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Cytology > Microscopy and prep > Flashcards

Microscopy and prep Flashcards

1
Q

Modern cell theory concepts (3)

A

-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)

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2
Q

general cell composition

A

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

!! solutes and solvents together form COLLOIDAL SYSTEM

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3
Q

Law of Driesch

A

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

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4
Q

Law of Levi

A

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)

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5
Q

what is the factor determining max possible size of cells

A

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

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6
Q

what is the relevance of the nucleus: cytoplasm

A

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

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7
Q

what is the general process of specimen preparation

A
  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)
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8
Q

2 types of fixation and why it is necessary

A
  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

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9
Q

types of chemical fixation

A
  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
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10
Q

preparation of a sample POST FIXATION

A
  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
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11
Q

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

A

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

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12
Q

dye usually used for blood smears

A

giemsa

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13
Q

2 types of dyes based on character

A
  1. ACIDIC: stain cytoplasm and collagen fibers (acidophillic) EOSIN: negatively charged
  2. BASIC: stain ground substance, nucleic acids, RER (basophilic) HEMATOXYLLIN: positively charged
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14
Q

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

A
  1. fluids present in tissues or interstitial spaces (blood/lymph)
  2. lipids and fat droplets
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15
Q

PAS+ staining use

A

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

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16
Q

Sudan staining use

A

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

17
Q

Osmium staining use

A

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

18
Q

immunofluorescence purpose + eg

A

uses antibodies with fluorescent dyes to detect antigens in tissues

!! DAPI STAIN

19
Q

immunohistochemistry def

A

enxymes are used to catalyse a reaction that is colour producing

20
Q

ultramicrotomy for electron microscopes process

A

-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

21
Q

Magnification equation

A

M=image size/actual size

22
Q

resolving power def

A

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

23
Q

resolution def

A

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

24
Q

resolution of light and electron microscope

A

light: 0.2 micrometers

electron: 0.2 nanometers

!! smaller resolutions are better

25
Q

Abbe’s formula

A

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

OR

resolving power = light wavelength/ 2(numerical aperture)

26
Q

Numerical aperture definition and equation

A

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

27
Q

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

A

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)

28
Q

components of a microscope

A

-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

29
Q

3 cutting planes

A

cross section
longitudinal
oblique

30
Q

Phase contrast microscopy

A

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

31
Q

interferance contrast (DIC)

A

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

32
Q

bright vs dark field microscopy

A

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)

33
Q

fluorescence microscopy

A

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

34
Q

confocal microscopy

A

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

35
Q

FISH

A

use of fluorescent probe to bind to complimentary parts of chromosomes

36
Q

how is confocal microscopy different to fluorescent microscopy?

A

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

37
Q

electron microscopy details

A

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

38
Q

TMA process

A

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

Cytology (8 decks)

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