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

How to examine cells and tissues Flashcards

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
Q

Considerations for microscopes

A

Fixation/preservation
Needs to be thin so its transparent
Needs to fit the equipment

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26
Q

Requirements for samples to be used in light microscopy

A

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

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27
Q

How to procure tissue

A

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

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28
Q

Staining methods

A 
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
Q

Fixation mixture

A 
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
Q

Paraffin wax embedding

A

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
Q

Preparing a frozen section

A

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
Q

Paraffin wax vs Frozen section

A

Pw Fs

Specimen Fixed Fresh

Making time 24-48hr 10-20min

Saving time permanent months

Morphology clarity opacity

Application Pathological intraoperative
diagnosis consultation

33
Q

Immunohistochemistry examples

A

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
Q

How does confocal microscopy work

A

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
Q

Pros of confocal microscopy

A

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
Q

How to culture cells

A

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
Q

Why are cells in culture useful

A

Allows for manipulation and experiments on cells/tissues to determine function

38
Q

Pros and cons of cell cultures

A

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
Q

How does dark field work

A

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
Q

Fixation artefacts

A

Tissues shrink
collagen may swell
Tissues may shrink or swell depending on water potential of fixation solution

41
Q

Frozen artefacts

A

Nucleus swells

42
Q

What are intrinsic proteins

A

Proteins that freely float within the bilayer

43
Q

What are extrinsic proteins

A

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
Q

What is the plasmalemma

A

Outermost bounding membrane (9nm)

45
Q

what is the glycocalyx

A

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
Q

Function of plasma membrane

A

Intercellular adhesion and recognition

Signal transduction

Compartmentalisation

Selective permeability

Transport of materials along and across the cell surface

Endocytosis

Exocytosis

47
Q

Nucleus, what does it contain

A

DNA, nucleoproteins. RNA

48
Q

Nucleus described by a TEM

A

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
Q

Do inactive cells have small nuclei

A

Yes because they are not doing RNA synthesis and contain heterochromatin.

50
Q

Do active cells have large nuclei

A

Yes because they are doing RNA synthesis and contain euchromatin.

51
Q

Are nuclei present in terminally differentiated cells

A

No

52
Q

Nucleolus job

A

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
Q

Nuclear envelope biography

A

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
Q

RER job

A

Protein synthesis(thanks to the ribosomes)
Proteins associate with RER and are transported of to cell membrane incorporation
organelles
cell exterior
lysosome

55
Q

Ribosome job

A

Protein synthesis,

Free ribosomes make proteins for the cytosol(liquid component of cytoplasm)

56
Q

SER biography

A

Cisternae not as flat and less scattered in cytoplasm compared to RER
Lipid biosynthesis(anabolism branch of metabolism)
Intracellular transport eg steroid production

57
Q

What is the endoplasmic reticulum

A

Interconnecting set of membranes, vesicles and cisternae(flattened sacs) (might be continuous through out cytoplasm)

58
Q

Golgi apparatus description

A

Saucer shaped stacks of cisternae

59
Q

Golgi apparatus job

A

Sort, concentrate, package

modify proteins synthesised by RER

60
Q

Golgi apparatus how it works

A

Protein enters cis face by transport vesicle and leave by trans face in a secretory vesicle

61
Q

Lysosomes contain? Where are they made? Something about their membrane?

A

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
Q

What are residual bodies

A

Lysosomes that contain indigestible remnants.

63
Q

peroxisomes biography

A

oxidises other substances using catalase H2O2

64
Q

What is phase contrast microscopy

A

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
Q

Pros of phase contrast

A

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
Q

What is a fluorescence microscope

A

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
Q

Fluorescence microscope advantages

A

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
Q

how much blood in a 70kg human

A 
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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