BIOL 2104 Lecture Notes - Lecture 2: Antigen, Centromere, Fluorophore

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Published on 13 Jan 2018
Course
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Module 2
Tools in cell biology
Tools in biochemistry
Model organisms
Concepts in compartmentalization
A sense of scale between living cells and atoms
Cells are around 20-50 micrometers
Tissue 0.2mm
You can visualize details within the mitochondria
o Look at protein synthesis
What can we see?
Minimum resolvable by unaided eye (0.2 mm)
Minimum resolvable by light microscope (200 nm)
Minimum resolvable by electron microscope (0.2 nm)
Atoms => molecules => organelles =>cells
Light Microscope
It can resolve details 0.2 micrometers apart
Objectives magnify the scale
A liquid is need to refract the light to achieve resolution
Light comes from bottom
Light passes through the specimen
Condenser takes light from the source and condenses the light
o Plant cells
o Bacterium
o Range is from ~1mm to ~1 micrometer
Inference between light waves
When waves are in phase
o Bright signales
Waves interfere (out of phase)
o Dim
o Out of color
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Numerical aperture
There are two lenses
o Objective = collects all light rays coming from image
o Condensers= focuses on cone of light rays onto each point of the specimen
Resolution
o Depends on wavelength of the light
o Formula = 0.61 (wavelength of light) / n (refractive index) * sign theta
o Also depends on the quality of lenses
Ways to obtain contrast in light microscopy
You can distinguish characteristics based on the kind of light
The white light is filtered and only one wavelength can pass through
One particular wavelength and you will only see the particular character
o In phase gives you a bright area
o Out phase yields to more of a dark area
Better resolution of inner structure with different phases
If you want more better resolution in terms of structure inside then you need to use a
different strategy
Sample processing:
Take individual tissues and put together and then stain it and it is mounted under a glass
cover slip
Resolution depends on the quality of the light source
Some molecules can be localized in cells by fluorescence microscopy
Some dyes are specific
Fluorescence microscope
Two sets of filters
1st barrier filter lets only blue light with wavelength between 450 and 490 nm
o This wavelength excites the dye and that dye emits a longer wavelength then that
goes through another wavelength
o Beam- splitting mirror reflects light below 510 nm but transmits light above 510
nm
2nd filter only lets emitted light to be shown
o Cuts out unwanted fluorescent signals
Things blocked is black
Things that filter in is colored
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Fluorescence probes: QD
Quantum dots tiny particles that are so small that the size of the particle gives physical
features
The size gives physical features to the dot
Size dot gives a different spectrum of color
Intensity of color fade then it’s gone
It’s a fast process
It can sustain the intensity of color for weeks
Ab
Antibodies are used
After primary antibody attaches to the antigen a secondary antibody comes in and it has a
sort of dye
Definition of Fluorescence
Visualize structures
Fluorescent component
Sensitivity and selectivity
Types of dyes
Ethidium bromide
Alexa fluor 350
Fluorescein
How it works
The fluorophore molecule absorption then it is in a excited state then it emits the light
waves out
When it returns to basic state (groud state) it emits light
The absorbed light and emitted light are two different wavelengths
The light wavelength of the light is different from light wave of emission
When light is in excitation state the energy goes up and the energy is lost and then it is emitted
Light energy emitted longer wavelength that the energy absorbed this means the color of
the light that is emitted is different from the color of the light that has been absorbed
Photobleaching
First sample is put in microscope
Light has been hidden that it exhaust the photo and it cannot show anymore
Change to another part of cover slip to visualize other cover cells
The picture is faded or destroyed
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