Microscopy

Question 1

describe the structure of compound microscopes

Answer 1

compound Ms have 2 lens, first the obective lens - near the specimen and next the eyepiece lens - used to view.

Question 2

How do compound light microscopes illuminate specimens

Answer 2

Using bright field microscopy, the sample is illuminated from underneath through multilpe lens, here the images appear dark against a bright background.
- for opaque specimens they are illuminated from above.

Question 3

How does the configuration of compound microscopes allow for a higher magnification

Answer 3

the objective/eyepiece lens allows for a higher magnification

Question 4

what is chromatin abberation and how do compound light microscpes reduce this problem.

Answer 4

chromatin abberation is the colour distortion that occurs around the edges of an object in a image.
- compound Ms use multiple lens elements - eg: achromatic lenses - which are designed to minimise the seperation of light wavelengths (colous) that occurs when light passes through a lens - resulting in more sharper and accurate images.

Question 5

how are sample preparation methods chosen

Answer 5

they are chosen depending on the nature of the specimen and the desired resolution.

Question 6

how are specimens viewed in dry mounts, and give examples of what can be viewed.

Answer 6

they are viewed whole (insect parts, pollen, hair, dust) or are sectioned into thin slices (muscle and plant tissue)
- they are placed on the centre of a slide with a cover slip on top.

Question 7

why is sectioning required

Answer 7

for light to pass through the specimens.

Question 8

give an example of when squash slides are used to view a specimen.

Answer 8

when looking at cell division in root tips, chromosomes and other cell stuctures.

Question 9

how are squash slides prepared

Answer 9

prepare a wet mount first, potential damage can be limited by using another slide to squash the sample
- so the cover slip doesnt break.

Question 10

how is a smear slide prepared and what specimens are they used for.

Answer 10

the edge of another slide at a 45 degree angle is used to smear the sample across it’s slide, then a cover slip is placed on top.
- used for blood smaples to view its cells

Question 11

how is a wet mount prepared and what specimens is it used to view.

Answer 11

the specimen is suspended into an immersion oil, and the cover slip is placed on top from an angle - thi reduces air bubbles from being trapped in the slide.
- for aquatic samples and other living organisms.

Question 12

define numerical aperture

Answer 12

it is the ability of lens to capture light and resolve fin detail.

Question 13

how does diffraction affect the numerical aperature of the microscopes resolution.

Answer 13

diffraction is when the light waves encounter an obstacle - like the edges of a specimen - when passing through the lens, this results in longer wavelengths which also interfere with each other - reducing the resolution of images.

Question 14

what is the refractive index, and describe it’s influence on diffraction

Answer 14

refractive index is the ability to bend light. A higher refrative index results in a shorter wavelength = lowered diffraction = increased resolution.

Question 15

describe refraction

Answer 15

it refers to how light bends as it passed from one medium to another with a different refractive index.

Question 16

give 3 examples of where refraction occurs in a light microscope

Answer 16

1. occurs between the glass lens and air
2. between the specimen and cover glass
3. when an immersion oil is used.

Question 17

link the use of an immersion oil in a wet mount to the resolution of the image

Answer 17

the oil will have to have a similar refractive index to the glass lens, so reduce the beding of light (lowers refraction) so less light is lost at the interference of the lens and specimen = incresaing resolution.

Question 18

what is the purpose of refrction in light microscopes

Answer 18

it is essential for focusing and directing light, contributing to light formation.

Question 19

why is staining important when viewing specimens through basic light microscopy.

Answer 19

light microscopy illuminates the whole sample. images have a lower contrast as most cells dont absorb a lot of light, the cytosol and other structures are usually transparent. so staining is needed to increase contrast - making structure identifiable.

-different components take up stains to different degrees.

Question 20

what is heat-fixing and why is it done to slides before being stained.

Answer 20

heat-fixing is when the prepared slides are air dryed and then passed through a flame. this is done to ensure the specimen adheres to the slide and takes up the stain.

• it is also done to kill microorganisms to stop their movement and metabolism whilst maintaining the specimens integrity for observation.

Question 21

what is the staining technique is used to differentiate myobacterium from other bacteria species.

• describe the process of the staining process

Answer 21

Acid-fast technique aka The Ziehl-Neelsen stain -

• a lipid solvent carried the dye carbolfuchin into the cells.
• the cells are then washed with a dilute acid-alcohol solution.

Question 22

describe the possible results when the acid fast technique is used
(aka Ziehl-Neelsen stain)

Answer 22

• myobacterium aren’t affected by the acid-alcohol solution, so they retain the carbolfuchin dye which is bright red.
• all other bacterium species lose the stain and are exposed to the methylene blue stain.

Question 23

what are myobacterium and what is the significance of using the Ziehl-Neelsen stain

Answer 23

they are a bacteria species which contain the pathogens that result in tuberculosis, they can enter the body through drinking contaminated water or when it comes into contact with a breakage in the skin at a wound. the pathogens can also inhaled.

• the Ziehl-Neelsen stain is a majour diagnostic tool for TB.

Question 24

list the 4 main steps in the production of pre-prepared slides.

Answer 24

1. Fixation
2. Sectioning
3. Staining
4. Mounting.

Question 25

what are the 2 ways to fixate a specimen during the pre-preperation of slides.

Answer 25

- chemical fixation includes using - formaldehyde and glutaraldehyde. - physical fixation includes heating, or cryopreservation (freezing)

Question 26

why is fixation of samples neccessary

Answer 26

needed to preserve the sample in as near-natural states as possible. (essentially dehydrating them) to stabalise their integrity.

Question 27

describe how sectioning is completed in the pre-preparation of slides.

Answer 27

the fixated specimens are then placed in a mould with wax or resin to form a hard block. -then thinly sliced with a microtome.

Question 28

what is the equation to calculate field of view

Answer 28

with the diameter given, find the radius (diameter/2) - this is usually and find the area using the area of the circle equation.

Question 29

what is needed to mesaure the sixe of sample under a microscope, and why does the magnification of lenses need to be calibrated.

Answer 29

an eyepiece graticule is needed. But the true magnification of different lenses can slightly vary from what is stated so they needed to be calibrated using a stage micrometer and the eyepiece graticule.

Question 30

why is resolution a limiting factor for light microscopes.

Answer 30

the tendancy of light waves to spread as they pass close to physical structures in the specimen (diffraction) and with the structures being close together, the light reflected from each individual structures overlaps, the structures then dont appear as seperate entities and detail is lost.

Question 31

what is the minimum wavelength that structures need to have between them to be resolved. in an optical microscope.

Answer 31

structures shouldnt be closer than half a wavelength of light to be seen seperately.

Question 32

why is resolution not a problem for electron miscroscopes. (compared to light microscopes)

Answer 32

electrons have much shorter wavelengths (around <1nm - about 1000 shorter than light) so when they pass through structures, they dont overlap as much so the effects of diffraction are lowered.

Question 33

what are the 2 types of electron microscopes

Answer 33

TEM - Higher resolution, 2D images, to see cell ultrastructure. SEM - 3D images, cell topography. Vacuum required for both.

Question 34

what is cell topography

Answer 34

the study of surface features, or landscapes of cells and tissues.

Question 35

being able to get super resolved images of cell ultrastucture is great with electron microscopes but what are some disadvantages

Answer 35

- expensive equipment - required a controlled environment - complicated slide preparation - artefacts arise and e- beams can damage the specimen.

Question 36

describe what aretefacts are, and how they are formed in light microscpes.

Answer 36

artefacts are visible structural details caused by the processing of specimens. they are basically just air bubles that get trapped under the cover slip.

Question 37

describe what aretefacts are, and how they are formed in electron microscpes.

Answer 37

the chemical processing of slides involved in e- microscopes, causes changes in the cells ultrastructure. - eg - loss of continuity of cell membranes, distortion of organelles and empty spaces in the cytoplasm.

Question 38

give an example of an artefact that was believed to be a cell component when viewed with an electron microscope. - what was it's purpose - and how did scientists realise they werent an oganelle.

Answer 38

Mesosomes - the invaginations of bacterias cell membrane, seen after the bacteria were chemically fixed. - their large SA made the prokaryotes organelles seem important in oxidative phosphorylation. - after techniques like chryofixation were used on the same bacterial cells, the mesosomes were no longer visible.

Question 39

why was chryofixation used to further analyse if mesosomes were organelles or just artefacts.

Answer 39

chryofixation is a non-chemical technique, so scientists could prove that the artefact mesosome was a result of chemical processing.

Question 40

mesosome like structures are found when some bacteria are treated with antibiotics, are mesosomes still artefacts or organelles.

Answer 40

the antibiotics causes physiological changes, resulting in the loss of cell membrane integrity. so components sometimes appearing as mesosomes are still artefacts with no functional role.

Question 41

why is chemical fixation important for electron microscopes.

Answer 41

it prevents the specimen from decomposing as it is dead unlike in light microscopes where they are alive still.

Question 42

how is staining in electron microscopes different to staining in light microscpes.

Answer 42

in e-Ms, staining is done with heavy metals like uranium. the metal ions create contrast by scattering or absorbing electrons, causing the denser areas to appear darker. - but light Ms use mostly organic (colourful) dyes to increase contrast and highlight specific cellular structures. both increasing visibilty.

Question 43

after fixation and staining of the slides in preparation for e- Ms,

Answer 43

the specimen needs to be dehydrated with solvents. this prevents vapourisation of water in the vacuum. as that would damage the specimen.

Question 44

list the 4 steps o slide preparation for e-Ms

Answer 44

1. fixation 2. staining 3. dehydraion 4.resin embedding (so the specimen can be cut into thin slices)

Question 45

Laser scanning confocal microscopy (LSCM), an advancement in optical microscopy. - How does it work.

Answer 45

it uses a laser instead of light for higher intensities, allowing for improved ilumination. - only thin slices can be examined.

Question 46

name and describe the illuminating technique used in LSCM and what types of images are produed.

Answer 46

spot-illuminating technique - where a single spot of light is focused across a specimen. - producing 2D images.

Question 47

Describe flourescence exitation and how it causes more magnified images.

Answer 47

flourescene is the absorption and re-radiation of light. Fluorophores (molecules in a dye that emit light when excited by a specific wavelength) in the focused spot are excited, causing them to emit light at a longer wavelength with a lower energy = more magnified image.

Question 48

what is the purpose of the beamsplitter in the LSCM

Answer 48

the beamsplitter is a dichoic mirror, that selectively seperates the exitation light from the emittted flourescene light from the sample. The laser beam, used to excite fluorescence in the sample, is directed towards the objective lens. After the sample emits fluorescence, the beam splitter directs this fluorescence light towards the detector, while the excitation light is diverted away. - reducing background noise and producing clearer images.

Question 49

where are the 2 pinholes found in a laser scanning confocal microscope. - whats the general purpose of both pinholes.

Answer 49

one pinhole at the detector (the confocal pinhole) and one pinhole in the excitation path (the illumination pinhole) - so unwanted light isnt detected = reduced blurring = increased resolution.

Question 50

what is the illumination pinhole in a LSCM used for.

Answer 50

placed near the laser source. - it shapes the laser beam before it passes through the objective lens and reached the sample. (the exitation path) - helps to create a small focused spot of light on the sample.

Question 51

what is the detector pinhole, and where is it placed.

Answer 51

placed in the detection path, after the objective lens and before the detector. it filters out out-of-focus flourescence light - and only light emitted from very close to the focal plane is detected. - this enhances the contrast and resolution of the image. - shapes the emission light pathway.

Question 52

why is the position of both the pinholes in a LSCM significant.

Answer 52

the position of both pinholes means the laser's light folllows the path as the waves radiated when the sample flouresces. - having the same focal plane = confocal.

Question 53

how can 3D images be formed using an LSCM

Answer 53

by creating images at different focal planes.

Question 54

list 3 benefits + future advancements of the uses of the laser scanning confocal microscope.

Answer 54

1. the flourescing proteins make it a non-invasive technique, which can be used in eye disease diagnosis and endoscopic procedures. 2. can be used to see the distribution of molecules = useful for development of new drugs. 3. could be used for virtual biopsies especially for suspected skin cancer.

Question 55

How does an atomic force microscope work? (AFM)

Answer 55

a mechanical sharp tip - probe on a canteliver is used to 'feel' the surface of the specimen. - generates 3D images. - used only for cell topography.

Question 56

what is a cantilever in an AFM

Answer 56

a lever supported at one end.

Question 57

how does the probe obtain information of the surface of the specimen when it 'feels' it. in an AFM

Answer 57

van der waals interactions are formed between the probe and the surface which causes detections. - this is mesaured by using a laser beam which is reflected into the detector.

Question 58

what is the difference between an AFM and e-Ms

Answer 58

fixation and staining arent needed for AFM - normal conditions are maintained with no damage to the cells - so living systems can be observed.

Question 59

what is the resolution that is achieved by atomic force microscopes.

Answer 59

approx, 0.1nm vertically and a lateral resolution of around 1mm.

Question 60

what is an important application of atomic force microscopes.

Answer 60

needed in the pharmaceutical industry to identify potential drug targets on cellular proteins and DNA - it gives a better understanding of drug interactions. - they can test if compounds found in the natural world have medical applications - usually takes ages, but faster and cheaper with an AFM .