Lab 10 Flashcards
Anatomy of microscope
- Ocular lens
- Objective lens
- Condenser
- Light source
What is most important component of microscope?
Objective lens
What are the two most relevant features of the objective lens?
- Magnification
- Numerical Aperture
What is numerical aperture?
Measure of the angle at which light can be collected
First microscopes vs present day microscopes
First microscope only had an objective lens and now an ocular lens
What is differential Interference Contrast
Exploits differences in density of different cellular compartments (nucleus, large organelles, cytoplasm)
Why fluorescence imaging?
Fluorescenece microscopy achieves higher contrast than other techniques
- Potential of highlighting localization among millions of other molecules
4 important elements of the fluorescence microscope
- Light source (broad spectrum)
- Filter cube
- Objective lens
- Sensitive camera
What is a dichroic mirror?
Reflects on wavelength and transmits another
Why are CCD cameras commonly used in modern microscopy?
- High sensitivity
- Large dynamic range
- Converts photons to electrical charges that can be recorded as intensities
- Monochromatic
- Determine sensitivity of microscope system
Steps in microscope analysis
- Localizing molecules
- Linking molecules
- Capturing diffusive states
- Classifying molecules based on movement
What is an F-test?
Compares variance between groups
- Null hypothesis that average value is same between two different samples
How does a spectrofluorometer work?
Used to quantify properties of fluorescent molecules
- Samples excited at a specific wavelength and properties and intesnity can be identified
What is special about spectrofluorometers?
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
3 parts of lab 10
- Excitation and emission spectra of mNG variants - sample prep
- Fluorescence of mNG variants as a function of pH
- Data analysis
Excitation scan
Excitation wavelength varies, wavelength at which data is collected is fixed
Emission scan
Excitation wavelength is fixed, wavelengths of light collected varies
What are we trying to answer in part 1?
Where mNG mutants are:
- More or less fluorescent
- Whether they have distinct excitation and emission spectra
compared to wildtype mNG
How might different mutations affect fluorescent properties?
- Shift in excitation/emission spectra
- Change of fluorescence intensity
- Increase/decrease of the stability of a fluorescent protein
What else are we loading into wells with the purified protein?
Buffer-only sample that serves as a reference
Too high vs too low mNG concentrations
- Too low: won’t detect
- Too high: saturate detector, resulting in inaccurate measurements
What is the pKa of mNG?
5.7.
Why might we lower the pKa of mNG?
It is useful for its use in acidic environments
What question are we trying to answer in part 2?
Whether fluorescence pKa of mutants is distinct (less or more acific-senstive)
