Fluorescent Materials

Fluorescence imaging is an imaging technique that uses fluorescent markers to visualize biological processes in living organisms. Fluorescence is produced when atoms and molecules absorb light of a given color and then gives off light of another color. The light that is given off or emitted is called fluorescence.

The color of light that people can see is made up of a rainbow of colors. Each color has a corresponding wavelength.

Source: PennState

  • Violet: 380 - 450nm

  • Indigo: 420–440 nm 

  • Blue: 450–495 nm 

  • Green: 495–570 nm 

  • Yellow: 570–590 nm 

  • Orange: 590–620 nm 

  • Red: 610–710 nm 

Fluorescent materials absorb light of a given wavelength and emit fluorescence that is a longer wavelength. For instance, if a fluorescent material or organism is observed under ultraviolet (UV) or blue light, then it may emit green, yellow, orange or red light because those colors have a longer wavelength.

The difference in the wavelength of the light absorbed by the fluorescent material and the light it gives off (ie: its fluorescence) is known as Stokes shift. Every fluorescent compound has a unique stokes shift that is specific to it.

In healthcare, fluorescence helps doctors detect specific biomarkers associated with diseases, enabling early diagnosis and monitoring. For example, disease-causing forms of proteins can be detected by its binding to compounds that are brightly fluorescent under UV light.

Did you know?

  • Fluorescence was 1st discovered in 1845 by Fredrick W. Herschel who found that UV light can excite a quinine solution to emit blue light.

  • Sir George G. Stokes built upon this observation, noting that fluorescence emissions were of longer wavelengths than the original UV light used to excite them.

Design Challenge:

Become a Biomolecular Scientist and demonstrate fluorescence produced by everyday objects and biological materials.

  • What color did your solution appear under UV light?

  • Why do you think the color is different than when the tube is viewed in room light?

  • How is the color of the fluorescence related to that of the UV light that you used to produce the fluorescence?

Materials included in kit:

  • Safety Googles

  • Disposable Gloves

  • Bowl + Spoon

  • Blacklight

  • Test Tubes

  • Pipette

  • Transparent Paper

Materials Not included in kit:

  • 2 - 3 Green Leaves (ie: Spinach or another green plant leaf)

  • 1 tsp - Rubbing alcohol (70% isopropanol)

  • 1 - Fluorescent Highlighter (Not Required - for bonus activity)

Build Instructions:

Build instructions provided by Biophysical Society

Green Leaf Experiment:

  1. Add one or two leaves (we used two medium-sized spinach leaves) to the bowl and about a teaspoon (4-5 ml) of rubbing alcohol (70% isopropanol). See Figure 1.

  2. Make a pulp from the leaves by using your spoon to mash it up. See Figure 2.

    • Chlorophylls, the molecules that give plants their green color, are soluble in isopropanol and can be extracted from green leaves by breaking the cells open so they release their contents.

  3. After you make a pulp from the leaves, transfer the mixture to your plastic tube using a pipette. See Figure 3.

  4. Notice the color of the solution under room light – it appears green, like the leaves from which it was made.

  5. Put on your UV safety glasses and use the black light to view the tube. Notice the color of the solution under the UV light. See Figure 4.

    • What color does it appear? Why do you think the color is different than when the tube is viewed in room light? How is the color of the fluorescence related to that of the UV light that you used to produce the fluorescence ?

  6. Repeat steps 1 - 5 with a different type of green leaf. Did your results differ? Explain.

Answer: The chlorophyll fluorescence appears red. Red is a longer wavelength of light than the colorless UV light the chlorophylls absorb. This agrees with the Stokes shift theory.

The color of the fluorescence observed for different fluorescent substances is a property of the molecules they contain that absorb light and emit fluorescence. The pyranine dye in the fluorescent yellow marker and the chlorophylls in the spinach leaves are very different molecules with different light-absorbing and light-emitting properties – because of this, their fluorescence properties differ, including the color of fluorescence they emit.

Figure 1: Add leaves and rubbing alcohol

Figure 2: Make a pulp from the leaves

Figure 3: Mixture in plastic tube

Figure 4: Color of the solution under the UV light

BONUS Activity:

Fluorescent Highlighter Experiment:

  1. Using a fluorescent highlighter, draw a picture on the transparent plastic sheet and fill in the picture with the marker.

  2. To test for fluorescence, put on your pair of UV safety glasses and use the black light to view the picture of yellow ink. What color of the light is given off by the picture?

  3. Now turn off the UV light. Can you still see the fluorescence?

Answer: The pyranine dye in the fluorescent yellow ink is absorbing the colorless UV light and fluorescing green.

 

Source: Biophysical Society

 

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