Lecture 2- Electron Microscopy

Question 1

Difference in magnification of light microscope and electron microscope

Answer 1

Light microscope: 1000x

Electron microscope: 20000x (SEM) ; 100000x (TEM)

Question 2

What type of illumination is used in light microscope? In electron microscope?

Answer 2

Light: Visible or UV

Electron: beam of electrons

Question 3

What type of lenses do Light microscope use? Electron microscope use?

Answer 3

Light: glass or quartz

Electron: electromagnets

Question 4

Detection of light vs electron microscope

Answer 4

Light: eye or camera

Electron: camera

Question 5

What are the two types of electron microscope?

Answer 5

1. Transmission electron microscopy(TEM)
2. Scanning electron microscopy (SEM)

Question 6

Transmission electron microscopy (TEM)

Answer 6

beam of electrons passes through specimen revealing a 2D image

Question 7

Scanning electron microscopy (SEM)

Answer 7

Beam of electrons bounces off specimen revealing a 3D image

Question 8

Preparing specimens for SEM

Answer 8

1. Choose metal atom
2. Coat specimen with metal atoms (must be uniformly coated)

Question 9

Why do we need to use a heavier metal (ie., Gold) to cover our specimen in SEM?

Answer 9

So that electrons will bounce off all of it.

Question 10

Preparing specimens for TEM

Answer 10

1. Ultrathin sectioning + metal atom staining
2. TEM tomography
3. Ultrathin sectioning + metal atom staining + gold-labelled antibodies
4. Negative staining
5. Shadowing
6. Freeze-fracturing + shadowing

Question 11

TEM tomography

Answer 11

several ultrathin sections can be assembled to make a 3D model

Question 12

Ultrathin sectioning + metal atom staining + gold-labelled antibodies

Answer 12

instead of attaching the antibody to a fluorescent molecule, we’re attaching it to a very large gold ball

Question 13

Negative staining

Answer 13

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

Question 14

Shadowing

Answer 14

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

Question 15

Freeze-fracturing + shadowing - specimens are:

Answer 15

• frozen in neon
• fractured through their membranes
• shadowed

Question 16

Which of these microscopes is used with freeze-fractured specimens?

a. brightfield
b. widefield/ confocal fluorescence
c. SEM
d. TEM
e. phase contrast/ DIC

Answer 16

d. TEM

Question 17

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

Answer 17

D. red and green ; yellow

Question 18

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

Answer 18

B. TEM and confocal fluorescence microscopy

Question 19

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.

Answer 19

• 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

Question 20

What are some similarities and differences between widefield fluorescence microscopy and confocal fluorescence microscopy?

Answer 20

Similarities:
- both use fluorescent stains and fluorescent probes to see the object
- you can not see the whole cells

Question 21

What are some similarities and difference between SEM and TEM?

Answer 21

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