Microscopy

Question 1

What is microscopy?

Answer 1

It’s when we use microscopes to view objects/specimens that are not visible to the naked eye.

Question 2

What are the fundamental parts of a microscope?

Answer 2

DETECTOR (PMT, CCD) – allows us to see the result of what we are looking at (e.g. naked eye, camera, photomultiplier that transfers info to computer)

OBJECTIVE (± immersion medium) – like a magnifying glass (can go through air, liquid) to zoom in

SPECIMEN (cover glass)

LIGHT CONDITIONING SYSTEM (Kohler illumination, phase ring, Wollaston prism and polarisers, filter cubes for fluorescence) – do we want whole length, choosing specific wavelength, reflecting light, etc

LIGHT SOURCE (Halogen, XBO, etc)

Question 3

We can observe specimens that are dead or alive.

How would we upkeep a living specimen during microscopy?

Answer 3

Live imaging boxes are used in the investigation of live specimens – it lets us control the temperature and CO2 to keep the sample alive and the conditions for the microscope as constant as possible.

The tightly controlled conditions keep the specimen alive.
It involves the use of an incubator box combined with a precision air heater to ensure temperature of the specimen and the microscope remain equilibrated and tightly controlled.

Even small changes in ambient temperature can lead to thermal extension/contraction of microscope stand, and the stage and objective can change their plane of focus.

The maintenance of the CO2 atmosphere:

• the controller is used to adjust air flow and CO2 percentage
• an air tight table top encloses the live cell culture devices – it’s used in very small samples as the box too big; it helps us better to control conditions in microenvironment, e.g. cells.

Question 4

What are some things to consider when looking at experimental timescales?

Answer 4

It’s important to consider several things when looking at cells/structures over time – e.g. cell motility, cytoskeleton, cell differentiation.

Shorter experiments require a higher level of resolution and acquisition time (faster capturing of images)

So, the system must be designed to ensure the viability of the sample for the amount of time needed.

Question 5

What is the triangle of frustration?

Answer 5

It’s a compromise between three factors, so consider what you are trying to investigate!

TEMPORAL RESOLUTION:
- how long and how fast images need to be taken

SPATIAL RESOLUTION:

• pixel number (bigger cube , so image taken faster but lower quality; low resolution OR smaller cube, so image taken slower but higher quality, so high resolution)
• consider what you are trying to investigate and compromise (e.g. if main aim is to look at movement of particle, high resolution not needed; but if looking at how particle looks, then needed.

SENSITIVITY:
- the ability to pick up image in lower light conditions (quality of image)

Question 6

List some markings on an objective (magnifier) and what they mean.

Answer 6

• Coverslip Thickness: lists its maximum thickness
• Immersion Medium: which medium this objective has been calibrated for
• Magnification: how many times the objective magnifies the image
• Numerical Aperture: a measure of the objective’s ability to gather light and resolve fine specimen detail at a fixed object distance

Question 7

What is the difference between numerical aperture and resolution power?

Answer 7

Due to the numerical aperture, we get resolution power.

The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.
- it concentrates light in a specific way to give a ‘crisper’ image

The higher the numerical aperture, the better the resolution power of the objective.

Question 8

Describe light microscopy.

Answer 8

It can view samples ranging from tissues to cells.
The full light can be modified through rings and filters – it doesn’t alter wavelength, but instead the way it goes through.

• BF (bright field) – no filter
• DIC – able to contrast background and sample (has some 3 dimensionality)
• Ph (phase contrast) – useful for tissue and cells that are changing shape, create refringement area to enable observations on whether sample is changing shape, as we can see the cells in context to each other.

Question 9

Describe the histology you can observe using light microscopy.

Answer 9

LASER CAPTURE MICRODISSECTION:

• once the area is detected, we’re able to remove area of interest using laser that cuts through selected area (e.g. separating stroma and epithelium)
• the advantage: area of interest can be cut out and reused for other investigations (can be re-dyed with other dyes, etc)

IMMUNOHISTOCHEMISTRY:

• an additional technique that allows us to identify presence or absence of protein of interest in sample (as histological sample itself only gives us an idea of distribution)
• you get the identification of pattern of protein in tissue or cell using antibodies that are attracted to that protein antigen

Question 10

Describe phase contrast in light microscopy.

Answer 10

You can select which intensity of light goes through the sample – wavelength is not changed, but how much is reflected and how much is refracted is.

It’s important when looking at where cells or tissue stays

(e.g. phase contrast microscopy culture on intact and denatured collagen, the cells change shape trying to align to collagen)

Contrast allows us to see this!

Question 11

Describe the time-lapse aspect of light microscopy.

Answer 11

The box life imaging method is used (control CO2 and temperature).

Heart cell differentiation can be observed.

Cell migration, e.g. crawling leukocyte chasing bacteria, can also be observed.

Question 12

Describe electron microscopy.

Answer 12

An electron source is used instead of light – a beam of electrons go to sample, and gives us a dark image of the areas sampled.

TRANSMISSION EM –
this is not 3D; a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through

SCANNING EM –
the sample is treated with specific reagents, then we scan a beam of electrons through sample at a particular angle, this creates a 3D image

(the key difference is with transmission!)

The disadvantage is that we cannot use a live sample.

Question 13

Describe fluorescence microscopy.

Answer 13

Here, we are controlling the wavelength of light – selecting if light going through is red, green, etc.

We are able to modify proteins in the sample to respond to specific wavelengths.
The structure is similar to other microscopes, but the light source is a fluorescent light source.

The ocular can be the eyes, a camera or a photomultiplier.

Question 14

Describe the mechanism of a fluorescence microscope.

Answer 14

The specimen is exposed to light; it then absorbs light (excitation), and releases energy (emission).

It emits light at a specific wavelength. Several rounds of this cycle will eventually lead to energy loss and the molecule getting destroyed, leading to no more fluorescence.

Question 15

What is stokes shift?

Answer 15

You’ll have 2 peaks: the excitation peak (energy given to molecule), and the emission peak (molecule releases energy) .

Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light.

Question 16

Describe photobleaching.

Answer 16

High intensity illumination may destroy fluorophores and cause them to permanently lose their ability to emit light.

You should work with reduced light intensity, use shorter exposure times or use anti-bleach in mounting media to avoid this.

Question 17

Fluorescent proteins can be fused with other proteins and introduced in cells via transfection. This allows the live study of fluorescent tags in living cells/organisms.

What are two ways in which we can achieve that?

Answer 17

• using antibodies

- protein fusion (tag the gene)

Question 18

Describe using antibodies to fuse fluorescent proteins.

Answer 18

Molecules are normally able to recognise specific proteins (antigens) – they can attach to a molecule that gives off colour (fluorophore).

The advantage: it’s specific (can know exactly where fluorescence is, so antibody with specific fluorescent marker can bind to target molecule in wanted area, e.g. protein in nucleus).

The disadvantage: it can only be applied to a fixed sample if you want antigen to bind to sub-cellular structures (as the antibody is big and can’t go past cell membrane).

Question 19

Describe protein fusion with fusing fluorescent proteins.

Answer 19

You use plasmids to incorporate a gene that causes fluorescence.

It’s incorporated into undifferentiated ES (embryonic stem) cells, which can eventually form fibroblasts.

The advantage: it can be used in live cells.

The disadvantage: it lacks specificity, and you don’t know if the molecule can be kept (cell can undergo apoptosis if they recognise the inserted gene is exogenous).

Question 20

Describe confocal light microscopy.

Answer 20

If we are able to control the depth that the source of light was going through sample, we’d be able to see through thinner portions of sample.

Thus, this involves stopping the beam of light at certain levels, so you can see different levels of sample, e.g. in seeing an epithelial cell (seeing specifically only apical or basolateral side).

The light source is a laser – so we are able to control how it goes through sample.

The advantage: better Z resolution, better specifics as we can see through several layers; it also allows live imaging (control temp and CO2).

The disadvantage: only a small volume can be visualised by confocal microscopes at once bigger volumes more time consuming, as more sampling and image reassembling is needed (widefield is better for bigger volumes!).