• Students will compare types of light.
• Students will describe scientific literature connected with Green Fluorescent Protein.
• Students will examine objects under different magnifications.

This will introduce students to Sanford Research resources and highlight the need for interdisciplinary problem-solving. Students will explore light sources, learn about microscopes, and understand the limits of light microscopy and advancements in viewing technology.

Teacher Preparation

• Print out one Microscopy Student Guide per student.
• Print out one Light Sources per student. This document will need to be printed one sided.
• Each student will need scissors and clear tape.
• Consider the sources of light (laser pointer, light bulbs, flashlights, optical mice, led light sources, LCD, …) that you have in the classroom and be prepared to share some of them with the students.

Lesson

Start with the Modern Microscopy Slideshow. 

Slide 1
Teacher Note: This lesson is to introduce the awesome techniques scientists are using in 2024 to try to better understand what is happening in the human body. This slide show is going to include a few videos and some abstractions of what is happening in a microscope.

Ask students: Record answers for the following questions in Microscopy Student Guide before discussing:

1. What are some tools that we use to observe the world around us?
2. What are some small things that you might be interested in viewing?

Teacher Note: Topics that might come up in a classroom might include nanotechnology, microbiology, ocean life (ameba, coral, zooplankton) Botany, geology, anatomy, physiology.

Slide 2
Play the Phenomenon Video Modern Microscopy: The run time is 4 seconds. Repeat the video 10 or 15 times.

• In the video I notice that…
• I wonder if the Green represents…
• I wonder if the Red represents…
• I wonder if in nature this process might be used to….
• Record any additional wonders or notices you had

Teacher notes: If you have R/G color blind students remind them that there is a right and a left video the left half is Green and the right half is Red. All data collected in this type of experiment is collected as light intensity for these types of experiments. The color comes from the computer, adding color to the samples afterward. Typically, the assigned color is based on the wavelength of light that is collected, but a person could use any color they could think of to color the sides. This statement is helpful sometimes to remind folks that scientists have lots of ways to work around things that may get in our way.

Slide 3
Ask students: Please share with the class something you noticed or wonder about the video on the previous slide.

Slide 4
Teacher notes: Information from this slide should be recorded in Microscopy Student Guide. Students should take a minute to write their own ideas as to what each piece of the microscope does before you share a more formal answer using the slide.

Slide 5
Tell students: There is an entire field of science dedicated to the study of light. Microscopy is only one branch of the field of Optics.

Slide 6
Tell students: Light is part of the electromagnetic (EM) spectrum, which consists of waves with different wavelengths. These wavelengths range from large radio waves, which can be larger than a planet, to microwaves about the size of a centimeter (e.g., 12.5 cm microwaves), and extremely small waves like cosmic rays, measuring as tiny as 10^-22 meters, or even smaller, beyond our ability to measure.

For this lesson, all we need to know is that there is a section near the middle of the spectrum that the human eye can detect, ranging from 700 nanometers to 400 nanometers.

Slide 7
Teacher notes: Depending on your class, this activity might be done better with some manipulatives cut out or maybe as a classroom discussion. You could have each student cut out the shapes and put them together at their desk, or you could come up with some way to organize these things around the room.

Students will need to have a copy of Light Sources, a pair of scissors and a glue stick

Tell students: Work through the Light Sources Sorting Activity section

1. Organize the light sources into some categories. (There is no right way to sort these light sources. As long as students can provide a rationale for why they are sorted in a specific way they will be on the right track.)
2. Write a short description for why each light source belongs into a given category.
   1. Why are items included?
   2. Why are items excluded?

If you finish early:

1. What are some types of light that are not pictured?
2. Are there additional groups that could be formed by the light sources you added?

Slide 8
Tell students: Microscopes use a variety of lamps and lasers along with a number of light bending tricks (diffraction, refraction, reflection) that let a person get clear pictures of small things. The rest of this presentation is going to try to help a student be able to understand more about how super resolution florescent microscopy can be used to help better understand what is happening in the human body.

Slide 9
The slide displays four types of light paths commonly used in microscopy:

• Phase Contrast Microscopy: This method uses a combination of pinholes and compound lenses to improve the visibility of living cells without the need for staining.
• Bright Field Microscopy: A bright light is shone through the sample, allowing you to see the cell's outer edges, though internal structures remain difficult to observe.
• Dark Field Microscopy: By blocking some of the light and utilizing light refraction and diffraction, this method reduces interference within cells, making internal structures like the nucleus and chloroplasts more visible.
• Fluorescence Microscopy: This technique filters specific wavelengths of light to interact with the sample and then captures the emitted fluorescent light through a camera or eyepiece. The light path is often bent or reflected to guide light from a specialized source (such as a laser or bulb) to the sample and back to the detectors, making these microscopes highly mechanized and expensive due to the intricate use of mirrors and prisms.

Slide 10
Teacher note: Spectra play an important role in microscopy and are a complicated part of how light interacts with matter. They are related to elements and their electron shells or energy levels. Basically, a photon hits an element and depending on the characteristics of the element it will sometimes give off certain colors of light. There is a very simplified online activity that looks at very few elements and what happens when they are hit by a photon. The NIST website is an additional resource you that allows you dig into how this data is collected and how a scientist might think about things. Encourage students to learn about the spectra associated with the elements of life (CHNOPS). (The phet software is intuitive while the NIST.gov is not. If you decide to use the .gov site, try to run a short tutorial on how to navigate websites (5 minutes) and 10 minutes for students to explore either option.)

Tell students: Different types of light sources produce specific wavelengths (spectra). Some, like the sun, emit a broad range of wavelengths, while others, such as LEDs, mercury lamps, and incandescent bulbs, produce a more limited range. Certain sources, like lasers, emit only a single wavelength. When light from these sources interacts with matter, it can excite the material, causing it to emit light in return. Scientists can identify materials based on the spectra they absorb or emit. In microscopy, researchers have discovered proteins that absorb one wavelength of light and emit another. This technique allows scientists to capture images of specific wavelengths, providing valuable insights into objects like planets, stars, nebulae, or even tiny proteins in the human body.

Slides 11
Tell students: Green florescent protein (GFP) is an important discovery when it comes to the world of biomedical (biological) science. This protein and its analogs, derivatives, and counterparts have driven many studies. GFP can be used in so many ways.

Teacher note: Have the students use their Microscopy Student Journal to record some information about a quick literature review related to GFP:

Tell students: Try to search for GFP in Google Scholar or in your school library’s database. (give students time to do this or bring up a new window and do the search). Record in your Microscopy Student Journal:

• Number of search results
• What is one article that had an interesting title if you skim the top 20 hits?

Slide 12
Tell students: GFP is an important molecule in modern research. These are some examples of some additional molecules that exist to fluoresce into different colors. One common use of the additional colors is to help study multiple things at once.

Teacher note: At Sanford we only scratch the surface of all the uses of GFP and its counterparts.

The students now have the basic information to describe what is happening in the video from the earlier slides. A basic description for the video at the beginning of the lesson:

1. Nucleus of the cell was ruptured.
2. The green stuff was the nuclear envelope and the endoplasmic reticulum which are continuous with each other.
3. The green protein stain helped observers to see the nucleus while the scientist was rupturing it.
4. There was a red marker (like GFP but red) used called mCherry to identify a second protein cGAS which binds to the DNA that spills out of the nucleus as part of the rupture repair processes.

Described by the scientist as:
“This is a 100x (magnification) movie of a live cell expressing the ER protein Sec61beta-GFP (green), and the cytoplasmic DNA-binding protein cGAS-mCherry (red). The ER is continuous with the NE, which you can notice forms around the nucleus. When the NE is compromised by mechanical forces, it can break open (see the gap that forms on the NE), which results in the exposure of DNA to the cytoplasm, where cGAS binds and enriches too. This rupture was especially catastrophic, as a substantial amount of DNA is emitted into the cytoplasm.”

Slide 13
Tell students: GFP can be used in many ways.

1. Immunohistochemistry: A GFP molecule is attached to an antibody and then the antibody targets a specific protein in the cell.
2. Fusion tag: The cell is given a specific set of DNA or RNA that tells the cell to produce a protein of interest that is connected to the GFP molecule.
3. There are many other ways that this protein can be used.

These molecules have led to many ways to look at cells and even ways to get to super resolution microscopy usable on living cells.

Slide 14
Tell students: Watch this video to learn how Elizabeth (Ellie) Menzel from Dr. Loukil’s lab uses innovative microscopy techniques to study cellular organelles.

Teachers note: Play the In the Microscope Room video (video length is 4 minutes and 30 seconds). Afterward, have students complete the “Figuring Out Figures” section on the last page of the Microscopy Student Guide. This exercise helps students interpret the images Ellie shared and develop skills for understanding advanced scientific literature.

Slide 15
Teachers note: Dr. Abdelhalim Loukil’s team at Sanford Research studies primary cilia and is a leader in using the SORA (Spinning Disk Super Resolution by Optical Pixel Reassignment) technology. His lab has been instrumental in creating this lesson. For more on his work, visit the Loukil Lab.