Microscopy and prep Flashcards
Modern cell theory concepts (3)
-all living things made up of cells (basic unit of life)
-all cells arise from cell division of pre existing cells
-energy flows within cells (metabolism)
general cell composition
75-80% water
10-20 proteins
nucleic acids
inorganic material
carbs
!! solutes and solvents together form COLLOIDAL SYSTEM
Law of Driesch
the volume of a cell is approx constant for each type in the same species and independent of the organism size
Law of Levi
EXCEPTION TO DRIECH’S LAW:
larger the size of the animal the larger the size of corresponding cells (which aplies to nerves muscles and lens fibers)
what is the factor determining max possible size of cells
SA:V RATIO
-cells need to stay small enough that they have a high SA:V ratio and uptake via diffusion in feasible
what is the relevance of the nucleus: cytoplasm
-needs to be maintained constant
-achieved through cell division and cell adaptations
what is the general process of specimen preparation
- tissue fixation (killing of sample to preserve morphology)
- tissue inclusion (use paraffin to harden sample for slicing)
- cut tissue (using microtomes)
- stain tissue (reagents able to absorb diff wavelengths)
2 types of fixation and why it is necessary
- PHYSICAL: exposure to very low/high temps
- CHEMICAL: exposure to chemical reagents
PURPOSE:
-stabilise tissue elements
-preserve topographic relationships
-inhibition of autolysis
-resistance to manipulation
types of chemical fixation
- IMMERSION: embed in a fixing reagents
- IN VAPORS: exposure to vapours produced by heating fixative solutions (eg. formaldehyde - high volatility)
- PERFUSION: fixative passed through vascular system of animal models to distribute fixative throughout tissues
preparation of a sample POST FIXATION
- DEHYDRATION: tissue passed through increasingly concentrated alcohol solutions, removing water
- CLEARING: immersion in solvent used for inclusion
- INFILTRATION: placed in inclusion agent until it is completely infiltrated
- EMBEDDING: in paraffin or pure resin to make ti hard enough to cut
- CUTTING: form serial sections (ribbons) of tissue using microtome
- DEWAXING + REHYDRATION: dewaxed by xylene, rehydrated with decreasing alcohol concs (relaxes sample and removes paraffin)
- STAINING AND MOUNTING: adding stain and placing coverslip over sample
how is the mounting of a blood smear different than a usual tissue
due to liquid state a second slide is used to smear the blood across the slide to thin it out
dye usually used for blood smears
giemsa
2 types of dyes based on character
- ACIDIC: stain cytoplasm and collagen fibers (acidophillic) EOSIN: negatively charged
- BASIC: stain ground substance, nucleic acids, RER (basophilic) HEMATOXYLLIN: positively charged
what parts of the stain dont stain with either eosin or hematoxylin
- fluids present in tissues or interstitial spaces (blood/lymph)
- lipids and fat droplets
PAS+ staining use
-stains carbs and carb rich macromolecules
-glycogen, basement membranes
Sudan staining use
-detects lipid rich structures of cells (bcos its lipid soluble)
Osmium staining use
-based on tissue lipid oxidation
-forms black or dark brown substances
immunofluorescence purpose + eg
uses antibodies with fluorescent dyes to detect antigens in tissues
!! DAPI STAIN
immunohistochemistry def
enxymes are used to catalyse a reaction that is colour producing
ultramicrotomy for electron microscopes process
-method of cutting tissue into extremely thin slices of 50-100nm for TEMs
-sections cut are placed on grid
-sections are cross colored via heavy metal salts to make sample electron dense
-observation under TEM
Magnification equation
M=image size/actual size
resolving power def
ability of microscope lens or optical system to produce separate images of closely positioned objects
resolution def
the smallest distance between two distinct objects that can be visualised as two different points
resolution of light and electron microscope
light: 0.2 micrometers
electron: 0.2 nanometers
!! smaller resolutions are better
Abbe’s formula
Resolving power = light wavelength/ 2(refractive index) sin(half of aperture angle)
OR
resolving power = light wavelength/ 2(numerical aperture)
Numerical aperture definition and equation
NA = nsina where a is the half angle of the light beam and n is refractve index
!! this formula indicates max aperture of light beam picked up by the object
what is the variable we try change when we want to change the resolving power?
refractive index (n)
-increase n for increased resolving power
-done using IMMERSION OIL bcos it has a larger n than air (1.5 vs 1)
components of a microscope
-field diaphragm: controls amount of light arriving at condenser (by changing diameter)
-condenser: coverges light beams from light source into single focal point
-stage: where spacimen placed and secured
-objective lens: fathers light passing through specimen to produce image with magnifixation
3 cutting planes
cross section
longitudinal
oblique
Phase contrast microscopy
-used differences in refractive index of diff tissue regions enabling their visualisation without staining
interferance contrast (DIC)
modification of phase contract miscroscopy useful to analyse surface properties of cells
bright vs dark field microscopy
BRIGHT: use of lght source passing through thin sample to be seen by lens
DARK: only light diffracted by structures in specimen reaches lens (useful to examine crystals and bacteria motility)
fluorescence microscopy
allows staining with fluorophores that appear coloured under lens
-fluorescence emitted by the triggering of excitation by laser
!! FLUOROPHORES are excited with colour of wavelength A, and then drop in energy to emit colour of wavelength B
confocal microscopy
-deep tissue visualisation and 3D images
-used to visualise dynamic cell processes
FISH
use of fluorescent probe to bind to complimentary parts of chromosomes
how is confocal microscopy different to fluorescent microscopy?
in confocal, the light dource is converged to a single focal point (not entire specime) and the emisison is detected from these focal points only –> leads to an increased resolution
electron microscopy details
uses electron beams to form image via transmission or scattering
TEM - 2D: heated tungsten generated electrons that pass through sample and are detected
SEM - 3D: electron beam scanned across surface (doesnt pass through) and are diffused by specimen
TMA process
Tissue micro array
-analyses several tissue samples simultaneously
-formation of cDNA
-hybridisation with the probes
-colour results depending on whether hybridisation has occured or not
