Microscopy

Question 1

uses of microscopes

Answer 1

view objects/specimens not visible to the naked eye

Question 2

parts of a microscope

Answer 2

• light source: e.g. sunlight, halogen lamps
• light conditioning system: how we allow light source to reach the specimen, e.g. Kohler illumination
• specimen: on cover glass
• objective: magnifying glass
• detector: e.g. eyes, cameras, photomultiplier tubes to increase signal

Question 3

what needs to be kept constant in light microscopy?

Answer 3

cover slip needs to be between 0.17 & 0.18 mm

Question 4

purpose of incubator box when imaging live specimens

Answer 4

• prevents temperatures from changing

- maintains O2 & CO2 to keep specimen alive

Question 5

examples of experimental timescales

Answer 5

• if looking at embryonic development, will capture image for days
• if looking at motility of microtubules, need to capture images within seconds

Question 6

problems with timescales

Answer 6

need to take images fast for short timescales

need to monitor conditions for longer timescales

Question 7

what is the triangle of frustration?

Answer 7

a compromise between 3 different factors

1. Temporal resolution: how long and fast images need to be taken
2. Spatial resolution: pixel number
3. Sensitivity: pick up images in lower light conditions

Question 8

what is numerical aperture?

Answer 8

the ability of objective to resolve two points that are very close together

• higher the aperture - greater the resolution

Question 9

what is working distance?

Answer 9

distance objective can work from the sample

Question 10

immersion mediums

Answer 10

light travels differently through different mediums, if the objective says oil but you put it in water, light may not travel through

Question 11

light microscopy

Answer 11

modify the intensity & direction of the light source

Question 12

Brightfield

Answer 12

ensures all the light reaches the sample in light microscopy

Question 13

DIC

Answer 13

condense the light through a smaller area, which allows for 3D in light microscopy

Question 14

Phase contrast

Answer 14

uses a phase ring to play with contrast; less detail but more definition

Question 15

Uses of microscopy

Answer 15

• Histology
• Phase contrast
• Time-lapse microscopy

Question 16

Using light microscopy for histology

Answer 16

pros: general idea of tissue
cons: lacks details, hard to distinguish between cells

Question 17

phase contrast light microscopy

Answer 17

helps you see margins of cells where collagen has been denatured

Question 18

time-lapse light microscopy

Answer 18

can see cell migration

Question 19

electron microscopy

Answer 19

• light source is an electron source

- collect the energy from the electrons and transform it into an image

Question 20

transmission electron microscope

Answer 20

• not 3D

- beams of electrons passed through ultra-thin specimen, interacting with the specimen

Question 21

scanning electron microscope

Answer 21

• sample treated with reagents

- 3D

Question 22

fluorescence microscopy

Answer 22

• modify proteins to respond to specific wavelengths

- specimen absorbs light & releases energy

Question 23

what is stokes shift?

Answer 23

the differences between excitation and emission wavelengths

Question 24

what is photobleaching?

Answer 24

• The amount of light that you put in the excitation may break the continuous cycle of absorption, excitation & emission

Question 25

uses of fluorescent proteins

Answer 25

can be fused with other proteins & introduced in cells via transfection, allowing the live study of flourescent tags

Question 26

how to use antibodies to see a protein of interest?

Answer 26

o Secondary antibodies recognise the primary antibody
 The secondary antibody has a fluorophore that that is excited and then emits at a particular wavelength, so you can see your protein of interest in colour

Question 27

difference between conofocal and widefield microscopes

Answer 27

• light source for confocal is a laser that can emit the wavelength
• emission will be enhanced by photomultiplier
• therefore higher resolution & reduced out-of-focus blur
• images are crisp and clear
• only small volumes can be visualised