Lectures 4, 5, 6 Flashcards
What are microscopes used to visualize? (Lenses and size)
Objects down to 0.2um (so if you can see them on a microscope, they’re too big to be involved in sterilization)
2 lenses in communication can magnify an object 2000x
What are the 4 things the quality of image dependant on?
Thickness of section- we can control (L4S3)
Lighting- amount and angles
Quality/cleanliness of lenses
Staining
Study the anatomy of a microscope L4S4 and L4S9
Okay
How do you focus a microscope? (9 steps)
- Turn on light, open diaphragm, raise condenser
- Use lowest power lens (bigger depth of field)
- Put slide on stage
- Raise stage to highest position
- Position slide so something is in sight
- Use both eyes (adjust eyepiece)
- Slowly lower slide until object of interest comes into focus
- Wiggle slide to find object of interest
- Select next objective, use only fine focus to adjust microscope
What is magnification and resolution?
What is empty magnification?
Magnification- a bigger image is useless without more detail
Empty magnification is making something bigger without adding any detail
Resolution- to increase the amount of detail in an image is to improve resolution
Smallest resolvable distance between 2 objects
What is the equation for resolution?
How can it be improved?
R=0.61λ/NA
NA= numerical aperture of the lens
NA= nSinθ
L4S7
Resolution can be improved so smaller distance between objects (smaller resolution is better)
Or change wavelength of light by changing colour since red and blue light have different wavelengths, red is longer than blue so use blue for smaller R value
What is the point of immersion oil?
It reduces light that bends and scatters from glass by getting it all into the lens
Must have a lens that is close enough to make continuous glass-oil-lens contact and does not react with oil
L4S8
What is spectrophotometry?
spectroscopy + photometry
Spectroscopy= study of absorption and emission of radiation
Photometry= measurement of intensity of radiation
The study of the interaction of light on a sample can tell us what compounds may be present and in what quantities
Shine light, see how much gets through and how much is blocked
What does higher energy and loess energy mean for wavelengths?
What is fluorescence?
Higher energy= shorter wavelengths (blue)
Lower energy= longer wavelengths (red)
In fluorescence, light hits an object, takes electrons, increases them to higher orbital which creates new photon that creates different wavelengths
Photons of correct wavelength can bump up electrons
Study basic spectrophotometer on L4S12
Ok
What is cuvette, transmittance, absorbance, blank, and standard curve in spectrophotometry?
Cuvette- tube that holds sample
Transmittance- proportion of light that the sample allows to pass (in percent) 0-100%
Absorbance- amount of light the sample blocks calculated as A=log(1/T) 0-2 values are typical
T is transmittance as a decimal value
Blank- comparison sample with none of the measured substance (Ex: 100% transmittance)
Standard curve- an series of known samples used to generate comparison data
L4S14
What are the 2 things that can block light on a cuvette?
What needs to be zeroed on a spectrophotometer?
Finger prints and condensation
Some cuvettes cant be sued for certain wavelengths
Proper blank must be zeroed, clean cuvette to tell machine what is 100% transmittance and 0% absorbance
What is the warm up time of a spectrophotometer?
20 mins
Lights getting brighter during that period which could result in getting over 100% transmittance if using blank too early
What is the absorption spectrum?
Measuring a sample at different wavelengths and plotting a curve of the absorbance
Used to determine the optimal wavelength for measuring absorbance
L5S4-5
What is the beer-lambert law equation involving absorbance, molar absorptivity, path length, and concentration?
The taller the glass, the darker the brew, the less light that gets through
A=elc
A- absorbance
e- molar absorptivity in L/mol cm (specific to certain wavelengths and certain compounds)
l- path length in cm (distance in cuvette usually 1cm standard)
c- concentration in mol/L (more concentrated, more light blocked)
L is usually constant, A is measured, if e is known then c can be calculated, and vice versa
What are the wavelength standard setting in a spectrophotometer for bacterial cells, DNA or RNA, and proteins?
Bacterial cells- light scattering measured at 595-600 nm
DNA or RNA- absorb UV light at 260 nm (peak)
260 finds how much DNA or RNA is in a sample (all DNA is same doesn’t matter the AT to CG pair ratio)
ABS x dilution factor x 50= [DNA] in ug/mL
Proteins- directly absorb UV light at 280 nm (peak)
Proteins have a lot of variability cause of the difference in amino acid chains (even two exact proteins at different temps will absorb diff wavelengths)
What happens when comparing the wavelengths for DNA and protein (260 nm, 280nm)?
What is A260/A280 compared to A260/A280?
By comparing the A260/A280 ratio we can get an idea of the quality of a DNA or protein isolation
260/280 for pure DNA is 2.0 (DNA absorbs twice as much at 260 compared to 280)
260/280 for pure protein is 0.55
L5S8-9
What is the Bradford Assay? (Indirect protein assays)
How does the amount of proteins affect absorbance reading?
Measurement of colour change when Coomassie blue binds protein Uses dye (CB), that changes wavelength of light it absorbs when it is bound to a protein compared to when it is floating More protein, less absorbance (ABS can vary by aa composition and protein structure)
L5S10-12
What is the Biuret Assay? (Indirect protein assay)
Measurement of colour change when Copper II Sulfate (CuSO4) reacts with proteins under alkaline conditions
Solution turns purple (Amax=540nm)
Reaction takes time (15 mins) as copper reacts with peptide bonds
L5S13
What is technological miniaturization?
Cuvette-less spectrophotometry
Sample 5uL-0.5uL placed on sensor then light head flipped down and measurement taken at 0.2mm to 0.5mm path length
Can do Abs spectrum from 200 to 900nm in 5 secs
Sample can be recovered
L5S14
How do we quantify amounts for direct assays on graphs?
To quantify amounts to use, looks for absorption peaks that are isolated from other absorption peaks
L6S5
