2. How To Examine Cells And Tissues Flashcards
(80 cards)
1
Q
What is a tissue?
A
Group of similar cells acting together to perform specific functions.
2
Q
What are the different types of tissue?
A
Connective, epithelial, nervous, muscle.
3
Q
Where is epithelial tissue found?
A
- Often on the edge of other tissues and surround other tissues
- Sometimes in clusters within other tissues (glands)
4
Q
What is the structure of epithelial tissue?
A
They have a basement membrane on the basal level and an apical surface.
They’re made up of lots of epithelial cells joined tightly together.
5
Q
How are epithelial cells held together?
A
They’re held together by strong anchoring proteins
6
Q
How do epithelial cells communicate?
A
They communicate through junction in their lateral and basal surfaces
7
Q
What is connective tissue?
A
A body tissue that provides support for the body and connects all of its parts.
8
Q
What are the components of connective tissue?
A
Consist of cells and extracellular proteins/glycoproteins and ‘gels’
9
Q
What are the main cells in connective tissue?
A
- Fibroblasts
- Chondrocytes
- Osteocytes/osteoblasts/osteoclasts
- Stem cells/progenitor cells/bone marrow/blood/adipocytes
10
Q
What are the main products of connective tissue?
A
- Fibres (many different types)
- ‘Ground substance’
- Wax and gel-like materials
11
Q
What are the structures of nerve cells?
A
- nerves can be micrometers short or cm long
- cells congregate into nerve fibres which congregate into nerves
12
Q
What is the longest nerve?
A
Sciatic nerve from middle of spine to posterior of leg
13
Q
What types of muscle are there?
A
skeletal, cardiac, smooth
14
Q
What types of muscles are involuntary?
A
smooth and cardiac
15
Q
What type of muscle is voluntary?
A
skeletal
16
Q
What does muscle tissue do?
A
Major Function: to contract!
• Movement (the organism)
• Stability (the organism, organs and tissues)
• Movement of tissue contents
Minor Function: to secrete hormones
• Natriuretic factor(s)
• Myostatin(s)
17
Q
How are cells measured? And in what unit?
A
micrometers, using a measurement graticule
18
Q
What is the only type of cell that can be seen with the naked eye?
A
Oocytes
19
Q
Define limit of resolution
A
Smallest distance two objects can be separated and still be distinguishable as two separate objects.
20
Q
How is the limit of resolution calculated?
A
The limit of resolution (m) is calculated by dividing the wavelength (m) by 2 x NA (numerical aperture)
21
Q
What is NA when measuring limit of resolution?
A
Numerical aperture- represents different angles a lens has and its ability to capture light - no dimension
22
Q
Features of a light microscope
A
- transmitted through specimen
- can view images in natural colours
- large field of view (2mm max diameter)
- cheap and easy prep
- living, moving specimens
- magnification x600
- resolution 0.25 micrometers
23
Q
Features of an electron microscope
A
- vacuum
- light bounces off and collected by detector
- only monochrome images can be seen
- limited field of view (100 micrometer diameter)
- difficult and expensive prep - samples are often coated in gold
- dead and inert specimens
- magnification X500 000
- resolution 0.25 nm
24
Q
describe the differences in preparation of TEM abd SEM samples
A
- both are fixed with glutaraldehyde and embedded in epoxy resin
- both are stained (osmium tetroxide)
- TEM: use microtome with diamond knives to slice samples
25
What is the difference between transmission and scanning electron microscopes?
Transmission: electron beam fired through the specimen and then focused by coils to form an image.
Scanning: electrons are reflected from the surface and received by a cathode ray tube. For viewing surface of specimen.
26
Why do electron microscopes have higher resolving power than light microscopes?
Electron microscopes have higher resolving power due to shorter wavelength of electrons.
27
What is freeze fracture EM?
Tissue frozen to -160C and fractured by hitting with a knife edge.
Fracture line passes through plasma membrane exposing its interior which can then be imaged.
28
Why is tissue frozen in the presence of glycerol?
To stop water crystals forming.
| Ice expands and destroys tissue.
29
What is important to remember when using microscopy?
- fixation and preservation to prevent putrefaction (rotting)
- thin samples (2-20 micrometers)
- ability to fit the microscope slide
30
Why does the specimen need to be very thin?
Sample being examined needs to be translucent to allow light to pass through it (less than 20 micrometers thick). Otherwise diffraction of light occurs and the produced image will be blurry.
31
When is preservation of the cells not required?
When the cells are being used in cell culture as a diagnostic frozen section
32
What are the stages of histology procedure?
Fixation, Tissue processing (dehydration, clearing), Embedding, Sectioning, Staining.
33
Why is fixation important?
Preserves biological tissue from decay due to autolysis or putrefaction.
Terminates biochemical reactions, increases tissue mechanical strength and stability.
34
What are the common fixatives for Light and Electron microscopy?
Light: Formalin solution(10% buffered neutral) (this is a 37% aqueous solution of formaldehyde combined with 0.9% sodium chloride solution)
Electron: Glutaraldehyde
35
How can fixation lead to formation of artefacts?
Specimens should only be left in the fixative for 24-48 hours. Any longer results in shrinkage by dehydration and cause fixation artefacts.
36
How do the fixatives work?
Aldehydes are cross-linking fixatives, they act by creating covalent bonds between proteins (primarily in Lysine) in tissue. Cross links proteins by putting methyl groups on to hold/ fix them together
Also anchors soluble proteins to cytoskeleton which lends additional rigidity to the tissue.
37
What occurs during tissue processing?
Dehydration: specimen immersed in increasing concentrations of alcohol to remove water and formalin.
Clearing: organic solvent (e.g. xylene, toluene) used to remove alcohol to allow infiltration with paraffin wax during the embedding stage.
38
Why does the use of Xylene not allow examination of fats and lipids?
Xylene strips lipophilic molecules out of the tissue so neutral fats and lipids are not detectable by this method.
39
Why is embedding important?
The solidified block produced provides a support matrix that allows very thin sectioning.
40
What common embedding agents are used in light and electron microscopy?
Light: Paraffin wax embedding
Electron: Epoxy resin
41
What is the procedure for paraffin wax embedding?
Specimen is immersed in hot dissolved paraffin wax overnight, it will infiltrate into any spaces.
Orientate tissue to desired position and more paraffin wax added.
Allow to cool to room temperature and ease out of mould.
42
How long does a paraffin embedded specimen last?
Permanently.
43
How is tissue sectioned?
Microtome is used to slice extremely thin sections, which can be of preset thickness (usually 5 micrometres).
Placed into warm bath where they float, can be scooped up onto a slide placed below water surface.
Dried upright at 37C to melt excess paraffin.
44
Why are stains important?
Most cells are transparent and almost colourless when unstained.
Staining provides contrast to tissue sections and make structures more visible.
45
What are the common types of stains for light and electron microscopy?
Light: Haematoxylin and Eosin
Electron: osmium tetroxide
46
What does haematoxylin stain?
stains acids blue (nuclei)
47
What does eosin stain?
stains proteins pink ( cytoplasm and extracellular matrix)
48
What does massons tichrome stain?
- Red keratin and muscle fibres
- blue/green collagen and bone
- light red/pink cytoplasm
- dark brown/ black nuclei
49
What does period acid-Schiff stain do?
Identifies anything with sugar attached e.g. glycocalyx
50
Describe common biopsy techniques and give examples of tissues which
can be sampled by each method
In order to obtain the biopsy, several methods can be applied;
(1) surgery and then later dissection by the histopathologist
(2) the use of scraping methods (i.e. curettes, scalpel scrapes)
(3) Sharp needles (i.e. Needle biopsy, pipelle, trephine, punch biopsy)
(4) direct venepucture (i.e. for blood smears and haematological disease assessment).
(5) Bodily fluids can also be examined (i.e. sputum or semen), but these rarely need to be fixed and processed for histology.
51
How is a frozen section obtained?
* 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
52
What does a cryostat do?
Maintains a low temperature
53
Pros and cons of paraffin wax embedding
```
Fixed tissues
Making time 24-48 hrs
Saving time is permanent
Clear morphology
For pathological diseases
```
54
Pros and cons of frozen sectioning
```
Fresh
10-20 mins making time
Saving time of months
Opaque morphology
Used for intraoperative consultation
```
Rapid but not as good quality as traditional techniques
Poor reflection of the stain
55
Why do red blood cells not stain in the middle?
Red blood cells are biconcave discs. They have no protein or DNA/RNA in the middle so light passes straight through the middle and appear white even with stains.
Stains around the outer edge which has proteins.
56
When are frozen sections most commonly used?
The principal use of the frozen section procedure is the examination of tissue while surgery is taking place (allow the use of fresh tissue - does not need to be fixed).
This is because preparation is much more rapid (10 minutes). However, the quality is not so good.
57
What is Immunohistochemistry?
Method of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens.
58
How does Indirect IHC work?
Unlabelled primary antibodies detect antigens. Secondary antibodies labelled with Horseradish Peroxidase (HRPO) detects and binds to primary antibodies.
Diaminobenzidine (DAB) added which produces a coloured product in presence of HRPO, which precipitates over the site.
59
How does Immunofluorescence work?
Fluorescent tag attached to primary antibody which when hit by a lazer allows you to see the presence of the antigens.
Commonly used in conjunction with confocal microscopy.
60
What are the different types of light microscopy?
Compound, Dark field, phase contrast, confocal, fluorescence.
61
How does confocal microscopy work?
Laser excites a fluorescent molecule and electrons in the dye are raised to higher energy level.
As the electrons relax back to ground state a light with higher wavelength is emitted.
Light is reflected through a pinhole screen to a CMOS detector.
Only focused light reaches the detector so images are very sharp.
Allows entire depth of specimen to be examined.
62
What is confocal microscopy?
a light microscope that uses fluorescent stains and laser to make two- and three-dimensional images
63
How are 3D images formed?
Images are taken 1-10 micrometers apart.
Computer lines then up and creates a z-stack.
Resulting image can be examined from all three dimensions.
Commonly used in evaluation of various eye diseases.
64
How are live cells prepared?
- cut/ dice cells to break down extracellular matrix and connective tissue and DNA which is released from broken nuclei as it's too sticky to remain
- collagenase and DNAse enzymes
- centrifugation steps on basis of cell density
- put cells in growth medium to maintain O2, CO2, salt, electrolytes, nutrients, waste, pH, temp, volume and pressure of fluid and cell compartments
- culture cells
- view under phase contrast microscope which is used to see live cells e.g. during cell division
65
How are cells cultured?
- harvest cells
- isolate with enzymes
- apply to growth media in culture dish
- put in cell incubator
(Subculture cells to obtain a pure culture/ bypass some problems like senescence - deterioration with age)
- verify cultured cells are the cells of interests
66
Why are cells cultured? Give an example
Allows manipulation of the cells and experiments to determine the cells and tissue function
E.g. propranolol —> used in treatment of heart disease and hypertension as it induces apoptosis in infantile hemangioma endothelial cells (benign tumour of blood vessels)
67
Advantages of cell culture
- control over physical environment
- homogeneity of sample
- less need for animal models
68
Disadvantages of cell culture
- hard to maintain (infection prone)
- grow small amount of tissue at a high cost
- dedifferentiation of cells into the wrong type due to instability
- aneuploidy - abnormal number of chromosomes in a haploid set
- 3D architecture lost
- influence of other cells/ tissues not maintained
69
What is dark field microscopy used for?
- observing living, unstained organisms; attempts to not disturb the organisms
- can observe transparent samples
- illuminates sample with light that's not collected by the objective lens thus wont form part of the image
70
How is dark field microscopy carried out?
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
• It can also be used in electron microscopy images (with the correct filters)
71
What is the smallest organelle in an H&E stained cell that can be seen under a light microscope?
Nucleolus
72
Why is fat not stained by H&E staining?
Water and fat don't mix so the fat has been left in the solution when it is removed before adding the paraffin wax
73
What is the smallest organelle that can be visualised using an electron microscope?
Ribosomes
74
Why are opposing basal and apical surfaces polarised?
Different charges at either side to stick cells together by gap junctions
75
Outline the advantages conferred by phase contrast, darkfield, fluorescence, and confocal light microscopy.
Dark field:
- Specialised technique can be used on living cells
- It illuminates the sample with light and produces a dark background with bright objects on it
Phase contrast:
- Living cells can be examined in their natural state without previously being killed, fixed, and stained.
- It allows us to biological processes in high contrast with sharp clarity and detail
Fluorescence:
- Can be used to study living cells and tissues
- It allows you to find the location of a specific protein in the specimen.
- It offers a magnified and clear image of the cellular molecules in the specimen.
Confocal light:
- It's the ability to produce thin optical sections through relatively thick fluorescent specimens
- This technique allows the examination of both living and fixed specimens under a variety of conditions with enhanced clarity.
76
What does the cytoskeleton consist of?
Thick microtubules, thin microfilaments, medium intermediate filaments
77
Microtubules structure
- spare subunits dissolved in cytoplasm
- made of tubulin subunits
- can be assembled and disassembled by microtubules organising centres in cells near the nucleus (centrosome)
78
How are microtubules involved in cell division?
- centrosome produces spindle fibres
- centrioles located in centrosome - 9 microtubules triplets forming edge, duplicated as cells divide
- in cells with cilia/ flagella the centrioles multiply and move to the cell surface to form basal bodies. 10 pairs of microtubules grow from each basal body - one in the centre and 9 at the edge to form cilia/ flagellum, gaps for motor proteins to cross and walk along adjacent microtubules pairs to force them to bend which together creates the movement of cilia/ flagella for sperm movement
79
How are microtubules involved in the movement of vesicles?
Motor proteins attach to receptor proteins of vesicles and walk the vesicle via a network of microtubules extending throughout the cytoplasm
80
What is dark field microscopy used for?
- observing living, unstained organisms; attempts to not disturb the organisms
- can observe transparent samples
- illuminates sample with light that's not collected by the objective lens thus wont form part of the image
