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body logistics (nirin) > How to examine cells and tissues > Flashcards

How to examine cells and tissues Flashcards

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1
Q

Define what a tissue is

A

Tissue (latin word for woven) a group of similar cells that perform a function.

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2
Q

What are the 4 tissue classification

A

Epithelial
Connective
Muscle
Nerve

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3
Q

Where is epithelial tissue found

A

On the edges or surrounding other tissues, sometimes clustered with glands

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4
Q

When are they polarised

A

When they are on the surfaces.

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5
Q

Structure of epithelial tissue

A

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

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6
Q

How does epithelial tissue communicate

A

Junctions at the lateral basal surface

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7
Q

What does connective tissue consist of

A

Extracellular proteins/ glycoproteins and gels

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8
Q

Main cells in connective tissue

A

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

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9
Q

3 types of muscle tissue

A

Smooth
cardiac
skeletal

(all under neuronal control)

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10
Q

Functions of muscle tissue

A

Movement
Stability
Movement of tissue contents

Secrete hormones:

  • natriuretic factor(produced and stored in the heart)
  • myostatin inhibit muscle cell growth
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11
Q

What is natriuretic hormone

A

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.

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12
Q

Properties of nerve tissue

A

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.

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13
Q

What is the standard measurement of a cell

A

The micron um

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14
Q

What do we use for sizing a cell

A

Graticule

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15
Q

What do enlarged red blood cells indicate

A

vasculitis

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16
Q

Define limit of resolution

A

Minimum distance by which 2 objects can be separated and distinguished as separate objects

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17
Q

Resolving power diagram(try to remember diagram)

A

a) d is reached
b) d is improving
c) d is low

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18
Q

Light microscopes points?

A 
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
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19
Q

Electron microscope points?

A 
monochrome
Limited field of view
Difficult and expensive preparation
Only dead and inert objects can be viewed
Magnification x500,000
Resolution 0.25nm
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20
Q

What does ‘fix’/fixation sample mean

A

Preservation of biological tissues from putrefaction

works by adding cross links to proteins

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21
Q

Preparation for TEM smaple

A

Fix with glutaraldehyde
Embed in epoxy resin
Stain (eg osmium tetroxide)
Use microtome with diamond knife

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22
Q

Preparation for SEM smaple

A

Fix with glutaraldehyde
Embed in epoxy resin
Stain (eg osmium tetroxide)

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23
Q

Preparation for Freeze fracture EM

A

Tissue is frozen to -160 and fractured by hitting with a knife edge which splits plasma membrane allowing interior to be imaged.

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24
Q

Why do we use fixation and preservation

A

prevent putrefaction (breakdown/rotting)

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25
Considerations for microscopes
Fixation/preservation Needs to be thin so its transparent Needs to fit the equipment
26
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
27
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
28
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 ```
29
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 ```
30
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
31
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)
32
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
33
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
34
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.
35
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.
36
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
37
Why are cells in culture useful
Allows for manipulation and experiments on cells/tissues to determine function
38
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
39
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
40
Fixation artefacts
Tissues shrink collagen may swell Tissues may shrink or swell depending on water potential of fixation solution
41
Frozen artefacts
Nucleus swells
42
What are intrinsic proteins
Proteins that freely float within the bilayer
43
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)
44
What is the plasmalemma
Outermost bounding membrane (9nm)
45
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
46
Function of plasma membrane
Intercellular adhesion and recognition Signal transduction Compartmentalisation Selective permeability Transport of materials along and across the cell surface Endocytosis Exocytosis
47
Nucleus, what does it contain
DNA, nucleoproteins. RNA
48
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
49
Do inactive cells have small nuclei
Yes because they are not doing RNA synthesis and contain heterochromatin.
50
Do active cells have large nuclei
Yes because they are doing RNA synthesis and contain euchromatin.
51
Are nuclei present in terminally differentiated cells
No
52
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
53
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
54
RER job
Protein synthesis(thanks to the ribosomes) Proteins associate with RER and are transported of to cell membrane incorporation organelles cell exterior lysosome
55
Ribosome job
Protein synthesis, | Free ribosomes make proteins for the cytosol(liquid component of cytoplasm)
56
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
57
What is the endoplasmic reticulum
Interconnecting set of membranes, vesicles and cisternae(flattened sacs) (might be continuous through out cytoplasm)
58
Golgi apparatus description
Saucer shaped stacks of cisternae
59
Golgi apparatus job
Sort, concentrate, package | modify proteins synthesised by RER
60
Golgi apparatus how it works
Protein enters cis face by transport vesicle and leave by trans face in a secretory vesicle
61
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
62
What are residual bodies
Lysosomes that contain indigestible remnants.
63
peroxisomes biography
oxidises other substances using catalase H2O2
64
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
65
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.
66
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
67
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
68
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 ```

body logistics (nirin) (3 decks)

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