How to examine cells and tissues Flashcards
Define what a tissue is
Tissue (latin word for woven) a group of similar cells that perform a function.
What are the 4 tissue classification
Epithelial
Connective
Muscle
Nerve
Where is epithelial tissue found
On the edges or surrounding other tissues, sometimes clustered with glands
When are they polarised
When they are on the surfaces.
Structure of epithelial tissue
Top consists of the apical(free) surface (which often secretes stuff),
the bottom of the epithelial cells is the basal layer
Under that is the basement membrane that consists of
basal lamina
reticular lamina
Under that is connective tissue
Epithelial tissue is held together by strong anchoring proteins
How does epithelial tissue communicate
Junctions at the lateral basal surface
What does connective tissue consist of
Extracellular proteins/ glycoproteins and gels
Main cells in connective tissue
Fibroblast-make collagen and also help with healing
Chondrocytes- makes cartilage, also important for endochondral ossification (helps with bone development)
Osteocytes/Osteoblasts/Osteoclasts- bones
Stem cells/progenitor cells/bone marrow/blood/adipocyte
3 types of muscle tissue
Smooth
cardiac
skeletal
(all under neuronal control)
Functions of muscle tissue
Movement
Stability
Movement of tissue contents
Secrete hormones:
- natriuretic factor(produced and stored in the heart)
- myostatin inhibit muscle cell growth
What is natriuretic hormone
natriuretic factor(produced and stored in the heart) acts on the kidney to increase sodium excretion and GFR, to antagonize renal vasoconstriction, and to inhibit renin secretion.
Properties of nerve tissue
Made up of nerve cells
Nerve cells can be very short to very long
Main fast communication system in the body
Cells congregate into nerve fibres
Fibres congregate into nerve that are visible to the naked eye.
What is the standard measurement of a cell
The micron um
What do we use for sizing a cell
Graticule
What do enlarged red blood cells indicate
vasculitis
Define limit of resolution
Minimum distance by which 2 objects can be separated and distinguished as separate objects
Resolving power diagram(try to remember diagram)
a) d is reached
b) d is improving
c) d is low
Light microscopes points?
Can view image in natural colour Large field of view Cheap and easy preparation Can view living and moving objects Magnification x600approx Resolution o.25microns
Electron microscope points?
monochrome Limited field of view Difficult and expensive preparation Only dead and inert objects can be viewed Magnification x500,000 Resolution 0.25nm
What does ‘fix’/fixation sample mean
Preservation of biological tissues from putrefaction
works by adding cross links to proteins
Preparation for TEM smaple
Fix with glutaraldehyde
Embed in epoxy resin
Stain (eg osmium tetroxide)
Use microtome with diamond knife
Preparation for SEM smaple
Fix with glutaraldehyde
Embed in epoxy resin
Stain (eg osmium tetroxide)
Preparation for Freeze fracture EM
Tissue is frozen to -160 and fractured by hitting with a knife edge which splits plasma membrane allowing interior to be imaged.
Why do we use fixation and preservation
prevent putrefaction (breakdown/rotting)
Considerations for microscopes
Fixation/preservation
Needs to be thin so its transparent
Needs to fit the equipment
Requirements for samples to be used in light microscopy
Preserve tissue to avoid putrefaction eg formalin
Embed tissue in substance that allows it to be sliced very thin eg paraffin wax
Stain tissue to make organelles clear eg Haemoxylin and Eosin
How to procure tissue
Endometrial scratching technique:
-Endometrial biopsy-tissue removed form endometrium
for histological evaluation
-endometrium curettage- using a currete to gain
endometrium cells/tissue by scratching or scooping
Venepuncture-blood smear-put drop of blood on slide, pull blood drop with second slide to form a thin layer, stain and add cover slip
Bone marrow aspiration- insert jamshidi needle into illiac crest remove bone marrow
Cheek cell swab
Staining methods
Haematoxylin and eosin- identifies most things in cells/ tissue structures(good for finding tumours) Masson's trichrome -red keratin and muscle fibres -blue or green collagen and bone -red/pink cytoplasm -dark brown/black nuclei Periodic acid-schiff stain -identifies anything with sugar attached- glycocalyx
Fixation mixture
Formalin solution(10% buffered neutral): -Formaldehyde(37-40%) 100ml -distilled water- 900ml -NaH2PO4 4g -Na2HPO4(anhydrous) 6.5g Mix to dissolve Leave to soak for 24-48 hours any more will lead to shrinkage and artefacts
Paraffin wax embedding
After fixation:
-Dehydrate sample in different concentrations of alcohol
-Immerse in hot dissolved paraffin wax for 6 hours
-Orientate tissue in mould and add more wax
-once cooled to room temperature gently remove from mould.
Cons- solvents used to remove paraffin wax also removes lipids
Preparing a frozen section
Freeze specimen on a metal disc rapidly to -20 to -30c
Cut specimen with a microtome in a cryostat freezer
Stain with haematoxylin and eosin
Quicker to prepare (10min vs 6hours)
Paraffin wax vs Frozen section
Pw Fs
Specimen Fixed Fresh
Making time 24-48hr 10-20min
Saving time permanent months
Morphology clarity opacity
Application Pathological intraoperative
diagnosis consultation
Immunohistochemistry examples
Indirect Immunohistochemistry:
- primary antibody binds to complimentary antigen
- labelled secondary antibody binds to primary
- label interacts with an enzyme producing a precipitate
- more sensitive than the other one, can identify organelles
Immunofluorescence:
-labelled(fluorescent tag) primary antibody binds to
complimentary antigen
-Fluorescent tag emits signal when in contact with light
used for prognosis of a disease
How does confocal microscopy work
Laser excites a fluorescent dye and electrons are raised to higher energy levels
When the electron returns to ground state a photon with a higher wavelength is emitted
Photon is sent through mirrors and a pinhole screen to a CMOS detector.
Pros of confocal microscopy
Only 1 photon that is in focus reaches detector produces a sharp image.
Motorisation allows full section scanning, which allows full examination of the cell/tissue
Also allows for 3D images, useful for various eye diseases.
How to culture cells
Harvest cells
Isolate cells using appropriate enzymes eg collagenase and DNAse as well centrifugation on basis of cell density
Apply isolated cells on to appropriate growth medium in a culture dish
Subculture cells to obtain a pure culture/ bypass problems(senescence)
Verify cell culture is the right type of cell
Why are cells in culture useful
Allows for manipulation and experiments on cells/tissues to determine function
Pros and cons of cell cultures
Pros:
- Absolute control over physical environment
- Homogeneity of sample
- Reduce need for animal models
Cons:
- Hard to maintain
- Growing even small quantities cost a lot
- dedifferentiation
- instability, aneuploidy
- 3d architecture is lost
- influence of other cells/tissues not maintained
How does dark field work
Illuminating sample (living cells) with light that will not be collected by objective lens therefore will not be part of the image.
This produces a black background with bright objects( cells) on it.
Good because you can see things with a higher contrast so you can see unstained sample that absorb a lot or transparent to light
Fixation artefacts
Tissues shrink
collagen may swell
Tissues may shrink or swell depending on water potential of fixation solution
Frozen artefacts
Nucleus swells
What are intrinsic proteins
Proteins that freely float within the bilayer
What are extrinsic proteins
Proteins that are loosely associated with the external surface of cells (may have trans membrane domains meaning parts of it are in the cell/organelle)
What is the plasmalemma
Outermost bounding membrane (9nm)
what is the glycocalyx
Glycoproteins and glycolipids projecting outwards from plasma membrane that confer immunogenicity (ability to trigger an immune reaction) to a cell to protect itself from the immune system
Function of plasma membrane
Intercellular adhesion and recognition
Signal transduction
Compartmentalisation
Selective permeability
Transport of materials along and across the cell surface
Endocytosis
Exocytosis
Nucleus, what does it contain
DNA, nucleoproteins. RNA
Nucleus described by a TEM
Heterochromatin(DNA tighly wrapped) electron dense dark blobs. DNA and associated nucleoproteins not active in RNA synthesis
Euchromatin electron-lucent light blobs contain dispersed nuclear material, active in RNA synthesis
Do inactive cells have small nuclei
Yes because they are not doing RNA synthesis and contain heterochromatin.
Do active cells have large nuclei
Yes because they are doing RNA synthesis and contain euchromatin.
Are nuclei present in terminally differentiated cells
No
Nucleolus job
ribosome synthesis(ish, just makes the subunits which are exported out of the nuclear pore for assembly on the rough endoplasmic reticulum)
Also disappear during cell division
Nuclear envelope biography
Double layer membrane around the nucleus, is a specialised endoplasmic reticulum.
Nuclear pores allow macromolecules to be transported, and allow micro molecules to diffuse without hindrance
RER job
Protein synthesis(thanks to the ribosomes)
Proteins associate with RER and are transported of to cell membrane incorporation
organelles
cell exterior
lysosome
Ribosome job
Protein synthesis,
Free ribosomes make proteins for the cytosol(liquid component of cytoplasm)
SER biography
Cisternae not as flat and less scattered in cytoplasm compared to RER
Lipid biosynthesis(anabolism branch of metabolism)
Intracellular transport eg steroid production
What is the endoplasmic reticulum
Interconnecting set of membranes, vesicles and cisternae(flattened sacs) (might be continuous through out cytoplasm)
Golgi apparatus description
Saucer shaped stacks of cisternae
Golgi apparatus job
Sort, concentrate, package
modify proteins synthesised by RER
Golgi apparatus how it works
Protein enters cis face by transport vesicle and leave by trans face in a secretory vesicle
Lysosomes contain? Where are they made? Something about their membrane?
acid hydrolases for digestion products are re-used (carbs, proteins and lipids) waste products excreted
golgi apparatus,
membrane proteins are highly glycosylated for protection from these enzymes
What are residual bodies
Lysosomes that contain indigestible remnants.
peroxisomes biography
oxidises other substances using catalase H2O2
What is phase contrast microscopy
Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image
Pros of phase contrast
living cells can be examined in their natural state without previously being killed, fixed, and stained.
e dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail.
What is a fluorescence microscope
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances
Fluorescence microscope advantages
live-cell observation and the structure elucidation of biomolecules in tissues and cells, allowing them to be studied in situ without the need for toxic and time-consuming staining processes
how much blood in a 70kg human
5L why? 70x0.6=42 42x1/3x1/4=3.5 3.5-0.5(transmembrane space(endothelial cell membranes)) =3L of plasma 3/0.6(%of blood that is placenta)=5L
