cours 2 - Microscopy Flashcards
name 4 type of light microscope
- Conventional (e.g. bright field, phase contrast etc.)
- Fluorescence microscope
- Confocal microscope
- Two-photon microscope
What type of microscope can see the smallest things?
electron microscope
What is a light microscope used for?
for live or fixed cell or tissue
what type of microscope is used for tissue?
upright microscope
what type of microscope is used for isolated cell?
inverted microscope
Name all the part of this microscope. What type of microscope is it?
light microscope
condenser lense: focus the light on the spacimen
objective lense: focus tthe image in the eye
Describe the 3 technics used withbright field microscope that manipulates light.
1) Phase conrast
- converts phase differences into changes in brightness. (2 beam of light adjacent)
- detection of sharp changes in reflective light
- no polariser no beam splitter no beam recombiner
- has halo
- Produce image by the incomplete separation of direct and diffracted light rays
- no 3d effect
- qualitative data
- not sensitive to specimen orientation
- low axial resolution (no sectionning possible
2) Differential
- detection of continuous changes of refractive index
- uses beam splitter, beam recombiner and polariser
- Produce image by the complete separation of direct and diffracted light rays
- no halo
- 3d effect
- qualitative and quantitative data
- sensitive to specimen orientation
- high axial resolution (so sectioning possible)
- expensive
3) Dark field
- lateral light source shows only scattered light.
- allows to observe transparent sample
- light goes in lense, a circle of light passes through the phase ring, a lense condense light through a sample, the condense image passing through the sample goes in a lense that focuses the image on the detector
What do we use dapi dye for?
Staining the nucleus
Name the microscope parts. What type of microscope is it?
fluorescent microscope
What does tissu preperation intail?
- To observe cells in tissue, in most cases tissues must be histologically prepared
-
Fixation: exposure to chemical reagents (aldehydes, acids, alcohols) to preserve and stabilize.
- May produce unwanted effects.
- cell become stable (preserve).
-
Embedding: plastic or polyethylene glycol.
- (solid resine) tissue dehydrate and is replace with solvent like alcool and then it will solidify.
- Sectioning: cutting of thin (1–10 µm) tissue sections on a microtome.
- Staining: if applicable, involves exposure to dyes, e.g. hematoxylin, eosin, antibodies.
describe the process used in immunofluorescence
- Antibodies are produced in host animal and collected.
- Fixed tissue is permeabilized with detergent (change the soluability of the membrane) and treated with primary antibody directed against a specific antigen.
- Antibody binds to antigen on or within cell.
- Secondary antibody conjugated with fluorescent marker binds to primary antibody.
- from different animals
- 2nd antibody is use to amplify the signal
- Indirect immunohistochemistry labels cell structures.
What are the advantages and disadvantages of Confocal Microscopy
advantages:
- Technique that provides clear images with reduced “background” signal.
- Particularly useful for applications involving thick sections or whole-mount preparations.
- can produce 3d image reconstruction
disadvantages:
- costly
describe the process of confocal microscope
same focal distance bw light source and object
uses high energy laser (pass through pinhole) and fluorescence
what is the problem with light microscope?
exibits refraction so the image resolution has a limit
resolution is the limit at which you can differentiate 2 seperate point
give the difference between an upright and a inverted microscope
inverted: lenses are underneath the specimen
upright: lenses are above the speciment
