Lecture 2- Electron Microscopy Flashcards
Difference in magnification of light microscope and electron microscope
Light microscope: 1000x
Electron microscope: 20000x (SEM) ; 100000x (TEM)
What type of illumination is used in light microscope? In electron microscope?
Light: Visible or UV
Electron: beam of electrons
What type of lenses do Light microscope use? Electron microscope use?
Light: glass or quartz
Electron: electromagnets
Detection of light vs electron microscope
Light: eye or camera
Electron: camera
What are the two types of electron microscope?
- Transmission electron microscopy(TEM)
- Scanning electron microscopy (SEM)
Transmission electron microscopy (TEM)
beam of electrons passes through specimen revealing a 2D image
Scanning electron microscopy (SEM)
Beam of electrons bounces off specimen revealing a 3D image
Preparing specimens for SEM
- Choose metal atom
- Coat specimen with metal atoms (must be uniformly coated)
Why do we need to use a heavier metal (ie., Gold) to cover our specimen in SEM?
So that electrons will bounce off all of it.
Preparing specimens for TEM
- Ultrathin sectioning + metal atom staining
- TEM tomography
- Ultrathin sectioning + metal atom staining + gold-labelled antibodies
- Negative staining
- Shadowing
- Freeze-fracturing + shadowing
TEM tomography
several ultrathin sections can be assembled to make a 3D model
Ultrathin sectioning + metal atom staining + gold-labelled antibodies
instead of attaching the antibody to a fluorescent molecule, we’re attaching it to a very large gold ball
Negative staining
staining the area next to a specimen
Specimen will appear bright while area on either side of specimen
specimen will appear dark because the metal atoms are preventing electrons from passing through
Shadowing
specimens are sprayed with metal atoms at an angle. We’re using the same device to prepare the specimen with SEM, but instead of covering the specimen on all sides, we’re only covering them on one side.
only getting one dark region on one side of the specimen
Freeze-fracturing + shadowing - specimens are:
- frozen in neon
- fractured through their membranes
- shadowed
Which of these microscopes is used with freeze-fractured specimens?
a. brightfield
b. widefield/ confocal fluorescence
c. SEM
d. TEM
e. phase contrast/ DIC
d. TEM
If you wanted to use fluorescence microscopy to demonstrate that two objects in a cell are at the same place you would use ___ and ____ for your objects because where they colocalize the color would be:
A. red and yellow ; green
B. red and blue ; green
C. yellow and green ; red
D. red and green ; yellow
E. blue and red ; yellow
D. red and green ; yellow
Tomography is when several 2D images can be assembled by a computer into a 3D image. Which microscopes are used to capture the 2D images?
A. TEM and widefield fluorescence microscopy
B. TEM and confocal fluorescence microscopy
C. SEM and widefield fluorescence microscopy
D. SEM and confocal fluorescence microscopy
B. TEM and confocal fluorescence microscopy
The number of mitochondria in an animal cell ranges from zero (human red blood cells) to 10 000 000 (frog eggs).
Using the microscopy techniques described in class, describe different ways to determine how many mitochondria are in a typical human fibroblast.
- Obtain a small amount of tissue containing fibroblasts
- Slice it up with a microtome
- Label the cells with a mitochondrial-specific fluorescent probe or antibody
-Use a confocal microscope to count the mitochondria present within some representative cells
What are some similarities and differences between widefield fluorescence microscopy and confocal fluorescence microscopy?
Similarities:
- both use fluorescent stains and fluorescent probes to see the object
- you can not see the whole cells
What are some similarities and difference between SEM and TEM?
Similarities:
-both of them use beam of electrons to illuminate objects
- both have electromagnet lenses
Difference:
-In TEM, beam of electrons PASSES through the specimen revealing a 2D image
-In SEM, beam of electrons BOUNCES off specimen revealing a 3D image
Resolution
-TEM have a greater resolution because the beam of electrons used to illuminate the specimen is narrower and the detector used in SEM microscope picks up electrons and X-rays given off by the object when the electrons strike it. These cloud the image somewhat
