BIOL 2104 Lecture Notes - Lecture 2: Antigen, Centromere, Fluorophore
20 views12 pages
Published on 13 Jan 2018
School
Department
Course
Professor
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
find more resources at oneclass.com
find more resources at oneclass.com
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
find more resources at oneclass.com
find more resources at oneclass.com
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
find more resources at oneclass.com
find more resources at oneclass.com