquired using a x40 microscope lens.

Stored in a multi-resolution file format and viewed in a web browser window with software capacity to “click and drag” and “zoom in” on the image.

Simulates the examination of glass slides with a real microscope, but with the added benefits of always having optimal focus and contrast, with orientation maintained and avoiding section-to-section variability.

Advantages

  • Common tissue - allows all students to examine the same scanned tissue section.
  • Annotation - can add your own labelled structures to help with revision.
    • These annotations can be shared with others.
    • Can see demonstrator annotations (if available).
  • Access - can access images from any internet connected computer.

Microscopy Methods

This Lab is an introduction to cell biology methods using microscopy. It includes a brief historic background and relevant modern technological advances. The focus is more on the application of these techniques in cell biology, rather than a comprehensive understanding of the physics and technology underlying the techniques.


"Diffraction inevitably limits the resolution of microscopy to around half the wavelength of light" Ernst Abbe (1873, German physicist)

This rule has recently been bent, not broken.....


Also take the time to look at the Textbook References and some of the Cell Biology Images on this Wiki. Later in the course we will be visiting the Confocal Microscope Facility, so spend some time reading about this technique.


MBoC Figure 9-8. Four types of light microscopy

Figure 9-2. Resolving power


Microscopy Timeline

  • 1665 - Robert Hooke publishes Micrographia, a collection of biological micrographs.
  • 1674 - Anton van Leeuwenhoek improved simple microscope for biological specimens.
  • 1833 - Brown published a microscopic observation of orchids, describing the cell nucleus.
  • 1898 - Golgi first saw and described the Golgi apparatus by staining cells with silver nitrate.
  • 1931 - Ernst Ruska first transmission electron microscope, TEM).
  • 1934 - Frits Zernike describes phase contrast microscopy.
  • 1957 - Marvin Minsky patents principle of confocal imaging.
  • 1953 - Frits Zernike receives the Nobel Prize in Physics for invention of the phase contrast microscope.
  • 1955 - George Nomarski theoretical basis of Differential interference contrast microscopy.
  • 1981 - Gerd Binnig and Heinrich Rohrer develop the Scanning Tunneling Microscope (STM).
  • 1981 - Daniel Axelrod develop Total Internal Reflection Fluorescence Microscope (TIRFM).
  • 1981 - Allen and Inoué perfected video-enhanced-contrast light microscopy.
  • 1986 - Ernst Ruska, Gerd Binnig and Heinrich Rohrer receive the Nobel Prize in Physics for invention of the electron microscope (ER) and scanning tunneling microscope (GB and HR).
  • 2000 - Hell and collaborators develop Stimulated Emission Depletion Microscopy (STED)
  • 2008 - Freudiger and Wei Min develop Stimulated Raman Scattering Microscopy (SRS)

Fixation

Techniques for tissue and cell fixation.

  1. Fresh Frozen - Cryostat Sectioning Movie
  2. Precipitation
  3. Aldehyde Cross-linked


File:Inverted Biological Microscope.jpg
Inverted Biological Microscope

Histology Stains

This table includes many of the common histology stains. Note that depending upon the stain used the same tissue can appear quite different. Check the Slide information panel to see the original histological stain.

Haematoxylin and Eosin

One of the most common staining techniques in pathology and histology. Acronym "H and E" stain. (H&E, HE)

Haematoxylin Eosin
  • Stains nuclei blue to dark-blue.
  • Stains the matrix of hyaline cartilage, myxomatous, and mucoid material pale blue.
  • Stains myelin weakly but is not noticeable if combined with eosin stain.
  • combined with Orange G (H & Or. G.) instead of eosin, specifically stains the granules of acidophilic cells of the adenohypophysis (anterior pituitary).
  • Stains cytoplasm pink to red; red blood cells are also bright red.
  • Common counterstain to hematoxylin.
  • Stain intensity varies with the formula as well as the fixative.
Histology Stains - Common Stains and Their Reactions
Stain
Nucleus
Cytoplasm
Collagen
RBCs
Other
Haematoxylin
blue
-
-
-
mucins - light blue
Eosin
-
pink
pale pink
bright red
colloid - pinkmuscle - red
Iron Haematoxylin
blue/black
-
-
-
Van Gieson
-
brown/yellow
red
yellow
muscle: yellow/browncartilage - pink
Verhoeff's Elastin
black
-
-
-
elastic fibres - black
Tartrazine
-
yellow
yellow
yellow
Silver Impregnation
-
-
grey/brown
-
reticular fibres - black
Methyl Green
dark green
light green
light green
green
Nuclear Fast Red
red
pink
pink
pink
Gomori's Trichrome
purple/red
purple
green
red
keratin - redmuscle - purple/red
Heidenhain's Azan
red
purple/red
deep blue
red
muscle - red
Osmium Tetroxide
-
-
brown
brown
myelin, lipids - black
Alcian Blue
-
-
-
-
mucins, - blue
Periodic acid-Schiff (PAS)
-
-
pink
-
mucins, glycogen, glycocalyx - magenta
Phosphotungstic Acid-Hematoxylin (PTAH)
blue
-
red
blue
muscle bands - blue
Masson's Trichrome
blue/black
red
green/blue
red
cartilage, mucins - blue or green; muscle - red
Luxol Fast Blue
-
-
-
variable
myelin - blue
Aldehyde Fuchsin
-
-
-
-
elastic fibres, mast cells - deep purple
Light Green
-
-
light green
-
Gallocyanin
dark blue
-
-
-
nucleic acids, Nissl granules - dark blue
Romanowsky (e.g. Leishman's)
blue
pink
acidophils - red, basophils - blue, azurophilic - purple
Aldehyde Pararosanilin elastic fibres - purple

Artefacts

  • Chatter artefact - fine parallel lines (Virtual slide - tendon)
  • Tissue cut - often a neat straight edge to tissue (where cut has been made) to allow the tissue to sit flat.
  • Tissue fold - darker well demarcated line with apparent disruption of the tissue structure (Virtual slide - artery)
  • Tissue tearing - irregular "tear" with apparent disruption of the tissue structure. Can be due to something intrinsic to the tissue that is hard.
  • Shrinkage - clear white spaces due to fixatives dehydrating tissues.
  • Uneven staining - Tissue has an "mottled" appearance that does not coincide with any tissue structure.


See also - Chatterjee S. (2014). Artefacts in histopathology. J Oral Maxillofac Pathol , 18, S111-6. PMID: 25364159 DOI.

Support Tutorial

2018 - Please note the Slide software has been updated and some of the original software descriptions below no longer apply.


UNSWTV - Tutorial Using the Virtual Microscope

This 15 minute video tutorial will take you through the organization and basic controls of the Virtual Microscope.


Virtual Slides Computer Requirements

  • Virtual slides requires a Flash Player (Adobe) plug-in to be installed in the browser application and will not function without this player.
  • Flash Player will not function on any Apple iPad (iPad or mini-iPad). It can function on their laptop and desktop computers.
    • Note - all online practical support pages will display on any computer, tablet (including iPad) and mobile phone platform capable of running a web browser.
  • All practical class computers have this player installed.
  • If you are using your own desktop or laptop computer this plug-in may need to be installed before virtual slides will display.
  • Ensure that you are downloading and installing the latest player version and only from the official Adobe website (http://get.adobe.com/flashplayer).
    • Note - you should also be using the latest available browser software.
  • If you install this plug-in yourself you may need to shutdown and restart the browser application before it will function.


Electron Microscopy

EM appearance and structure of: nucleus, nucleolus, nuclear envelope, nuclear pore, mitochondria, endoplasmic reticulum (smooth & rough), ribosomes, Golgi apparatus, lysosomes, microtubules, microfilaments, plasma (cell) membrane, microvilli, stereocilia, cilia, and junctional complexes.

(Stain - Osmium) used as the main staining technique for electron microscopy.

Cilia

Lung epithelium sem11.jpg Lung epithelium cilia em01.jpg
Cilia and microvilli on respiratory epithelium. (scanning EM) Cilia cross-sections showing microtubules. (EM)

Junctional Complexes

Epithelial junctions EM02.jpg

A series of junctional complexes between adjacent epithelial cells.

  • Tight junction - (zonula occludens), located nearest the lumen, extends from arrow 1 to arrow 2. The narrowing of the apparent intercellular "gap" (~90 A) is clearly visible, but the fusion line of the two apposed membranes cannot be clearly distin- guished at this magnification. Note that there is relatively little accumulation of dense cytoplasmic material along this part of the complex.
  • Adherens junction - (zonula adhaerens) intermediate junction extends from arrow 2 to arrow 3. A relatively wide intercellular space (~200 A) is maintained throughout the junction. Extensive condensation of cytoplasmic fibrils occurs as a fine feltwork along either side of the junction. This condensation is continuous with the terminal web (tw) into which the filamentous rootlets (r) of the microvilli penetrate. Plate-like densifications within the cytoplasmic feltwork can be seen along part of the junction, especially along the right side (pi).
  • Desmosome - marked by arrows 4 and 5. This element is characterized by a wide intercellular space (~240 A) bisected by an intermediate line (id). Bundles of cytoplasmic fibrils (fd), coarser (diameter ~80 A) and more distinct than those of the terminal web, converge into dense plates (pd) on each side of the desmo- some. These plates are separated from the inner leaflets of the cell membrane by a zone of low density. Similar fibrils (if) appear throughout the remainder of the field below the terminal web.

Desmosome 02.jpg Desmosome 01.jpg

Desmosome


Course Links

Histology Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | ANAT2241 Support | Histology | Histology Stains | Embryology Glossary
Common Histology Stains  
Histology Stains - Common Stains and Their Reactions
Stain
Nucleus
Cytoplasm
Collagen
RBCs
Other
Haematoxylin
blue
-
-
-
mucins - light blue
Eosin
-
pink
pale pink
bright red
colloid - pinkmuscle - red
Iron Haematoxylin
blue/black
-
-
-
Van Gieson
-
brown/yellow
red
yellow
muscle: yellow/browncartilage - pink
Verhoeff's Elastin
black
-
-
-
elastic fibres - black
Tartrazine
-
yellow
yellow
yellow
Silver Impregnation
-
-
grey/brown
-
reticular fibres - black
Methyl Green
dark green
light green
light green
green
Nuclear Fast Red
red
pink
pink
pink
Gomori's Trichrome
purple/red
purple
green
red
keratin - redmuscle - purple/red
Heidenhain's Azan
red
purple/red
deep blue
red
muscle - red
Osmium Tetroxide
-
-
brown
brown
myelin, lipids - black
Alcian Blue
-
-
-
-
mucins, - blue
Periodic acid-Schiff (PAS)
-
-
pink
-
mucins, glycogen, glycocalyx - magenta
Phosphotungstic Acid-Hematoxylin (PTAH)
blue
-
red
blue
muscle bands - blue
Masson's Trichrome
blue/black
red
green/blue
red
cartilage, mucins - blue or green; muscle - red
Luxol Fast Blue
-
-
-
variable
myelin - blue
Aldehyde Fuchsin
-
-
-
-
elastic fibres, mast cells - deep purple
Light Green
-
-
light green
-
Gallocyanin
dark blue
-
-
-
nucleic acids, Nissl granules - dark blue
Romanowsky (e.g. Leishman's)
blue
pink
acidophils - red, basophils - blue, azurophilic - purple
Aldehyde Pararosanilin elastic fibres - purple
Haematoxylin and Eosin
One of the most common staining techniques in pathology and histology. Acronym "H and E" stain. (H&E, HE).


Haematoxylin
  • Stains nuclei blue to dark-blue.
  • Stains the matrix of hyaline cartilage, myxomatous, and mucoid material pale blue.
  • Stains myelin weakly but is not noticeable if combined with eosin stain.
  • combined with Orange G (H & Or. G.) instead of eosin, specifically stains the granules of acidophilic cells of the adenohypophysis (anterior pituitary).
Eosin
  • Stains cytoplasm pink to red; red blood cells are also bright red.
  • Common counterstain to hematoxylin.
  • Stain intensity varies with the formula as well as the fixative.

Virtual slides

Moodle - ANAT2241 - 2018

Pages require student zpass to access.

The Virtual Microscope | Covering and Lining Epithelia | Glandular Epithelia | Connective Tissue Components | Connective Tissue Types | Bone, Bone Formation and Joints | Blood | Muscle Tissue | Nervous Tissue | Cardiovascular System | Respiratory System | Integumentary System (skin) | Liver, Gallbladder, and Pancreas | Gastro-Intestinal System I | Gastro-Intestinal System II | Lymphatic Tissue and Immune System | Endocrine System | Urinary System | Female Reproductive System | Male Reproductive System | Special Sense Organ: The Eye


Practical support

Pages can be accessed from any internet connected computer.

ANAT2241 Support Links: The Virtual Microscope | Covering and Lining Epithelia | Glandular Epithelia | CT Components | CT Types | Bone, Bone Formation and Joints | Blood | Muscle | Nervous | Cardiovascular | Respiratory | Integumentary | GIT Organs | GIT 1 | GIT 2 | Lymphatic and Immune | Endocrine | Urinary | Female Reproductive | Male Reproductive | Eye | Histology Stains | Histology Drawings | Practicals Health and Safety 2013 | Moodle - 2018


ANAT2241 This practical support page content is not part of the science practical class and provides only background information for student self-directed learning purposes.


Cite this page: Hill, M.A. (2018, December 13) Embryology ANAT2241 The Virtual Microscope. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2241_The_Virtual_Microscope

What Links Here?
© Dr Mark Hill 2018, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G