Chapter 10-Bioimaging

Question 1

Advantages of inverted microscope

Answer 1

• compatible with live cell imaging

Question 2

Features of phase contrast microscopy

Answer 2

• does not require cell staining
• special objective required
• based on the principle that diff organelles have diff refractive indices, resulting in diff phase shift and hence intensity diff in phase rings

Question 3

Define fluorescence

Answer 3

• process where substance absorbs photons and subsequently emits photons as emission light
• molecular: transition of electron from one molecular orbital (ground state) to another with higher energy

Question 4

Define stokes shift

Answer 4

Emission fluorescent light has a longer wavelength (lower energy per photon) than the absorbed light (due to energy loss between the time a photon is absorbed and emitted)

Question 5

Define photobleaching

Answer 5

A fluorophore permanently loses its fluorescence due to photon-induced chemical damage

Question 6

Why is detergent used in immunofluorescence labelling?

Answer 6

Low concentration dissolves part of the cell membrane to increase permeability of the cell to allow antibodies to enter

Question 7

Function of excitation filter in wide-field fluorescence microscopy

Answer 7

• select bandwidth of light to excite fluorophore

- as narrow as possible

Question 8

Function of dichroic mirror in wide field fluorescence microscopy

Answer 8

1. Reflect excitation light

2. Transmit emission light and suppress scattered excitation light

Question 9

Function of emission filter in wide field fluorescent microscopy

Answer 9

• pass fluorescence emission light and further block scattered excitation light

Question 10

Limit of light microscope resolution

Answer 10

d=200nm

Question 11

What does the numerical aperture (NA) determine?

Answer 11

• resolution of microscope system

- NA=nsina

Question 12

What is spatial resolution (d)?

Answer 12

• smallest distance of the 2 resolvable point light sources

- the smaller the d value, the higher the resolution

Question 13

Practical resolution of TEM

Answer 13

0.1nm

Question 14

TEM

Answer 14

• image area brightness is proportional to electrons collected by the detector
• transmitted electrons detected as light areas
• darker areas occur where electrons have been scattered or absorbed by sample

Question 15

Define FSC

Answer 15

• refracted by cell in the forward direction and in the same direction the light was already travelling
• used to determine cell size
• the smaller the cell, the less scattered light

Question 16

Define SSC

Answer 16

• refracted by cells in a diff direction than the laser beam’s axis
• used to measure granularity and complexity of cell
• the higher the complexity, the more the scattered light (higher intensity)

Question 17

Advantages of flow cell cytometry

Answer 17

1. Multiparametric analysis (physical and biochemical—scatter and fluorescence respectively)
2. High speed
3. Quantitative