How to examine cells and Tissue Flashcards
Definition of tissue
Tissue is a Latin word for WOVEN
What are the 4 broad tissue classifications
- Muscle
- Nerve
- Epithelial
- Connective
(7) things about Epithelial Tissue
- Often on the edge of other tissues and surrounding other tissues
- Sometimes in clusters within other tissues (glands)
- Polarised when on surfaces
- Always have a basement membrane on the basal (lowest surface)
- Often secrete something from apical surfaces
- Held together by strong anchoring proteins
- Communicate through ‘junctions’ at their lateral and basal surfaces
What does Connective tissue consist of
Consist of cells and extracellular proteins/glycoproteins and ‘gels’ - (glycoproteins attract water)
What are the main cells in connective tissue
The main cells are:
* Fibroblasts
* Chondrocytes
* Osteocytes/osteoblasts/osteoclasts
* Stem cells/progenitor cells/bone marrow/blood/adipocytes
What are the main products of connective tissue
The main products are:
* Fibres (many different types)
* ‘Ground substance’
* Wax and gel-like materials
what are the 3 types of muscle tissue?
3 types
* Skeletal
* Cardiac
* Smooth
What is the major function of muscle tissue
to contract!
* Movement (the organism)
* Stability (the organism, organs and tissues)
* Movement of tissue contents
What are the Minor Functions of muscle tissue?
to secrete hormones
* Natriuretic factor(s)
* Myostatin(s)
What do nerve cells consist of
Nerve cells (neurons) and several support cells
What is the size of a nerve cell
- Nerve tissues can be relatively short (μm)
- Can be very long (cm to m)
what is the function of the nerve cell
Main fast communication system in the body
how are nerve cells formed
- Cells congregate into nerve fibres
- Fibres congregate into ‘nerves’ that can be dissected and visualised ‘by eye’
what is the standard measurement
the standard measurement for a cell is the micron (μm) - Measurement graticule used for sizing
what is the definition of limit of resolution
Definition: The smallest distance by which two objects can be separated and still be distinguishable as two separate objects.
What are the methods of tissue procurement (7)
- Endometrial Scratching technique
- Pipelle
- Hysterectomy
- Venepuncture
- Blood Smear
- bone marrow
- DNA testing
What are the methods of tissue procurement (7)
- Endometrial Scratching technique
- Pipelle
- Hysterectomy
- Venepuncture
- Blood Smear
- bone marrow
- DNA testing
Fixation STEPS
- Formalin Solution (10% buffered solution)
- Formaldehyde ( 37-40% ) - 100ml
- Distilled Water - 900ml
- NaH2PO4 - 4.0g
- Na2HPO4 (anhydrous) - 6.5g
- Mix to dissolve
- After a day or two needs to be dehydrated and water and wax dont mix
Paraffin Wax Embedding (After fixation) STEPS
- Dehydrated in different concentrations
of alcohols - Immersed in dissolved paraffin wax
(hot) overnight - Tissue orientated in a mould and more
wax added - Allowed to solidify
- Gently eased out of mould
What do you do after Paraffin Wax Embedding
Using a microtone get a thin slice of tissue
What does Haematoxylin stain and what colour
ACIDS ( RNA/ DNA/ Nucleic Acids)
Haematoxylin stains the nucleus blue most strongly
What does Eosin stain and what colour
ALKALIS
Eosin stains the cytoplasm and extracellular matrix pink most strongly
What do H and E staining do
The nuclei are clearer and there is more detail in the cytoplasm
Two other staining methods other that H and E
Masson’s trichrome
* red keratin and muscle fibres,
* blue or green collagen and bone,
light red or pink cytoplasm
* dark brown to black cell nuclei
Periodic Acid-Schiff stain
* Identifies anything with sugar attached - glycocalyx
( anything with sugar - Glyco….)
How does immunohistochemistry work
- Tissues and cells present different proteins (antigens) on surface
- Primary antibody binds to the antigen of interest
- Secondary antibody detects primary antibody
- Enzyme that is attached to the secondary antibody takes dye and converts it to a coloured product
- coloured product stays with the enzyme at the desired area
how does immunoflourence work
- primary antibody attached to the protein on the tissue surface
- a florescent tag is attached to antibody
- light hits the tag forming a signal
Frozen Section preparation steps
- Surgical specimen on a metal disc frozen rapidly to –20 to –30°C - ‘Rock hard’
- The frozen specimen cut with microtome in cryostat in a freezer.
- Stained with hematoxylin and eosin.
- Sample preparation more rapid than with traditional histology technique
Paraffin wax formalin-fixed vs. Frozen section (5)
Paraffin wax formalin-fixed
- fixed tissue
- 24-48 hours to prepare - longer
- permenant (lasts longer)
- better clarity
- Can detect pathological diagnosis
Frozen Section
- Fresh Tissue
- 10 - 20 min - shorter
- Months (Last LESS long)
- Less Clear
- Intraoperative Consulation ( eg check for cancer tissue during operation)
Summary for preparation for histology (4)
- Tissue Procurement
- Tissue Processing:
- Fixation & Preservation of tissue (with formalin to prevent putrefaction) OR * Frozen Section
- Make sure that sample is very thin/transparent (2 to 20 μm) & small enough to fit the equipment
- Embedding tissue (with paraffin wax) * Microtome
- Tissue Staining (if applicable)
- H&E and other staining methods
Tissue preparation for Light Microscope
Light Microscope
* Fix with Formalin
* Embed in paraffin wax
* Stain (e.g H&E/methylene blue)
Tissue preparation for electron microscope
Electron Microscope
* Fix with Glutaraldehyde
* Embed in Epoxy resin
* Stain (e.g. Osmium tetroxide)
Comparison of light and electron microscope (6)
LIGHT microscope
- Can view in natural colour
- Larger field of view (2mm) diameter
- Cheap and easy preparation
- Can view living/ moving objects
- magnification x600
- Resolution - 0.25uM
Electron Microscope
- Only monochrome images
- Limited field of view ( 100uM) diameter
- Difficult and expensive
- Can only view dead or inert cells
- Magnification - x500,000
- Resolution - 0.25 nm
Scanning Electron Microscope preparation
Fix with Glutaraldehyde
Embed in Epoxy resin
Stain (e.g. Osmium tetroxide)
can be viewed from an eyepiece directly
Transmission electron microscope
Fix with Glutaraldehyde
Embed in Epoxy resin
Stain (e.g. Osmium tetroxide)
Use microtome with diamond knives
the image goes to a collector
How does a confocal microscope work
- Laser excites a fluorescent dye and electrons are raised to higher energy level
- As the electron relaxes back to its ground state (lower energy level), a light with higher wavelength emitted
- Emitted light is sent through mirrors and a pinhole screen to a CMOS detector (like the one in your mobile phone’s camera)
three benefits of the confocal microscope
- Because only one part of light (that bit that’s in focus) reaches the detector, the images are very sharp
- Motorisation allows full section scanning
- Means the entire depth of the cell/tissue can be examined
what is the preparation of live cells steps
- ‘Cutting’ and ‘dicing’
- Collagenase and DNAse
- Centrifugation steps on basis of cell density
- Put cells into the appropriate growth medium
- Culture cells
- View under phase contrast microscope
benefit of cell culture for live cells
- Allows manipulation of the cells and experiments to determine cells and thus tissue function
adv and dis adv of cell culture
Advantages:
* Absolute control over the physical environment
* Homogeneity of sample
* Reduced need for animal models
Disadvantages:
* Hard to maintain (skilled worker!)
* Only grow small amount of tissue at high cost * Dedifferentiation
* Instability, aneuploidy
* 3 dimensional architecture is lost
* Influence of other cells/tissues not maintained
what is dark field and how does it work
- Very specialised technique used with living cells
- It works by illuminating the sample with light that will not be collected
by the objective lens and thus will not form part of the image - This produces the classic appearance of a dark, almost black, background with bright objects on it
describe the relationship between milli micro and nanometers
1 millimeter (mm) = 1000 micrometers (μm)
1 micrometer (μm) = 1000 nanometers.
how does tissue processing lead to shrikage or other formation of other artifacts
Tissue shrinkage may occur as the section undergoes alcohol dehydration. therefore might lead to decay
In addition, during the embedding stage, the tissue may be compressed if the paraffin is too warm and undergo extra shrinkage during the cooling process.
once removed from body, Organs tissue and cells no longer have protection of bodies immune system so can be digected by microbes.
advantages of dark field microscope
Its dark background offers a high degree of contrast, making it easy to see samples on difficult backgrounds. This technique is easily accessible since many brightfield lab microscopes can be configured for darkfield illumination.
advantages to fluroscene
they can be studied in situ without the need for toxic and time-consuming staining processes.
why are electron microscopes capable of finer resolution than light microscopes
This is because electons have a smaller wavelength that light thus they can reveal the tiny details
