Midterm 1 study Q's Flashcards
(42 cards)
What are the 4 types of CONVENTIONAL Light Microscopy?
(and conventional is one of 4 types of light microscopy.)
1) Bright Field
2) Phase Contrast
3) Differential Interference Contrast
4) Dark Field
How does Bright Field microscopy work?
Bright field microscopy is one of the simplest and most common types of optical microscopy. It works by passing visible light through a specimen and into an objective lens, which magnifies the image for observation.
The image is typically very bright in value, with slightly darker bits representing shapes of the specimen.
How does Phase Contrast Microscopy work?
It converts phase differences into changes in brightness.
How does Differential Interference Contrast microscopy work?
polarized light is separated and then recombined.
result is a more 3d looking image with ‘shading’.
how does Dark Field Microscopy work?
light source is lateral and it only shows scattered light.
What are the 4 types of LIGHT microscopy?
- Conventional
*Bright field, Phase contrast, dark field, Differential interference contrast (DIC). - Fluorescence Microscopy.
- Confocal Microscope.
- Two-photon microscope.
What is Fluorescence Microscopy?
and how does it work?
- type of light microscopy
-The point is to tag a part of a cell with a specific coloured fluorescent protein, and then we illuminate the sample with a light of specific wavelength (a specific colour light), and then the proteins will emit light in a certain colour, so we will see a coloured image.
-Commonly uses GFP (green fluorescent protein).
-(the steps to prepare the tissue are on another cue card!)
Steps of Tissue Preparation for Fluorescence Microscopy.
1: Fixation ~ expose tissue to chemical reagents like aldehydes, alcohols, etc. to preserve and stabilize it.
2: Embedding ~ tissue placed in a medium like a plastic resin (epoxy, etc.) to maintain its shape. This firms it up for the next slicing step. vv
3: Sectioning ~ The tissue is now sliceable, to we slice it thin for use in the microscope.
4: Staining ~ Immerse the tissue in the staining solution, ensuring the stain is chosen to specifically bind to the desired proteins or structures within the tissue.
What is Immunofluorescence?
A specific type of fluorescence microscopy, that specifically colours & deals with ANTIBODIES.
so, we are trying to locate an antigen (an identifying molecule specific to a pathogen’s cell membrane, that the immune system will use to recognize the bacteria.) or an antibody (antibody is from immune system and binds to the antigen), and so we use fluorescently probed “secondary” antibodies that will bind to the first antibody and we can then locate our target!
What is the Confocal Microscope, what does it do?
it’s a type of Light microscope.
unfortunately it is costly $$.
“Confocal” refers to the fact that the light source and the detector are always the SAME DISTANCE away from the object.
utilises fluorescence and high focus lasers.
a Pinhole focuses light on a specific area, which will decrease background noise.
*Only light that is actually FOCUSED at the point of the detector will go through.
so, confocal microscopy can produce a much clearer image than fluorescence microscopy.
What is Two-Photon microscopy?
a type of Light microscopy.
Very costly
Allows deep tissue imaging (1mm).
We use longer wavelengths of light, such as red or near-infrared light, because they are not absorbed by tissues or scattered as easily as blue and ultraviolet light. This allows the light to penetrate further into the tissue.
rapid, high-energy laser pulses.
does NOT need a pinhole (and confocal does).
What is electron microscopy?
What are the 2 types?
It’s a different type of microscopy that uses bombardment of electrons (rather than beams of light as seen in light microscope).
The two types are:
1. Transmission Electron Microscope.
2. Scanning electron Microscope.
Unfortunately, very time-consuming preparation.
Instead of lenses as used to focus light beams in light microscopy, what does Electron Microscopy use instead?
An electron microscope uses magnets to focus the electron beams in the same way that a lens does for light.
Tell me about the preparation of tissues for Electron Microscopy.
*Very long prep, can take days to weeks.
Tissue must be fixed in Glutaraldehyde, then dehydrate it, then fix it in a plastic resin.
(to preserve and keep structure).
Sections must be ULTRA THIN so electrons can go through the sample. (Must be cut with a DIAMOND knife).
–> (Sections cannot be handled directly)
Sections are placed on a copper grid.
Why do we fix the tissue in glutaraldehyde, dehydrate it, then use plastic resin for TEM (transmission electron microscopy)?
We fix the tissue with glutaraldehyde, then dehydrate it and infiltrate it with a plastic resin, because that makes the tissue firm and “plastic-like” enough to actually cut slices this thin. (as you can imagine, if we tried to do this without fixing the tissue, the ‘slice’ would just be mush.
Transmission electron microscopy (TEM) can ONLY be used for thin sections, because for this to work, we have to send the electrons THROUGH the sample.
What is Scanning Electron Microscopy?
How is it different from TEM?
Used to study whole cells and tissues rather than sections of intracellular structures.
Sample is coated with HEAVY METAL.
Prep similar to with TEM.
We can see things like detailed insect heads, or even a bacteriophage.
So this is different than Transmission electron microscopy because here, we are NOT sending electrons THROUGH the sample. Instead, we are bombarding the sample with electrons, and we will see how the electrons deflect off, and this gives us the information.
Notice that the detector is NOT below the specimen, and that’s because the electrons will not be going through the specimen.
–> Instead the detector is off to the side to receive the bounced light.
What is Ion Imaging?
Sperm entering egg video example.
So, we send a beam of ions at a sample (“primary ions”), and then they smack other ions off of the sample (“secondary ions”), and then we have equipment that analyzes these ejected ions and forms images.
What is X-Ray Crystallography?
Rosalind Franklin used this to generate Photo 51.
X-ray crystallography is a technique used to determine the atomic and molecular structure of a crystal by scattering X-rays off the crystal’s atoms.
–>When applied to biological molecules like DNA, researchers can still refer to it in terms of crystallography because the process involves creating a crystalline form of the substance (in this case, DNA) to analyze its structure.
–> So, we turn DNA into a solid crystal structure.
What is X-Ray Crystallography used for compared to microscopy?
Microscopy is used for imaging tissues and cells at the cellular level, giving images of the overall composition.
X-ray crystallography essentially shows the smaller scale detailed structure of a sample such as Rosalind franklin’s Photo 51 of DNA.
–> X-ray crystallography and microscopy are both tools used to study structures, but they serve different purposes. X-ray crystallography is primarily used to determine the atomic structure of crystals, including biological molecules like DNA and proteins, by analyzing how X-rays scatter through a crystallized sample. In contrast, microscopy is used to visually observe cells, tissues, and smaller structures in real-time, often at lower resolutions. Microscopy can work with live or fixed samples, while X-ray crystallography requires the sample to be crystallized.
What are filament proteins?
Actin & myosin, cytoskeleton etc.
These are long, thin, rod-like proteins.
3 types of Filament proteins?
and quick definition of each.
1: Actin ~ (“Microfilaments”) Found in muscle fibres and in the cytoskeleton.
2: Myosin ~ usually associated with actin, it’s a motor protein whose heads interact with actin in order to generate cellular movement (and muscle contraction in the case of muscle cells).
3: Intermediate Filaments ~ anchoring and structural support for cell.
What conformation are filament proteins constructed in, so that they are stable?
Lego is a good analogy here.
These structures cannot be built just end-on-end, because that would be easily broken.
So instead, we build them in sheets so that each filament protein is bonded to multiple others to hold it in place.
When we build them in a sheet, the subunits deep inside are much more stable.
What is Tubulin?
Heterodimer = 2 different proteins. but considered “1 subunit” of tubulin.
–> (they are alpha and beta tubulin). (each binds GTP).
–> Alpha tubulin is not actually used as part of the cytoskeleton.
Has a plus and minus end.
Actin structure?
Uses ATP
each actin monomer in the filament will be touching 3-4 other monomers.
(see diagrams if you don’t remember how it looks).
