Microscopes

Question 1

What are microscopes?

Answer 1

Microscopes are instruments that allow us to magnify an object/specimen thousands of times, making visible the individual cells which make up multicellular organisms allowing us to see how their structure relates to their function.

Question 2

Brief history of microscope

Answer 2

Light microscopes invented in 16th/17th century
Cell theory developed in 19th century

Question 3

What does cell theory state?

Answer 3

Botha animal and plant tissue is composed of cells
Cells are the basic units of all life
Cells only develop from existing cells

Question 4

Advantages/disadvantages of Light Microscope

Answer 4

Relatively cheap
Portable
Used out in the field
Dead or alive specimens can be viewed
Relatively easy preparation
Natural colour
BUT - low magnification/resolution

Question 5

Robert Hooke?

Answer 5

1663 examined a sectioned (thinly sliced) piece of cork through a microscope and then described the honeycomb structure seen with each compartment being a cell

Question 6

What is a compound light microscope?

Answer 6

It is an instrument that uses both an objective (near specimen) and eyepiece (near the eye) lens to magnify an object using a reflected beam of light

Question 7

Advantages of compound light microscope?

Answer 7

Higher magnification
Reduced chromatic aberration- reduction of dispersion of all wavelengths of light allowing better resolution and focus

Question 8

What is resolution?

Answer 8

The ability of a microscope to distinguish between two close points as separate entities ; shortest distance between two points that are seen as separate objects

Question 9

Describe a dry mount

Answer 9

Solid specimens are viewed (whole/sectioned) and placed on the centre of a slide covered by a cover slip ; hair/dust can be viewed whole and muscle tissue/plants are sectioned

Question 10

Describe a wet mount

Answer 10

Specimens are suspended in a liquid such as water or an immersion oil - cover slip placed at a 45* angle (to push out any air bubble - artefact) and aquatic organisms can be viewed

Question 11

Describe a squash slide

Answer 11

A wet mount is first sent up with a lens tissue gently pressing down on the cover slip (to prevent it from breaking it) ; can prevent damage to cover slip by squashing sample between two slides (MUST BE A SPFT SAMPLE) - this means the cover slip will not break and root tips can be squashed to look at cell division

Question 12

Describe a smear slide

Answer 12

Edge of a slide is used to smear the sample creating a thin, even coating on another slide - place cover slip over sample to then be viewed

Question 13

What is brightfield microscopy?

Answer 13

Light passes through bottom of sample and is observed from above

Question 14

Widefield microscopy

Answer 14

This is when the whole sample is illuminated at once

Question 15

Why is staining required in cells?

Answer 15

This is because the light alone results in a very low contrast as most cells do not absorb a lot of light (resolution is limited by diffraction and the tendency of light to bend as it passes through physical structures) - the aqueous cytosol of cells is transparent therefore stains INCREASE contrast as different organelles absorb stains to varying degrees creating an identifiable difference

Question 16

What must happen before the sample is stained?

Answer 16

Must be heat fixed -
Sample is first allowed to air dry - to fix bacterial specimens on the slide so it does not wash away with a subsequent staining procedure (removes excess water and ensures the smear is thin enough to stain)
Then is passed through a flame allowing the specimen to adhere to the slide and will therefore take up stains

Question 17

Positively charged stains

Answer 17

CRYSTAL VIOLET + METHYLENE BLUE - positively charged stains attracted to negatively charged materials in cytoplasm leading to staining of cell components

Question 18

Negatively charged stains

Answer 18

NIGROSIN + CONGO RED - this is repelled by negatively charged cytosol and stains the background leaving the cells untouched (allows us to study the morphological shape)

Question 19

What is differential staining?

Answer 19

Staining in order to distinguish between two types of organisms/different organelles within an organism within a sample

Question 20

How does Gram Staining work?

Answer 20

It is used to separate the bacteria into gram positive and gram negative bacteria - Crystal violet (positively charged) is applied to the sample alongside iodine (which forms a complex which fixes the dye) ; the slide is then washed with alcohol (this dehydrates the peptidoglycan cell wall layer and tightens it) meaning that the Crystal violet-iodine complex is trapped in the cell in the gram positive bacteria (thick cell wall). BUT with gram negative bacteria the thin cell wall is degraded and thus the gram negative cells are unable to retain the stain and the colour is lost. Instead they are stained with a counter stain (application of second stain which contrasts colour of sample) sacra in which stains the gram negative bacteria RED. Gram positive bacteria are susceptible to penicillin (which inhibits cell wall growth) and gram negative bacteria are immune because of much thinner cell wall

Question 21

Acid-fast technique

Answer 21

Differentiate species of micro bacterium from other bacteria ; lipid solvent carries carbofuchin dye which are then washed with dilute acid-alcohol solution - microbacterium are not affected by acid/alcohol and retain the carbolfuchin dye (bright red) and other bacteria are exposed to methylene blue and are stained blue

Question 22

Production of slides

Answer 22

Fixing - formaldehyde preserves the specimen to a near natural state
Sectioning - dehydrate with alcohols and moulded with resin to then be sliced with a microtome knife
Staining - create contrast
Mounting - secured with cover slip

Question 23

Risk Managment with stains

Answer 23

Many chemicals are toxic irritants - GLEAPIS advises students on reducing risk and many schools buy slides which are pre-stained/prepared ; must use Goggles and impervious gloves

Question 24

What is magnification?

Answer 24

How many times the image through a microscope is enlarged compared to the actual size of the specimen

Question 25

What must you do to increase magnification?

Answer 25

Must also increase resolution (focusing knobs)

Question 26

What is resolution limited by?

Answer 26

It is limited by the diffraction of light and it’s tendency to spread as it passes close to physical structures such as those present in the specimens ; THEREFORE because structures in specimen are very close together light reflected from individual structures may overlap due to diffraction thus they are no longer seen as separate entities and detail is lost

Question 27

Rule for resolution

Answer 27

If structures are closer than half the wavelength of light they cannot be resolved (seen separately)

Question 28

How can resolution be increased?

Answer 28

By using an electron microscope which have a wavelength of thousands of time shorter than light THEREFORE individual beams can be much closer before overlapping therefore objects much closer together and smaller can be seen without diffraction blurring the image

Question 29

Magnification formula

Answer 29

Magnification = image size/actual size

Question 30

When were electron microscopes invented?

Answer 30

In the mid 20th century - they revolutionised the study of cells and enabled biologists to see deep inside structures that were previously invisible

Question 31

Limiting factor light microscope vs EM

Answer 31

Light microscope - resolution is limiting factor
EM - wavelength of beam of electrons < 1nm and thus more detail like cell ultra structure can be seen ; produces magnified images as well as clear resolution

Question 32

Magnifications of microscopes

Answer 32

TEM - x1000000
SEM - x500000
Light microscope - x2000

Question 33

Disadvantages of electron microscopes

Answer 33

Very expensive
Cannot be used on the field
Not portable
Very complex preparation (thus more problems with artefacts - structures that are produced as a result of the preparation process)
Specimens must be dead
Specimens may be damaged by electron beam

Question 34

How does TEM work?

Answer 34

A beam of electrons transmitted through specimen and are focused to produce an image - like light microscope with highest resolution and magnification

Question 35

How does SEM work?

Answer 35

Electrons are sent across surface of specimen and reflected electrons are then collected to produce 3D images of surfaces (NO NATURAL COLOUR - ALL ARTIFICIAL IN ELECTRON MICROSCOPES)

Question 36

Resolution of microscopes?

Answer 36

TEM - 0.5nm
SEM - 3-10nm
Light microscope - 200nm

Question 37

Sample preparation of EM

Answer 37

Vacuum inside EM to ensure electrons travel in straight line - requires long preparation
1) Fixation : chemicals (can produce artefacts/distortion) or cryofixation to preserve in a near life like state to prevent decay and stabilise organism’s macrostructure
2) Sectioning - embed in resin and thin slices (solidified - thin semi-transparent cuts)
3) Stained with heavy metals - scatter/absorb electrons (to create contrast) that would otherwise be projected onto lens
4) Dehydration - remove water and replace with ethanol to preserve (must appear living)

Question 38

What are artefacts?

Answer 38

A visible structure/distorted detail caused by processing the specimen and not a feature of the specimen
In light - air bubbles that get trapped under the cover slip as you prepare a slide
EM - changes in the ultra structure (lack of cytoplasm/loss of continuity of membrane)
EXPERIENCE enables scientists to distinguish between an artefact and true structure

Question 39

Mesosomes?

Answer 39

They were invagination of cell membranes that were observed using electron microscopes after bacterial specimens had been chemically fixed
BUT when using non chemical fixation method (more recent) - mesosomes no longer visible as they were artefacts (membrane damaged by chemical) - scientific community accepts ideas based on evidence available at the time and as more evidence becomes available (disproved earlier theory)

Question 40

Fluorescent microscope

Answer 40

High light intensity to illuminate a specimen that had been treated with a fluorescent dye - absorption and re-radiation of light therefore light of a longer wavelength and lower intensity is emitted and used to produce a magnified image ; low resolution because of heavy diffraction of light

Question 41

Laser scanning confocal microscope

Answer 41

This moved a single spot of focused light across a specimen and causes fluorescent from the components labelled with a dye and emitted light is filtered through a pinhole aperture with only light emitted from very close to the focal place (distance that gives the sharped focus) is DETECTED (rest is filtered out as it would cause blurring and therefore does not pass through the pinhole) - laser instead of light as higher intensity

Question 42

Why does laser scanning confocal microscopy have a high resolution?

Answer 42

Light is removed from elsewhere and the sample is transparent (very thin) - spot illuminating the specimen is moved to create 2D images ACROSS the specimen but if positioned at different focal places it can produce 3D images

Question 43

Why is laser microscopy useful and where is it used?

Answer 43

It is non-invasive (not involving introduction of instruments into the body) and used in diagnosis of eye procedures and endoscopic procedures (biopsies of skin cancer in future)

Question 44

What is a dichroic mirror?

Answer 44

They only reflect one wavelength (from mirror) but allow other wavelengths from sample to pass through and position of pinhole means light waves from laser follow same path as re-radiation from sample therefore they have the same focal plane (hence the term confocal)