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BMSC 240 > Lectures 4, 5, 6 > Flashcards

Lectures 4, 5, 6 Flashcards

1
Q

What are microscopes used to visualize? (Lenses and size)

A

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

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2
Q

What are the 4 things the quality of image dependant on?

A

Thickness of section- we can control (L4S3)
Lighting- amount and angles
Quality/cleanliness of lenses
Staining

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3
Q

Study the anatomy of a microscope L4S4 and L4S9

A

Okay

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4
Q

How do you focus a microscope? (9 steps)

A
  1. Turn on light, open diaphragm, raise condenser
  2. Use lowest power lens (bigger depth of field)
  3. Put slide on stage
  4. Raise stage to highest position
  5. Position slide so something is in sight
  6. Use both eyes (adjust eyepiece)
  7. Slowly lower slide until object of interest comes into focus
  8. Wiggle slide to find object of interest
  9. Select next objective, use only fine focus to adjust microscope
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5
Q

What is magnification and resolution?

What is empty magnification?

A

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

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6
Q

What is the equation for resolution?

How can it be improved?

A

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

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7
Q

What is the point of immersion oil?

A

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

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8
Q

What is spectrophotometry?

A

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

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9
Q

What does higher energy and loess energy mean for wavelengths?
What is fluorescence?

A

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

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10
Q

Study basic spectrophotometer on L4S12

A

Ok

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11
Q

What is cuvette, transmittance, absorbance, blank, and standard curve in spectrophotometry?

A

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

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12
Q

What are the 2 things that can block light on a cuvette?

What needs to be zeroed on a spectrophotometer?

A

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

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13
Q

What is the warm up time of a spectrophotometer?

A

20 mins
Lights getting brighter during that period which could result in getting over 100% transmittance if using blank too early

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14
Q

What is the absorption spectrum?

A

Measuring a sample at different wavelengths and plotting a curve of the absorbance

Used to determine the optimal wavelength for measuring absorbance

L5S4-5

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15
Q

What is the beer-lambert law equation involving absorbance, molar absorptivity, path length, and concentration?

A

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

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16
Q

What are the wavelength standard setting in a spectrophotometer for bacterial cells, DNA or RNA, and proteins?

A

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)

17
Q

What happens when comparing the wavelengths for DNA and protein (260 nm, 280nm)?
What is A260/A280 compared to A260/A280?

A

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

18
Q

What is the Bradford Assay? (Indirect protein assays)

How does the amount of proteins affect absorbance reading?

A 
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

19
Q

What is the Biuret Assay? (Indirect protein assay)

A

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

20
Q

What is technological miniaturization?

A

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

21
Q

How do we quantify amounts for direct assays on graphs?

A

To quantify amounts to use, looks for absorption peaks that are isolated from other absorption peaks
L6S5

BMSC 240 (9 decks)

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