Microscopy

Question 1

What is microscopy ?

Answer 1

Using microscopes in order to see specimens which are not visible to the naked eye

Question 2

What are the different parts of the 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

Describe how we mount the specimen.

Answer 3

Cover glass (0.17mm)
Sample surrounded by embedding medium
Glass slide

Question 4

We can observe specimens that are dead or alive.

How would we upkeep a living specimen during microscopy?

Answer 4

We use a device called The Box.

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 5

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

Answer 5

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 6

What is the triangle of frustration?

Answer 6

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 7

What is intensity resolution?

Answer 7

This is defined by bit so the more information we have the more intensity we can have.
This means we can have more intensity resolution and distinguish details.

Question 8

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

Answer 8

• 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 (number x)
• Numerical Aperture: a measure of the objective’s ability to gather light and resolve fine specimen detail at a fixed object distance
  Application (What applications microscope can be used for )

Question 9

What is the objective resolution power ?

Answer 9

This is determined by the numerical aperture.
(NA)
This is important as it gives an idea of how much detail the objective can give us.
The higher the NA =the higher the resolution by that objective.