Microscopy and Spectrophotometry Flashcards
(16 cards)
basic microscopy
- used to visualize objects down to 0.2 um
- 2 lenses in combination can magnify N OBJECT 2000X (1000X for bacteria)
- although a 200x lens is comparable in price to a full standard microscope
- quality of image is dependant on: thickness of selection, lighting, quality / cleanliness of lenses and staining
magnification and resolution
- the bigger image is useless without more detail - empty magnification
- to increase the amount of detail in an image is to improve the resolution
- formally defined, angular resolution = the smallest resolvable distance between 2 objects
how can resolution be imporved
- large NA, gives better resolution
- smaller distance = better resolution ( R or wavelength as small as possible)
- can change light colour (more blue ) for better resolution
refractive index and oil immersion
- to use oil immersion you must have a lense that:
- is close enough to make continous slide oil lens connected
- doesnt react with oild
- usually 100
spectrophotometry
- spectroscopy + photometry
- sp = the study of absorbtion and emission of radiation
- ph = the measurement of intensity of radiation
- in a solution every component may interact with a light (electromagnetic radiation) source
- a study of the interaction of light on a sample can tell us what compounds may be present, and in what quantities
light
- made up of protons
- higher energy = shorter wavelength = blue
- lower energy = longer wavelenght red
- light interacting with matter can pass through or interact depending on the compound and the wavelenght
- most often interactions generate heat, but they can also react with some compounds (or make them reactive)
- electrons in molecules exist in orbits/shells)
- photons of the correct wavelength, with the right compounds can cause releases of less energetuc photons = fluorescence
cuvette
- a tube that holds the sample
- round test tubes will reflex light differently so it must be square containers
transmittance
- the proportion of light that the sample allows to pass (usually %) - if sample is .9 it means ()% of light passes through
- problem because absorbance of light isnt standard
absorbance
- the amount of light the sample blocks, calculated as A=log (1/T)
- as it is a log scale, values 0-2 are typical
blank
a comparison sample with none of the measured substances
standard curve
- a series of known samples used to generate comparison data
general considerations
- clean cuvette - fingerprints and condensation block light
- square cuvettes have to be lined up carefully
- calibrations ( what needs to be zeroed) the blanks to make sure theres 0 absorbtion
- warm up time? - 20 minutes before turn it on so it cannot get any brighter during experiment
- ensure adequate volumes in cuvette
- seat the sample, shut the door - fully nothing blocking
- is the cuvette compatible with wavelength
spec use: absorbance spectra and identification compounds
- which wavelengths are absorbed and how much is a function of the molecular structure of a compound
- measure a sample at various wavelengths and plot a curve of the absorbance to produce an abs spectrum
- also useful to determine the optimal wavelength for measuring abd for determining amount
direct spectrophotometric measurements
bacterial cells - light scatters measuring at 595-600nm
- there are different conversation factors go from abs to cells depending on the species
DNA/RNA - absorb light the same, reguardless of source, a single conversion factor can be used
- the same goes for DNA and RNA
protiens - directly absorbs light at 280nm
- extent of absorbance varies by conformation and aa complement, so a single conversion factor cant be used
- more accurate and robust assays use indirect measurements
bradford assay
- measurement of colour changes when coomassie blue dye binds protein
- unbound dye abs max = reddish brown
- bound dye abs = blue
- ## strongest interaction with basic amino acids - abs can vary by aa composition and protein structure
biuret assay
- measurement of colour change when copper 2 sulfate reacts with protiens under alkaline conditions
- solution turns purple (Amax=540 nm)
- reaction takes time (15 minutes) as copper reacts with peptide bonds
