4.2 Excitation and Emission Spectra

Emission and Absorption Spectra of Fluorescence Microscopy

Figure 4.4: Emission and Absorption Spectra of Fluorescence Microscopy

Both spectra are normalized with the following properties:

  1. Absorption spectrum

    These plot absorption (i.e., y-axis) against wavelength (i.e., x-axis).

  2. Emission spectrum

    These plot flourescence intensity (i.e., y-axis) against wavelength (i.e., x-axis).

Emission spectra are independent of their absorption spectra due to the Stokes’ shift. Both spectra’s curves also appear like mirror images of one another.

4.2.1 Small Molecule Fluorophores

Examples of Fluorophores Used in Fluorescence Microscopy

Figure 4.5: Examples of Fluorophores Used in Fluorescence Microscopy

Professor Lu Lei lists two examples of fluorophores and their emission and absorption spectrums.

4.2.1.1 Aequorea victoria’s GFP

Tertiary Structure of a GFP

Figure 4.6: Tertiary Structure of a GFP

Note that GFP is short for Green Fluorescent Protein.

The GFP in this jellyfish (i.e., Aequorea victoria) can generate fluorescence on its own (i.e., no co-factors or enzymes are required). The fluorophore of the GFP is protected within the \(\beta\)-sheet barrel.

In Biology, GFPs are typically used to label a protein and is highly compatible with live cell imaging3.

4.2.2 Fluorescence Labelling Biological Samples

A Sample Immuno-Fluorescence Slide

Figure 4.7: A Sample Immuno-Fluorescence Slide

After a cell is grown on a glass coverslip and fixated to it, the following steps can be performed:

  1. Permeabilization

    This allows the antibody to enter the cell. Detergent is used in this step to make “holes” in the cell.

  2. Adding Primary Antibodies

    These antibodies bind to a specific antigen and often sourced from mice, rabbits, and humans.

  3. Washing

    This removes unbounded antibodies.

  4. Adding Secondary Antibodies

    Primary and Secondary Antibody Labelling

    Figure 4.8: Primary and Secondary Antibody Labelling

    These label the antibodies; goat antibodies conjugated with fluorophores are used here.

  5. Washing

    This serves the same purpose as before.

  6. Mounting

    This immobilizes the glass coverslip. The refractive index of the mounting medium should be the same as that of the immersion medium’s.

Multi-Color Labelling in Fluorescence Microscopy

Figure 4.9: Multi-Color Labelling in Fluorescence Microscopy

With the above technique, multi-color labeling is also possible:

4.2.3 Pros and Cons of Fluorescence Microscopy

Some pros include:

  1. It is highly specific (i.e., allows the viewer to only see an organelle or object of interest).
  2. It has a high signal-to-noise ratio.

  1. Though, note that many different fluorescence proteins are also available!↩︎