Lecture 2 – Visualizing Cells pp. 579-598; 604-611
May5th
How do we study these subjects that are so small?
Human eye can only see a fraction of the scale needed. To probe down all to the way of
the atoms, microscopes are used to see/resolve structures that our naked eye cannot.
Light microscope
Uses photons of light. Because light travels in a wave, it causes it to behave funny
ways when exiting water to air, creating a bend in the light.
Leaving certain medium causes bend, and this can be manipulated, to stretch a point of
light (a point has multiple rays of light hit it, and these rays are bent to cover a larger
area, and making a larger image to view via the objective/ocular lense)
- white light is used. A condenser lense is used to focus the light onto the object
(it concentrates light onto the specimen, in order to make it bright)
- the light interacts with specimen, and then emitted, and the compound
objectives (objectives + eyepiece) capture the light from the specimen and
spread it out from one tiny little dot into a larger spreaded area.
o Again, this uses the bending property of the light to magnify the image
There are four major components that are crucial to viewing small/larger objects from
distance.
1. Detection: You can detect something is there, but you have no idea what it is.
2. Magnification: Increases the size to it is more clear, but very little
information can be gleaned from how blurry it would look.
3. Resolution: increases the number of pixels per one (we are resolving each
one pixel into 15 pixels). Seeing more detail of the object. More pixels per
space being averaged out.
4. Contrast: Proper illumination is required. Ability to look at side by side
pixels and tell them apart from each other.
Resolution: The ability to be able to look at two objects side by side and be able to see
them as two different objects. Go any further, and you cannot separate things apart
anymore. Limit of resolution is a distance measurement
- Why is there a limit of resolution?
o Light interference: Two waves/rays of light travelling in synchrony
provide an emphasized/amplified total wave. If out of phase, the
waves cancel out, and create a smaller amplitude, making for a
dimmer image.
o Light diffraction: The wave length of the light is larger than the
particles being measured. The wavelegnths are diffracted and create
undulating wave patterns due to interference.
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o Light is a wave that has it’s own resolution
▪ Resolution depends on wavelength (i.e., blue light is better than
red light to view small things. The refractive index of the medium
it travels through is important too, because it determines the
angle of spreading of the light. The halg angular width of the
cone of light collected by objective lense plus the other two are
the factors involved for the resolution. Small numbers for
resolution are better. Always choose the smallest wavelength to
find the best resolution.
▪ Numerical aperture = n sin theta
• This measurement is directly proportional to your
resolution. Larger NA = smaller things you can resolve =
good!
• But it does limit the depth, meaning that stuff below and
above will be more blurry.
▪ R = (0.61 lambda)/NA
▪
-
Bright field microscopy
Take some cells, shine some light, see what you got (low contrast)
Manipulation of light for imaging can be done in a variety of ways. i.e., differential
interference contrast
- interference
- phase-shifting
- DIC creates 3D images.
- Phase contrast vs DIC is that DIC uses polarized light (all in the same
direction, so interference can be looked for)
o Lightwaves in phase before hitting cell, but once travelling through the
cell, gets out of phase, creating different contrasts via amplitudes of
light.
- Dark field microscopy involves light shining from the side, instead of coming
directly from middle. The result gives either darkness, or a strange wave of
light that is diffracted, which is visible to eye. Used for dense stuff
Colour can be manipulated, by processing the sample with dyes and then emits only the
colour which the dye permits (if the sample latches to the dye).
- this requires loads of processing.
- Histology requires loads of fixing. (meaning that the samples become “frozen”
or still in motion) Embedding, (water is removed and wax is added to stabalize
and harden the cell) Sectioning (thin slices of the tissue), and Stain
*it’s important to know where the location of the proteins are, to determine the
functioning in the body. If not in the right place, can cause disease
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Fluoresence Microscopy:
Flourescent compounds capture photons of specific wavelength and emit another
wavelength that is observed in the microscope via filter blocks. There is a loss of energy,
giving a lower energy wavelength emmitance.
e.g., FITC is one example fluorchrome that captures light at 488nm, 520nm is emitted,
which is a larger wavelength and hence lower energy!
Rhodamine absorbs 540nm, emits 630nm. (green light became red light!)
Excited by high energy…. Gives off low energy
The shift from excitation to emission is called the stokes shift (K) measured by x axis
shift of amplitude
Order of light emittance:
Light source is shined through the excitation filter, only letting a certain colour light
exit (specifically the light that will excite the fluorochrome), then the light goes and
bounces down from the dichroic mirror, hitting the object. The object’s fluorochromes
are excited, allowing the excited light to leave and travel through the dichroic mirror
and towards the emission filter. Light passes through the emission filter (making sure
only the emission light of coice is emitted through to eyes)
The point of emittance of the object also determines the resolution/quality of the image.
To solve this, computers can use image processing to see the final image. Confocal
microscopes can be used, spinning disc confocal miscroscope can be used (the last two
are super duper expensive)
Image deconvolution can be used, where multiple images are taken at different
depths (like an MRI). The computer compares all the images to figure out the origin of
the light from the image and creates a clearer image
Laser scanning confocal microscopes uses a laser (which has precision) that goes
through a constriction (i.e., a pinhole) in front of the laser. There is another pinhole
leading towards the objective, meaning that 95% of out-of-focus light is excluded from
the ocular. This is repeated for the image PIXEL BY PIXEL to create a plane.
(on the slide, the green shaded area is emission light while the blue is excitation light)
Immunocytochemistry
VDJ recombination > disease
The thing is you can make antibodies for anything you want, so use this to manipulate
studies of living organismal tissue
Blank genes given at birth, B cells alter the gene sequence of the blank in order to
modify the protein sequence made.
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- most of these get rid off, because it is useless (binds to self), or creates a
antibody that binds to something else (e.k.e a ligand)
- it would be useful for antibodies to bind to foreign things
- when the antibody is duplicated, it is used to make a working antibody.
- Antibodies can bind anything (including things of your own, which are
killed before used)
- Proteins you want to make antibodies to are called antigens (proteins to
molecules)
May 9 – recording starts at slide 34 (reviewed first)
th
How to make a antibody in lab thatis for a specific protein:
1. inject mouse with the protein that is an antigen of choice
2. take cells from the mouse that has mechanisms to create the antibodyDAPI
and combine it with the cancer cells
3. creates a forever creating antigen cell
How does Indirect immunocytochemistry work?:
- epitope is the site where the antibody recognizes and binds to on the B cell.
- Antigen = molecule
- Antibodies = bind to the antigen
- Epitope = specific surface to bind to (many epitopes on cell, each is unique to
an antigen)
o A population of antibodies that bind to only one epitope = monoclonal
o A population of different antibodies that bind to different locations =
polyclonal
- inject mouse with the protein that is an antigen of choice (with 2 antibodies,
primary, and the secondary, which binds to the primary antibody, where the
secondary antibody has a marker-coupled antibody. 2ndary antibodies carry a
molecule that makes them fluorescent ) this is called indirection
amplification .
- the secondary has loads of epitopes, to allow different antibodibodies to bond to
it. The primaries can be either mono or polyclonal, but tend to be monoclonal
- Bcell = 1 antibody = 1 epitope
In order to attach the fluorescent molecule onto the antibody, crosslinking is
required (either synthesized, use existing, or a probe)
- GFP in jellyfish allow for fluorescence. The barrel structure has a molecule in
the center of it that can be excited and emit a light as a result
- Recombination of gene A and gene GFP is used exactly side by side, where
protein A and GFP are made fused as one protein, and allows connection of
GFP to ANYTHING
- Holes do not have to be punched into the cell anymore, so that GFP can be
made indefinitely
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- But what about bulkiness of the attached molecule? Some fluorescent
molecules are super small while others are really big. Why do you need the
barrel structure to the GFP?
Fluorescent proteins are used in the cell to:
1. study gene expression (if proteins are expressed, or not)
2. see where proteins are in the cell
3.
less acidic = fluoresce changes of the GFP
in , the fluorescent proteins have been modified to express at different rainbow
sites, allowing colour coding
Fluorescent probes:
- fluorescent. Dyes
o Phalloidin can be chemically modded to bind to F-actin
o Intercalate (sticks into the membrane) into membranes. Allows one to
see into the plasma membrane
o Fura2 can bind to Ca, and causes it to be more fluorescent. Hence if you
have loads of Ca in sample, more Fura2 binds to it, and intensity of the
light in image describes the concentration of calcium in the cell
▪ In the image on slide 52, it is the neuron concentration of Ca in
the cell. Found at the dendrite ends of the cell
o Flruorescent is used as an indicator for pH
- How are they introduced into the cell?
o Tiny needles inject molecules (i.e., fluorescent ones) into the cell. But
not great to manipulate loads of cells
o Electroporation: shock the cell membrane, to allow for perforations to
appear in cell membrane. The substance x is then allowed to go into the
cell, but loads of cells can die from the shock
o Vesicles introduced (transfected) into the target cell
DAPI is a fluorescent dye, not the same as the protein modded or the gene
modded.
Take away: The whole point of microscopy is to locate and understand the cell
Electron Microscopes
What’s smaller than light waves? Electrons.
- allows for more detail to be viewed. Electron beams bombard the sample,
with magnets used instead of glass lens to view the specimen.
- Osmium tetroxide bind to decorate membranes.
Relisten to 70:00 about the heavy atom salts
- flash freezing to avoid crystallization to the cell sample
o darker shades = electrons deflected to somewhere.
o Lighter shades = electrons passed straight through the cell.
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- Likelihood of electrons being deflected allows the cell image to be formed.
-
- Function of clatharin
But how to tell which protein is which in electrons? Use immunocytochemistry Use
antibodies to generate stains of the cell, where the secondary antibody (covalently
modded with gold particles), which look dark underneath the EM. The gold bounces
off the electrons. Great for locating the proteins in the cell
In scanning electron microscopy, you can get a 3D image of the sample. By spraying the
sample’s surface with the heavy metal, to create different shades when electrons shot at
it from an angle.
How is EM used to see stuff?
- the advantage of TEM is that it can be used to see inside the sample, while the
SEM can get a 3D image of it.
- Skip electron tomography.
- Deep etching: you are creating a different layer. You are cracking and
fracturing the sample to reveal the inner layer to view (like a pistachio shell,
or a geode)
- Metal Shadowing: metal sprayed at an angle, onto the surface of the sample,
to strengthen the structure + make it visible by the electrons that bounce off
of it.
- Negative staining: like stencilling . the straight dark black line on the slide 72 Is
where the uranium bonded to the actin filament border. It gives contours of the
heavy metal studied.
- Cryoelectron microscopy: biotic stuff. looking for Sample suspended on
water, and then flash frozen in -160 degrees celcius, and also in a vaccum so
that the water molecules don’t have time to create hydrogen bonds (in which
case otherwise they would be spiky, and irregular, instead of smooth and flat,
which is easiest to see through). No fixing is required, and biotic stuff can be
viewed thanks to this
o Very expensive
o Thousands of images are taken, and computers reconstruct it to form a
3D structure for the sample
o
- The disadvantage is that the images could be distorted, but in cryogenic EM,
there is not as much a chance because only freezing is involved, so less margin
of error (less likely for the sample to be damaged)
-
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