WEEK 6 - VISUALISATION OF TISSUES AND CELLS Flashcards
VISUALISATION OF TISSUES AND CELLS
why is visualising tissue and cells important?
- discovery of cellular and molecular compositions
- discovery of cellular functions
- diagnosis of disease
size
eukaryotic 5-100um
prokaryotic 0.2-2um
light microscope 10nm-200nm
electron 100um-1nm
wavelength of radiation
- Light microscopy: visible light (400-700 nm)
- Fluorescence microscopy: ultraviolet(UV) light (200-400 nm)
- Electron microscopy: electron waves (0.01-0.001 nm –100,000 times smaller than visible light)
magnification
- The apparent increase in size of an object
- A beam of radiation refracts(bends) as it passes through a lens
*Glass lenses refract light
*Magnetic lenses refract electron beams - Magnification by a convex glass lens
*Thickness of lens
*Curvature of lens
*Speed of light in the len
light bends when entering a glass lens due to the glass being denser than air causing the wavelength to slow down. it bends again as it comes out from the convex lens
resolution
- Ability to distinguish between objects that are close together
- The smallest distance between 2 particles at which they can be seen as separate objects
- resolution distance = 0.61 x wavelength, you want low number / numerical aperture (N.A.) (a unitless number that measures the range of angles at which an optical system can emit or accept light), you want a high NA
on a microscope
160/0.17
100/1.25/OIL
0.17= coverslip thickness
100= magnification
1.25= numerical aperture
OIL= immersion medium
effects of immersion oil on resolution
without immersion oil, most light is refracted and lost
job of the oil is to increase the amount of light that the lens is able to collect as the oil has the same optical density as the glass
contrast
- Differences in intensity between two objects, or between an object and its background
- Important in determining resolution
- Staining increases contras
light microscope
- Most commonly used in laboratories
- White light: 550 nm
- Can magnify effectively to about 1,000 times the size of the actual specimen
- Maximal resolution 0.2 μm(200 nm)
- Can be used with live unstained cells and fixed stained specimens (>contrast & resolution)
- Compound microscope: uses a series of lenses for magnification
light microscope : components
- Ocular lens (eyepieces): Remagnifies the image formed by the objective lens
- Body: transmits the image from the objective lens to the ocular lens using prisms
- Objective lens: primary lenses that magnify the specimen
- Stage: holds the microscope slide in position
- Condenser: Lens (or lenses) focuses light through specimen
- Diaphragm: Controls the amount of light entering the condenser
- Illuminator: light source
- Light Microscope: ComponentsCoarse focusing knob: Moves the stage up and down to focus the image
- Fine focusing knob: Sharpens the image
fluorescence microscope
- Light source: UV light (200-400nm), has shorter wavelength than light
- Maximal resolution 10-30 nm
- Fluorescent substances absorb UV light and emit visible light
- Some cells are naturally fluorescent; others must be stained with fluorophores
- Used to identify cells and cellular components with high degree of specificity (antibodies, recognise a specific protein sequence and antibody is conjugated with a fluorophore)
- Can be used with fixed or live cells and tissue (biological processes)
fluorescence microscope function
- excitation filter: selects only the wavelength that excites a particular fluorescent dye
- emission filter: selects only the wavelength emitted by the fluorescent dye
overall same functions as light microscope
electron microscope
- To study subcellular structures(100 μm –1 nm)
- Use electrons instead of light
- The shorter wavelength of electrons (<1nm) gives greater resolution (up to 200 times better than LM)
- Very high magnification(100,000X)
- samples are fixed and stained or coated with electron dense material (gold)
- Two types:
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
electron microscope function
- has an electron gun, which is the source of electrons
- condenser is a magnet (unlike in light where its glass) and focuses the electrons on the specimen
- objective lens is also a magnet as well as the lens
- and final image is seen on fluorescent screen, since we cannot visualise electrons
preparing specimens for microscopy
- Fixation of tissue
- Embedding in a supporting medium
- Sectioning into thin sections
- Stainingsections to enhance contras
fixation
Aim:
* Preserves cells and tissue components maintaining their natural structure
* Prevents degradation caused by autolysis and bacterial attack
* Fixation makes cells permeable to staining reagents
Types of fixation:
* Chemical fixation: Use of cross-linking proteins via covalent bonds (chemicals: formalin, glutaraldehyde),
Dehydration reagents (ethanol, methanol)–denature, precipitation
* Physical fixation can preserve native structure (freezing-70ºC)
embedding
- Infiltrate fixed specimen with medium that hardens and supports tissue for sectioning
- Paraffin wax and resin (most common) or cryo protective medium
- dehydration - removal of water from tissue
- clearing - Replacement of alcohol with xylene, Tissue ready for embedding medium infiltration
- embedding - Tissue infiltrated & surrounded with liquid wax, Wax solidified by cooling or polymerization
sectioning
- Cut tissue into very thin transparent sections
- Thin sections allow light or electrons to pass through the specimen essential for identification of cellular details
- improves access of dyes and antibodies
- if media is too weak or strong deficits could occur
- paraffin wax embedded tissue goes in a microtome and is sliced with a sharp blade - section on slice is 4-10um thick
- frozen tissue goes in a cryostat (cold chamber -20 degrees) and is sectioned with a sharp blade - section on slice is 10-20um thick
staining
- Most biological material is colorless and invisible in a microscope
- Staining is needed to reveal cells and their components
Aim:
* Add contrast to the image
* Identify chemical components of interest
* Locate particular tissues, cells or organelles
staining methods
1.Histology
* Uses organic dyes with affinity for particular subcellular components
* Affinity depends on physical and chemical properties of dyes
* Basic dyes stain acidic structures
* Acid dyes stain alkaline structures
2. Histochemistry
* Identification of macromolecules (DNA, RNA, carbohydrates, lipids) & chemicals compounds (Ca2+, Na+) in cells & tissue using dyes
* allows identification of specific components in cells
3. Immunohistochemistry
* Uses antibodies to detect and localise specific proteins
histology
Haematoxylin & Eosin (H&E) :
* HAEMATOXYLIN–basic dye –binds acidic components of cells: nuclear DNA & RNA (blue round structures)
* EOSIN–acidic dye –binds basic components: proteins in cytoplasm (pink)
Masson’s Trichrome :
*Identify and quantity fibrous tissue (collagen), important with the heart
*Aniline blue: Collagen blue
*Biebrichscarlet: Cytoplasm red *Haematoxylin: Nuclei dark blue
histochemistry
Periodic Acid Schiff (PAS)
* Practical 4
* Detect carbohydrate macromolecules such as glycogen, and mucosubstances such as glycoproteins& glycolipids in tissues
* Periodic acid oxidises sugars and exposes aldehyde groups
* Aldehyde groups react with the Schiff reagent give a purple-magenta colour
immunohistochemistry
- Antibodies to detect specific proteins
- Antibody-antigen specific interaction
- Antibodies conjugated with fluorescent dyes
direct method - uses one step which is a primary antibody conjugated with a fluorophore that binds to the antigen of interest
indirect method - primary antibody step which doesn’t carry the fluorophore and binds to the antigen of interest and a secondary antibody that carries the fluorophore and binds to the primary antibody
indirect would give a high fluorescence signal under the microscope because there’s more fluorophores sticking to the antigens in the primary antibody
what is it called when light bends through a lens
refraction (difference in optical density)
light microscope gives a
large inverted image
using a light microscope you can see..
cells (molecules too small)
the lens in the light microscope is called
ocular lens
the lens in the eyepiece has a magnification of
10x
what controls how much light passes through the stgae
diaphragm
what is the function of the condenser lens…
focuses light through the specimen
what is the N.A. of an objective lens
ability to collect light
what is the max resolution of the light microscope
200nm
what is the max maginifctaion of light microscope
1000x
what is the first step of tissue preparation
fixation
what is the most common fixative
formalin
how does formalin preserve a specimen
by cross-linking proteins
after embedding the specimen what is the first thing you need to do before staining
cut into sections
eosin is used to stain
cytoplasm
what satin works well when staining carbohydrates
periodic acid Schiff (PAS) reaction
What technique uses labelled antibodies to localise specific proteins or other antigens?
immmunohistochemsitry
Name a fixative used in the preparation of samples for microscopy
Formalin, Glutaraldehyde, Ethanol, Methanol, Freezing
