Introduction to the Cell › Microscopy

Fluorescence Microscopy

Notes

Fluorescence Microscopy

Sections

FLUORESCENCE MICROSCOPY APPLICATIONS

Fluorescence recovery after photobleaching (FRAP)

Fluorescence resonance energy transfer (FRET)

  • Used to study protein-protein interactions

METHOD

  1. Light passes through excitation filter
  2. Excitation filter filters out undesired wavelengths of light
  3. Mirror deflects light downward toward sample
  4. Light passes through the objective lens and onto specimen of interest
  5. Molecules in sample absorb light & emit light with longer wavelength (fluoresce)
  6. Fluorescent light travels back upward and passes through mirror w/o deflecting
  7. Barrier filter above the mirror lets fluorescent light through
  8. Fluorescence observed

FRAP

  1. Tag membrane proteins with fluorophore (i.e. GFP)
  2. Irreversibly bleach portion of membrane with laser (photobleaching)
  3. Measure rate at which membrane recovers fluorescence (proportional to rate at which tagged molecules diffuse back into bleached area)

FRET

  1. Tag one protein with blue GFP and another with green GFP
  2. Shine violet light on sample

If the proteins interact (i.e they come in close proximity):

  • Blue light from blue GFP excites green GFP
  • Green light observed

If the proteins do not interact:

  • Blue light observed (not absorbed and reemitted by green GFP)

Full-Length Text

  • Here we will learn about fluorescence microscopy, in which proteins are labeled with a fluorescent protein and observed under a specialized microscope.
  • To begin, start a table to learn the two key experimental methods that use fluorescence microscopy.
  • Denote that they are:
    • FRAP, fluorescence recovery after photobleaching, which is commonly used to study membrane fluidity.
    • FRET, fluorescence resonance energy transfer, which is commonly used to study protein-protein interactions.

We will illustrate each of these processes in this tutorial.

First, let's learn the structure of a fluorescent fluorescence microscope, through which we can observe fluorescent molecules.

  • Draw a vertical rectangle and label it excitation filter.
    • Filters distinguish fluorescent microscopes from standard ones.
  • Indicate that there is a light source behind it.
  • Show that it emits different wavelengths within the visual spectrum of light.
  • Illustrate that only blue light (450 to 490nm) passes through the filter.
    • We use blue light in this example, but different filters can be used for different wavelengths.
  • Now, draw a mirror that deflects this light downward.
  • Show that once deflected, this light passes through the objective lens and onto the specimen of interest.
    • It illuminates molecules within the specimen that are tagged with a fluorescent dye.
  • Below our microscope, write that this "dye" is specifically green fluorescent protein (GFP), which can be fused with another protein to tag it.
    • GFP was first discovered in jellyfish, and fluoresces green when activated by blue light.
    • Many other fluorescent dyes now exist.
  • Indicate that the molecules within the specimen emit green light when they are illuminated.
  • Next, illustrate that this green fluorescent light can pass vertically through the mirror without deflecting.
  • Draw a rectangular barrier filter above the mirror.
  • Show that the desired fluorescent light passes through it, while unwanted fluorescent signals cannot.
    • An eyepiece sits just above this filter, and the transmitted fluorescent light can be observed at this point.

Finally, let's illustrate the two experimental methods that we noted in our table.

We will start with FRAP, which is used to measure membrane fluidity.

  • Draw a cell with a protein-rich membrane.
  • Now, redraw the membrane.
  • Here, tag each of the proteins with GFP.
  • Next, draw a third cell, again with the protein's tagged with GFP, but show that a small section of the membrane is bleached: it loses its fluorescence.
  • Now, show that this occurs when a laser focused on a small region of the membrane irreversibly bleaches the GFP's in this area.
  • Next draw a final cell and show that the bleached proteins have diffused laterally in the membrane, mixing with the fluorescent ones.
  • Use an arrow to show that this step is known as "fluorescence recovery."
    • The fluorescence in the bleached area increases as fluorescing GFP's diffuse into it and bleached proteins diffuse out.
    • Scientists use this technique to determine the rate of diffusion of a membrane protein.

How do they do this?

  • Draw a graph that records the fluorescence of the bleached region of the membrane over time.
  • Show that fluorescence is at its highest before bleaching.
  • Illustrate that it drops rapidly when it is bleached.
  • Finally, show fluorescence recovery as a rising curve: fluorescence slowly recovers.
  • Indicate that it never reaches the original level of fluorescence.
    • The irreversibly bleached GFPs do not regain their fluorescence; they merely mix with GFPs that were never bleached.
  • Write that the diffusion coefficient calculated from this data is proportional to the rate of fluorescence recovery.

Next, let's learn FRET, which measures protein-protein interactions.

  • Draw two distinct proteins, separated from one another.
  • Indicate that they do not interact.
  • Now, on one, bind a GFP variant that emits blue light.
  • Show that violet light excites it.
  • On the second protein, bind a GFP, which as we have seen emits green light and is excited by blue light.
    • Write that if we excite these proteins with violet light, we only see a blue emission.
  • Why?
    • Because the proteins do NOT interact; the second protein does not produce an emission.
  • Now, draw protein 1 and 2 bound to each other.
  • Excite the complex with violet light.
  • Show that the blue emission from protein 1 excites protein 2 when they are bound.
  • Now, show that protein 2 produces a green emission.
  • Write that when violet light excites the complex, it produces a GREEN emission.
  • The blue light produced by protein 1 excites protein 2.
  • Write that this proves that the proteins interact.
  • As a clinical correlation, write that FRET is used to test the membrane dynamics of HIV proteins that mediate viral fusion with host cells.

How? Let's illustrate this, now.

  • Synthesize a liposome.
  • Cover it in fluorescent proteins.
  • Adjacent to each fluorescent protein, place a protein that quenches emissions.
  • Indicate that as a result, the liposome does not produce fluorescent emissions.
  • Now, draw a vesicle covered in viral proteins.
  • Imagine that it fuses with the liposome.
  • Show that after fusion, the viral proteins diffuse laterally, and separate the fluorescent proteins from the proteins that quench their emissions.
  • Indicate that after fusion, when light is shown on the liposome, fluorescence occurs.