Microscopy and prep

Question 1

Modern cell theory concepts (3)

Answer 1

-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)

Question 2

general cell composition

Answer 2

75-80% water
10-20 proteins
nucleic acids
inorganic material
carbs

!! solutes and solvents together form COLLOIDAL SYSTEM

Question 3

Law of Driesch

Answer 3

the volume of a cell is approx constant for each type in the same species and independent of the organism size

Question 4

Law of Levi

Answer 4

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)

Question 5

what is the factor determining max possible size of cells

Answer 5

SA:V RATIO
-cells need to stay small enough that they have a high SA:V ratio and uptake via diffusion in feasible

Question 6

what is the relevance of the nucleus: cytoplasm

Answer 6

-needs to be maintained constant
-achieved through cell division and cell adaptations

Question 7

what is the general process of specimen preparation

Answer 7

1. tissue fixation (killing of sample to preserve morphology)
2. tissue inclusion (use paraffin to harden sample for slicing)
3. cut tissue (using microtomes)
4. stain tissue (reagents able to absorb diff wavelengths)

Question 8

2 types of fixation and why it is necessary

Answer 8

1. PHYSICAL: exposure to very low/high temps
2. CHEMICAL: exposure to chemical reagents

PURPOSE:
-stabilise tissue elements
-preserve topographic relationships
-inhibition of autolysis
-resistance to manipulation

Question 9

types of chemical fixation

Answer 9

1. IMMERSION: embed in a fixing reagents
2. IN VAPORS: exposure to vapours produced by heating fixative solutions (eg. formaldehyde - high volatility)
3. PERFUSION: fixative passed through vascular system of animal models to distribute fixative throughout tissues

Question 10

preparation of a sample POST FIXATION

Answer 10

1. DEHYDRATION: tissue passed through increasingly concentrated alcohol solutions, removing water
2. CLEARING: immersion in solvent used for inclusion
3. INFILTRATION: placed in inclusion agent until it is completely infiltrated
4. EMBEDDING: in paraffin or pure resin to make ti hard enough to cut
5. CUTTING: form serial sections (ribbons) of tissue using microtome
6. DEWAXING + REHYDRATION: dewaxed by xylene, rehydrated with decreasing alcohol concs (relaxes sample and removes paraffin)
7. STAINING AND MOUNTING: adding stain and placing coverslip over sample

Question 11

how is the mounting of a blood smear different than a usual tissue

Answer 11

due to liquid state a second slide is used to smear the blood across the slide to thin it out

Question 12

dye usually used for blood smears

Answer 12

giemsa

Question 13

2 types of dyes based on character

Answer 13

1. ACIDIC: stain cytoplasm and collagen fibers (acidophillic) EOSIN: negatively charged
2. BASIC: stain ground substance, nucleic acids, RER (basophilic) HEMATOXYLLIN: positively charged

Question 14

what parts of the stain dont stain with either eosin or hematoxylin

Answer 14

1. fluids present in tissues or interstitial spaces (blood/lymph)
2. lipids and fat droplets

Question 15

PAS+ staining use

Answer 15

-stains carbs and carb rich macromolecules
-glycogen, basement membranes

Question 16

Sudan staining use

Answer 16

-detects lipid rich structures of cells (bcos its lipid soluble)

Question 17

Osmium staining use

Answer 17

-based on tissue lipid oxidation
-forms black or dark brown substances

Question 18

immunofluorescence purpose + eg

Answer 18

uses antibodies with fluorescent dyes to detect antigens in tissues

!! DAPI STAIN

Question 19

immunohistochemistry def

Answer 19

enxymes are used to catalyse a reaction that is colour producing

Question 20

ultramicrotomy for electron microscopes process

Answer 20

-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

Question 21

Magnification equation

Answer 21

M=image size/actual size

Question 22

resolving power def

Answer 22

ability of microscope lens or optical system to produce separate images of closely positioned objects

Question 23

resolution def

Answer 23

the smallest distance between two distinct objects that can be visualised as two different points

Question 24

resolution of light and electron microscope

Answer 24

light: 0.2 micrometers

electron: 0.2 nanometers

!! smaller resolutions are better

Question 25

Abbe's formula

Answer 25

Resolving power = light wavelength/ 2(refractive index) sin(half of aperture angle) OR resolving power = light wavelength/ 2(numerical aperture)

Question 26

Numerical aperture definition and equation

Answer 26

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

Question 27

what is the variable we try change when we want to change the resolving power?

Answer 27

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)

Question 28

components of a microscope

Answer 28

-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

Question 29

3 cutting planes

Answer 29

cross section longitudinal oblique

Question 30

Phase contrast microscopy

Answer 30

-used differences in refractive index of diff tissue regions enabling their visualisation without staining

Question 31

interferance contrast (DIC)

Answer 31

modification of phase contract miscroscopy useful to analyse surface properties of cells

Question 32

bright vs dark field microscopy

Answer 32

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)

Question 33

fluorescence microscopy

Answer 33

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

Question 34

confocal microscopy

Answer 34

-deep tissue visualisation and 3D images -used to visualise dynamic cell processes

Question 35

FISH

Answer 35

use of fluorescent probe to bind to complimentary parts of chromosomes

Question 36

how is confocal microscopy different to fluorescent microscopy?

Answer 36

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

Question 37

electron microscopy details

Answer 37

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

Question 38

TMA process

Answer 38

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