BL10 L2 Examining Cells And Microscopy Flashcards
List the 4 types of tissues
- striated muscle
- nerves
- connective
- epithelial
Resolution definition
Smallest distance that two objects can be separated and seen as two different objects
Mili
1mm = 1x10^-3m
Micro
1um = 1x10^-6m
Nano
1nm = 1x10^-9
List 5 biopsy techniques
- surgery and later dissections
- scraping methods e.g. scalpel, scrapes, curettes
- sharp needles e.g. needle/ punch biopsy, pipelle
- direct venepuncture e.g for blood smear
- tranvascular: device travels through BVs to site of biopsy e.g. liver, lungs, brain, heart
Examples of scaring biopsy methods
Curettes
Scalpel scrapes
Examples of sharp needle biopsy techniques
Needle biopsy
Punch biopsy
Pipelle
What can a pipelle be used for?
Endometrial biopsy sample
Endometrium-Uterus lining
What does transvascular mean and what can it be used to take a biopsy of?
Device travels through BVs to biopsy site
Kidney, lungs, brain, heart
2 reasons for fixation
- samples need to be translucent + thin
- protect against microbes
How thin do samples need to be for fixation
2-10 micrometres
Why do samples need to be translucent and thin?
To avoid diffraction of light and prevent blurry image
Why do samples need to be fixated to protect against microbes?
Once of out the body, samples are no longer under the protection of the immune system
What fixative is used?
Formalin solution - 37% solution of formaldehyde
How long do you fixate for?
24-48 hours only
What happens if you fixate for too long?
Shrinkage occurs due to dehydration
Causes fixation artefacts
What causes fixation artefacts?
Fixation for longer than 48 hours
Causes shrinkage due to dehydration
How does paraffin wax embedding occurs?
- dehydrating in varying alcohol cones
- immersed in hot paraffin wax overnight
- tissue orientated in mould
- add more wax
- allow to solidify
- slice thinly using microtome
What machine is used to slice samples thinly?
Microtome
Frozen section process
- specimen frozen rapidly
- frozen specimen cut with microtone
- stained with H+E
Difference between paraffin embedded tissue section and frozen sample
Paraffin - fixed tissue, 24-48 hours to make, permanent, clear/detailed
Frozen sample - fresh tissue (preserves lipid containing organelle), 10-20 mins to makes, lasts a few months, lacks detail
What colour does haematoxylin stain and what does it stain?
Blue
Nucleus - binds to acidic things e.g DNA, RNA
What colour does eosin stain and what does it stain?
Pink
Cytoplasm, extracllular matrix
What colour does H+E stain and what difference does it make to only using one?
Purple
Nuclei stained clearer
Cytoplasm more detailed
Tissue prep for light microscope
- fix with formalin
- embed with paraffin wax
- stain e.g. H&E, methylene blue
Tissue preparation for electron microscope
- fix with glutaraldehyde
- embed with epoxy resin
- stain e.g. osmium tetroxide
What has a high resolution, light or electron microscope?
ELECTRO MICROSCOPE
Light- 0.25 MICROmetres
Electron- 0.25 NANOmetres
What has a higher magnification, light or electron microscope?
ELECTRON MICROSCOPE
Light- x600
Electron- x500,000
What can be viewed with light microscopes compared to electron microscopes?
Light- living and moving
Electron- dead and inert (have to be vacuumed)
Colours seen in light vs electron microscope
Light- natural colour
Electron- black and white
Financial costs of light vs electron microscopy
Light- cheap and easy prep
Electron- expensive and difficult prep
What type of image does SEM give?
3D
Black and white
What image does TEM give?
2D
Black and white
Advantage of cell culture
- complete control over physical environment
- homogeneity (the same) of sample
- reduced need for animal models
Disadvantages of cell culture
- hard to maintain so skilled worker needed
- high cost for small amount
- 3D is lost
- aneuploidy - abnormal number of chromosome in haploid organism
What can be visualised in dark field and how does it work?
- living cells
- illuminates sample with light that won’t be collected by objective lens so wont form part of image
- very dark background with bright objects on
How does confocal microscopy work?
- laser excites fluorescent dye
- electrons raised to higher energy level
- wavelength is emitted as electron returns to ground state
- emitted light is sent through mirrors and pinhole screen to CMOS detector
What can confocal microscopy be used for clinically?
Evaluation of various eye diseases
