Lab 10

Question 1

Anatomy of microscope

Answer 1

• Ocular lens
• Objective lens
• Condenser
• Light source

Question 2

What is most important component of microscope?

Answer 2

Objective lens

Question 3

What are the two most relevant features of the objective lens?

Answer 3

• Magnification
• Numerical Aperture

Question 4

What is numerical aperture?

Answer 4

Measure of the angle at which light can be collected

Question 5

First microscopes vs present day microscopes

Answer 5

First microscope only had an objective lens and now an ocular lens

Question 6

What is differential Interference Contrast

Answer 6

Exploits differences in density of different cellular compartments (nucleus, large organelles, cytoplasm)

Question 7

Why fluorescence imaging?

Answer 7

Fluorescenece microscopy achieves higher contrast than other techniques
- Potential of highlighting localization among millions of other molecules

Question 8

4 important elements of the fluorescence microscope

Answer 8

• Light source (broad spectrum)
• Filter cube
• Objective lens
• Sensitive camera

Question 9

What is a dichroic mirror?

Answer 9

Reflects on wavelength and transmits another

Question 10

Why are CCD cameras commonly used in modern microscopy?

Answer 10

• High sensitivity
• Large dynamic range
• Converts photons to electrical charges that can be recorded as intensities
• Monochromatic
• Determine sensitivity of microscope system

Question 11

Steps in microscope analysis

Answer 11

1. Localizing molecules
2. Linking molecules
3. Capturing diffusive states
4. Classifying molecules based on movement

Question 12

What is an F-test?

Answer 12

Compares variance between groups
- Null hypothesis that average value is same between two different samples

Question 13

How does a spectrofluorometer work?

Answer 13

Used to quantify properties of fluorescent molecules
- Samples excited at a specific wavelength and properties and intesnity can be identified

Question 14

What is special about spectrofluorometers?

Answer 14

Can be used to define a fixed wavelength of excitation and emission to measure fluorescence
- Can also scan wavelengths at which excitation and emission are the highest - useful for characterization

Question 15

3 parts of lab 10

Answer 15

• Excitation and emission spectra of mNG variants - sample prep
• Fluorescence of mNG variants as a function of pH
• Data analysis

Question 16

Excitation scan

Answer 16

Excitation wavelength varies, wavelength at which data is collected is fixed

Question 17

Emission scan

Answer 17

Excitation wavelength is fixed, wavelengths of light collected varies

Question 18

What are we trying to answer in part 1?

Answer 18

Where mNG mutants are:
- More or less fluorescent
- Whether they have distinct excitation and emission spectra
compared to wildtype mNG

Question 19

How might different mutations affect fluorescent properties?

Answer 19

• Shift in excitation/emission spectra
• Change of fluorescence intensity
• Increase/decrease of the stability of a fluorescent protein

Question 20

What else are we loading into wells with the purified protein?

Answer 20

Buffer-only sample that serves as a reference

Question 21

Too high vs too low mNG concentrations

Answer 21

• Too low: won’t detect
• Too high: saturate detector, resulting in inaccurate measurements

Question 22

What is the pKa of mNG?

Answer 22

5.7.

Question 23

Why might we lower the pKa of mNG?

Answer 23

It is useful for its use in acidic environments

Question 24

What question are we trying to answer in part 2?

Answer 24

Whether fluorescence pKa of mutants is distinct (less or more acific-senstive)

Question 25

What are two buffers we are making?

Answer 25

• Acid-based
• Basic disodium hydrogen phosphate-based buffer