Introduction to Microscopy

Question 1

What is microscopy

Answer 1

Using microscopes to view objects/specimens that are not visible to the naked eye.

Question 2

Name the parts of a microscope

Answer 2

• Light source
• Light conditioning system
• Specimen
• Objective
• Detector

Question 3

How is the light microscopic specimen prepared

Answer 3

• use a coverglass of thickness 0.17mm
• Sample is surrounded by embedding medium
• use a glass slide

Question 4

Explain how life imaging microscopy works

Answer 4

A living specimen is placed in a box to be examined with a microscope.

Prevent focus instability

The specimen must be kept alive so the temperature, CO₂ and O₂ levels are monitored and maintained.

Question 5

What problems may arise from using microscopes to examine specimens for different timescales

Answer 5

• When only measuring for seconds, there may be problems with artifacts in multichannel/4d imaging
• when measuring from hours to days there may be problems with the viability of the specimen, also the positioning of the capture may change

Question 6

Describe the triangle of frustration for signal detection

Answer 6

We can satisfy 2 of the three parameters when doing a measurement:
- Temporal resolution 1
- spatial resolution 2
- sensitivity 3

All detections have their benefits and limitations, what is best depends on the application requirments

Question 7

What is spatial resolution

Answer 7

The size of the smallest unit of an image capable of distinguishing objects.

Where the bigger the pixel area the smaller the resolution and vice versa

Takes more time to capture an image with more resolution

Question 8

What is Intensity resolution?

Answer 8

It refers to the number of intensity levels used to represent the image.

Where a higher bit value means larger intensity levels. (Shades of grey)

Takes more time to capture an image with more intensity

Question 9

What are the markings on objective lens’

Answer 9

• Magnification
• Application
• Coverslip Thickness
• Numerical aperture/ Immersion medium
• Working distance

Question 10

How does aperture determine resolution

Answer 10

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

RESOLUTION ≠ MAGNIFICATION

Question 11

How is the function of light and lenses in a light microscope

Answer 11

To illuminate and magnify

Question 12

How can light microscope be used to study cell histology

Answer 12

• look for proteins of interest using antibodies

Question 13

How can light microscope be used to study cell morphology

Answer 13

• look for intact and denatured collagen

Question 14

How can light microscope be used to study cell histology

Answer 14

• study the differentiation of cardiomyocyte-like cells derived from adipocytes
• study cell migration

Question 15

What are the parts involved in electron microscopy

Answer 15

• Electron source
• Electron beam
• specimen
• Electromagnetic lens
• Viewing screen

Question 16

What are the types of electron microscopes

Answer 16

• Transmission EM
• Scanning EM

Question 17

What is the difference between Bright field and Fluorescence mode microscopy

Answer 17

• Different light source
• Fluorescence uses a filter cube whereas bright field uses a normal Wollaston prism

Question 18

How does fluorescence work

Answer 18

• Fluorochrome absorbs energy and emits it at a different wavelength which shows up as a different colour

Question 19

What is photo bleaching

Answer 19

Bleaching of fluorochromes: due to high-intensity illumination the fluorophores might permanently lose their ability to emit light

Question 20

How can fluorescent proteins be used: i.e. GFP

Answer 20

• Naturally found in light-producing cells of cnidarians
• The proteins can be fused with other proteins and introduced in cells via transfection.
• Allows us to tag organisms for live studies

Question 21

What is the difference between confocal and widefield fluorescence microscopy

Answer 21

• A confocal setup uses a dichroic mirror, detector pinhole aperture and a photomultiplier detector
• a wide field setup uses CCD or Ocular to view the samples