Light microscopy

Question 1

why are some things not visible?

Answer 1

Not enough photons
Photons outside the visible spectrum for our photoreceptors
Objects too small to resolve
Objects do not interact with or emit visible light
Objects and surrounding material interaction with light the same way (same refractive index)
Objects bend light in a way that hides them

Question 2

how does a convex lens cause magnification?

Answer 2

refract light in a way that increases the angle between the top and bottom of the object, light spreads out from focal point, making object appear larger

Question 3

what is magnification and how is it calculated?

Answer 3

size of object : size of image
objective lens x ocular lens

Question 4

how do microscopes solve the problem of too little photos to view a sample

Answer 4

use of condenser lens to focus light onto the sample

Question 5

solution to wrong kind of photons

Answer 5

use detectors or cameras sensitive to other wavelengths

Question 6

solution to objects being too small for array of photoreceptors to resolve

Answer 6

compound lenses to magnify the sample

Question 7

resolution

Answer 7

smallest distance at which two objects can be distinguished from one object

Question 8

what determines resolution?

Answer 8

how much:
light is lost between specimen and lenses
light is gathered by lenses
wavelength used for illumination

Question 9

refractive index and resolution relationship

Answer 9

match refractive index to give a higher resolution
refraction at interface between glass and air = less light enters objective lens

Question 10

what is meant by the angular aperture of the objective lens

Answer 10

how much of the light is collected by the objective lens
higher angle = more light gathered

Question 11

relationship between wavelength and resolution

Answer 11

shorter wavelength = higher resolution

Question 12

limit of resolution for light vs electron microscopes

Answer 12

around 200nm vs 2nm

Question 13

phase contrast

Answer 13

boosts contrast resulting from differences in refractivity between cells and cellular structures

Question 14

DIC

Answer 14

uses polarised light to boost contrast from diffraction of light within the sample

Question 15

solution to objects not interacting with visible light

Answer 15

stains or labels

Question 16

fluorophores

Answer 16

molecules that emit energy in the form of photons of a longer wavelength when hit with specific shorter wavelength photons
eg quinine which is excited by UV light and emits blue light

Question 17

mechanism of fluorescence

Answer 17

photons of a particular wavelength carrying a particular amount of energy excite electrons in the fluorophore. as electrons go back to ground state, energy is released as emitted photon. less energy is released as light than was absorbed so emitted photons have longer wavelengths

Question 18

fluorescence microscopy

Answer 18

Light passes through objective lens to and from the sample
Goes through excitation and emission filters
Dichroic (‘two-colour’) mirror: shorter wavelengths reflected onto the sample, longer wavelengths pass through to detector

Question 19

DAPI and Hoechst

Answer 19

small molecules that bind to DNA, fluorescent labels
emit fluorescence in UV/blue range

Question 20

phalloidin

Answer 20

binds F actin

Question 21

fluorophore-conjugated molecules

Answer 21

can be any colour
eg phalloidin

Question 22

GFP

Answer 22

green fluorescent protein
derived from jellyfish
encodes fluorescent protein that can be incorporated into plasmids and genomes

Question 23

hybrid tagged protein

Answer 23

adding fluorescent protein sequence at beginning or end of another protein

Question 24

how gene expression can be monitored

Answer 24

adding FP gene after a promoter
when it is active, the fluorescent reporter fluoresces green

Question 25

how to improve resolution in fluorescence microscopy?

Answer 25

reduce out-of-focus fluorophore excitation
reduce out-of-focus emitted light collection

Question 26

confocal fluorescence microscopy

Answer 26

removes out of focus light

Question 27

two photon confocal microscopy

Answer 27

improves resolution in z axis by only exciting fluorophores where bean of light cross paths

Question 28

digital deconvolution

Answer 28

use of mathematical algorithms to improve images by reducing blurring effects caused by light diffraction

Question 29

super resolution microscopy

Answer 29

higher resolution than diffraction limit

Question 30

epitopes, monoclonal and polyclonal antibodies

Answer 30

part of antigen that the antibody recognises
monoclonal antibodies: bind one epitope
polyclonal antibodies: mixture of antibodies that recognise multiple epitopes on the same antigen

Question 31

direct immuno-labelling

Answer 31

primary antibody with fluorescent label attached binds to antigen

Question 32

indirect immuno-labelling

Answer 32

secondary antibody has the fluorescent label and binds to primary antibody
This allows for signal amplification as multiple secondary antibodies can bind to a single primary antibody, increasing the fluorescence intensity.