A Introduction To Microscopy

Question 1

What is microscopy?

Answer 1

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

Question 2

What are the essential parts of the microscope?

Answer 2

Detector
Objective
Specimen (cover glass)
Light conditioning system (aim to concentrate beam of light towards sample, avoid reflection and filter types of light)
Light source

Question 3

How is a light microscope specimen prepared?

Answer 3

Cover glass

Sample surrounded by embedding medium

Glass slide

Question 4

How can live specimens be viewed under a microscope?

Answer 4

Have a box or cube for the specimen

Small changes in temperature lead to thermal extension or contraction in the microscope stand,stage and objective - changing plane of focus

Incubator box and precision air heater ensures that the temperature of specimen and microscope remain equilibrated and tightly controlled

Question 5

How is a CO2 atmosphere maintained in a live specimen box?

Answer 5

• controller allows to control airflow + CO2%

OR

• a air tight table top encloses the liver cell culture device

Question 6

What is the problem with observing short and fast events under microscopy?

Answer 6

Artefacts in multichannel/4D imaging

Question 7

What is the problem with observing longer processes under a microscope?

Answer 7

• stability, visibility

Question 8

What is the “triangle of frustration”?

Answer 8

Temporal resolution - sensitivity - spatial resolution

Question 9

What is the relationship between pixel area and resolution?

Answer 9

The higher the resolution the smaller the pixel area

Question 10

What is the relationship between intensity levels and resolution?

Answer 10

With greater intensity levels and greater bits we will have greater resolution.

Question 11

What do the markings on objectives of a microscope mean?

Answer 11

Applications :DIC
Working distance (mm) :WD
Cover slip thickness: -
Numerical aperture/immersion medium: 1.30 Oil

Question 12

What is the relation between the numerical aperture and resolution?

Answer 12

The higher the numerical aperture the greater the resolution power of the objective.

Question 13

What is light microscopy?

Answer 13

Uses light and lenses to illuminate and magnify

Has a:
- light source
- condenser
- sample
- objectives

Question 14

What are the 3 types of light microscopy?

Answer 14

DIC
Phase
Colour brightfield

Question 15

What is the advantages and disadvantages of using full light microscopy in histology?

Answer 15

Advantages - Have a overview of the tissue and identify areas of interest

Disadvantages - unable to see how the cells interact with each other

Question 16

How are proteins of interest found under a light microscope for histology?

Answer 16

Using antibodies to identify and “dye” specific cells

Question 17

How can light microscopy be used to analyse cell morphology?

Answer 17

Microscopy of fibroblasts cultured in:

Intact collagen
Denatures collagen

Can observe structural changes

Question 18

When can light microscopy be used for time lapses?

Answer 18

Heart cell differentiation

Differentiation of cardiomyocyte-like cells derived from adipocytes

Or in cell migration

Question 19

What is electron microscopy?

Answer 19

Scanning and transmission

Components:
- electron source
- electron beam
- specimens
- electromagnetic lense
- Viewing screen

Question 20

What is fluorescence microscopy?

Answer 20

Components:
- fluorescence source of light
- fluorescence filter cube

Question 21

Explain the absorption and emission of fluorescence light

Answer 21

1. Excitation:
   Molecules absorb light and her excited
2. Loss of energy: (STOKES SHIFT)
   Molecules loose energy
3. Emission:
   Molecule emits light as it looses energy

Question 22

What is the stokes shift?

Answer 22

Due to the energy loss the emitted light is shifted to a longer wave length relative to the excitation light.

Absorption always has a slightly lower wavelength than emission

Question 23

What is photo bleaching?

Answer 23

Bleaching of fluorochromes due to high intensity illumination the fluorochromes might loose permanently they’re ability to emit light

Question 24

Name some common fluorochromes in light microscopy

Answer 24

ultraviolet/blue:
DAPI

Blue/green:
FITC
GFP
Cy2

Green/red:
TRITC
Cy3

Red/infrared:
Cy5

Question 25

Where are GFP fluorescent proteins found?

Answer 25

Naturally found in light-producing cells of cnidarians

Question 26

How to tag cells with fluorescence?

Answer 26

Antibodies vs protein infusion

Plasmid construction inserted into cells and fluorescent proteins expressed

Question 27

What are the two different fluorescence microscopes?

Answer 27

Confocal:
Laser
Detector pinhole aperture
Dichroic mirror
Photoplier detector (detects light emitted by fluorescence)

Widefeild:
Original set up

Question 28

What is the difference between widefeild and confocal fluorescence microscopy?

Answer 28

Confocal advantages:
higher resolution and reduced out of focus blur make images more crisper and clearer

Confocal imitations:
Only a small volume can be visualised by confocal microscopes at once
Bigger volumes needed time consuming sampling and image reassembling

Question 29

What can be observed using a confocal microscope?

Answer 29

Vaccinia virus uses actin tails just after crossing the plasma membrane of infected cells