Light Microscopes, Indirect Immunofluorescence (IMF), GFP

What is the difference between Light Microscopes, Electron Microscopes, and Indirect Immunofluorescence (IMF)?

What is a Microscope Dependent on?

A microscope is defined by its resolving poweror the ability to clearly tell two close objects apart from each other, and the maximum resolving power is dependent on the illumination’s wavelength.

microscope

  • Resolving Power: The ability to tell two close objects apart from each other
  • Maximum Resolving Power: Dependent on Illumination’s Wavelength

The illumination is incoming light, which can include visible light or even infrared light. For visible light, its wavelength is in the 400-700 nm range.

 

Resolving Power Differences between Light Microscope and Electron Microscope

Let’s consider two spots 100 nm or 0.1 um apart from each other. For the light microscope, its resolving power is too weak to be able to distinguish these two spots from each other, while for the electron microscope, the spots can be easily distinguished.

twospots

Type of Microscope Can distinguish two spots 100 nm apart?
Light Microscope No
Electron Microscope Yes
sizescales_and microscopes
Image retrieved from Essential Cell Biology.

Remember our lesson on biological size scales? The light microscope’s minimum resolvable is at 200 nm or 0.2 um, the size of the string of ribosomes. On the other hand, the electron microscope’s minimum resolvable is at 0.2 nm, the size of an atom.

  • Minimum Resolvable
    • Light Microscope: 0.2 um (string of ribosomes)
    • Electron Microscope: 0.2 nm (atom)

In order to overcome its low power resolution, light microscopes are often used with staining. Staining includes bright field, phase contrast, and interference contrast to enhance visibility of cells and organisms.

 

Indirect Immunofluorescence (IMF) with Light Microscopes

Let’s first know the difference between bioluminescence and fluorescence. The difference involves the source of the light. Biolouminescence’s source of light comes from the organism itself and an internal chemical reaction. The organism provides the mechansim to produce light. An example is fireflies.

Fluorescence’s source of light comes from an external source. The external source produces the light, which can be absorbed or reflected accordingly by the organism. An example is the glow-in-the-dark jellyfish.

This short ~one minute video explains the difference between fluorescence and bioluminescence: <How do animals glow? – Youtube>

ana_nucleolar_3

For indirect immunofluorescence, blue light enters, and green light is emitted. That’s why we often see green in experiments using indirect immunofluorescence. The sensitivity and specificity of indirect immunofluorescence are fluorescence and antibody, respectively.

  • Sensitivity: Fluorescence
    • This means that the glowing green we see is sensitive or caused by the fluorescence in work.
  • Specificity: Antibiody
    • This means that the indirect immunofluorescence is specific to the antibodies that attach to the targeted antigens.

 

How Does Indirect Immunofluorescence (IMF) Work?

Here are the simplified steps:

  1. Fix and permeabilize the cells. Fixing means crosslinking, and permeabilizing means adding detergent. This immobilizes the specific antigen to the specific cell or cell part.
  2. Add the Primary Antibody. The primary antibody binds to the antigen.
  3. Wash away unbound antibodies. (Now, we won’t have any excess floating antibodies)
  4. Add Secondary Antibodies. This secondary antibody contains a fluorescence marker. They attach to the primary antibodies which are attached to the antigen.
  5. Wash away unbound secondary antibodies.
  6. View them under the light microscope!

Note: By fixing and permeabilizing cells, the cells are DEAD.

It’s important to know what type of secondary antibodies and primary antibodies to use. If the primary antibody is from a rabbit (rabbit antibody), then the secondary antibody must be the anti of that primary antibody. In other words, it must be an anti-rabbit antibody to recognize that rabbit primary antibody.

Test Tip: Always use the anti-<organism> of the primary antibody’s organism for secondary antibodies.

 

Practice Question:

Bobby wants to use indirect immunofluorescence targetng antigen B. He wants the cells to glow green under the light microscope. He has the following supplies available:

Primary antibodies:

  1. Cow antibody A
  2. Cow antibody C
  3. Rabbit antibody B
  4. Sheep antibody B

Secondary antibodies:

  1. Green Anti-rabbit antibody
  2. Red Anti-sheep antibody
  3. Pink Anti-cow antibody
  4. Pink Anti-rabbit antibody

Which antibodies should Bobby use for his indirect immunofluorescence targeting antigen B?

A. Primary 1 AND Secondary 2

B. Primary 4 AND Secondary 4

C.Primary 3 AND Secondary 3

D. Primary 3 AND Secondary 4

*Please scroll to the bottom for answer and explanation.

 

How Does Green Fluorescent Protein (GFP) Work?

  • Sensitivity: GFP Fluorescence
    • This means that the glowing green we see is sensitive or caused by the GFP fluorescence in work.
  • Specificity: GFP Protein Fusion
    • This means that the indirect immunofluorescence is specific to the GFP protein gene that is fused with targeted gene of protein interest.

The advantage of GFP over IMF is that the cells are alive, and we can see the live processes and movements of cell mechanisms, movement, etc. The disadvantage of GFP is that we must know the gene code for the protein of interest, to allow the fusion of GFP with it. If we do not know the gene code, it is difficult to perform GFP. That is why scientists still use IMF in the biological field.

 

___________________________________________

Practice Questions’ Answers and Explanations:

The answer is D. We know that Bob is targeting antigen B. Thus, the primary antibodies must be of B type. This leads us to narrow down our primary antibody choices to 3 and 4. Bobby also wants to see pink fluorescence. Thus, we can narrow down our secondary antibodies to 3 and 4 as well. However, the primary antibodies 3 and 4 available are from a cow and a sheep. That means the secondary antibodies must either be anti-rabbit or anti-sheep. Only secondary antibody 4 fits this; it is anti-rabbit. Thus, the final answer is D. Primary 3 and Secondary 4.

 

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s