Teaching fluorescence of plants & algae in physics class to address climate change

In physics classes and general education classes, teaching the concept of fluorescence can be challenging, and it may seem too theoretical for some students. A short YouTube video titled ‘Seeing Photosynthesis from Space’ displays a global map of photosynthesis, and this is an excellent, attention-getting way to visually introduce fluorescence to students and also to address climate change. Therefore, three hands-on activities were designed using spinach chlorophyll ethanol extract and olive oil to observe the fluorescence emission; in addition, a smartphone spectrophotometer was employed to observe the spectrum of the emission. The class also addressed the issue of global warming because the absorption of CO2 by plants and algae is decreasing, which may cause serious climate change.


Introduction An Internet video titled: Seeing Photosynthesis from Space: NASA Scientists Use Satellites to
Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author (s) and the title of the work, journal citation and DOI.[1] demonstrates that plants emit fluorescence during photosynthesis that is barely visible to the naked eyes.Also, physics teachers have designed many interesting studies of fluorescence, such as the red fluorescence emitted in extra virgin olive oil when illuminated with the light from a green or blue-violet laser pointer; this results from the high chlorophyll (fluorophores) content of olive oil [2,3].In physics and general education classes, students can be taught about the fascinating fluorescence phenomenon of chlorophyll, which absorbs CO 2 during photosynthesis and emits red fluorescence.This phenomenon may be a key method for assisting scientists who are monitoring serious climate change issues via satellites.Chlorophyll is the most abundant and important natural photosynthetic pigment; moreover, it can help in environmental protection because techniques are now being developed and utilized to employ chlorophyll as a bio-solar clean energy source to reduce environmental pollution.Therefore, we designed a hands-on activity that uses fluorescent pigments obtained from spinach and algae to produce visualized images that should enhance students' interest and also teach the sustainability issues that the world is facing.

Concept of fluorescence
Fluorescence is the emission of light by a fluorophore that absorbs incoming light and then reemits (see figure 1).The fluorescence occurs when the light strikes electrons in the fluorophore molecule and raises to an excited state (figure 1 x), then some of the energy is lost via nonradiative processes (figure 1 y), and finally the fluorophore emits fluorescence and falls back to the ground state (figure 1 z) [4,5].Therefore, the fluorescence is emitted at longer wavelengths than those of the incoming light wavelengths [6].
There are some fluorophores that we can easily obtain from the kitchen, such as the curcumin that is in turmeric or used in curry.If we sprinkle the curry or turmeric powder into ethyl alcohol under blue-violet light illumination, we can observe a green-yellow fluorescence.This fluorescence can be explained by the fact that the electrons in curcumin absorb and gain energy from the incoming shorter wavelengths, blue-violet light, and move from their ground states to excited states.When the electrons return to their ground states, a green-yellow glow is emitted at longer wavelengths (figure 2) [7].

The role of natural photosynthetic pigments in climate changes
Plants and algae (including microalgae and cyanobacteria) contain chlorophylls, which are the most abundant natural pigments.Chlorophylls  capture light and convert water and CO 2 into glucose and oxygen during photosynthesis and also re-emit red fluorescence as part of the process.Photosynthesis is the absorption of a photon that stimulates the emission of a lower-energy photon with longer wavelengths (red light) [8,9].The red fluorescence emission during photosynthesis activity is about 1% of the intensity of the absorbed light, and it is re-emitted at a wavelength of approximately 670 nm.Because the emission intensity is so low, it is invisible to the unaided eye, but it is detectable by NASA satellites.Solar-induced chlorophyll fluorescence (SIF) provides a direct measure of how much oxygen is being released and how much carbon is being absorbed.Tracking red fluorescence from space demonstrates the effects of climate change and allows us to monitor the health of the earth's ecosystems [10,11].
The satellite observations shows the potential of combining fluorescence with atmospheric CO 2 to monitor and quantify the vegetation activity caused by the climate changes such as flooding, drought or heatwaves, providing early warnings of ecosystem degradation or potential crop failures [12,13].
The fluorescence is a proxy for photosynthetic activity, which is influenced by environmental factors such as temperature, precipitation, and atmospheric composition.By monitoring changes in red fluorescence over time, the health and productivity of Earth's ecosystems affected by the climate changes can be assessed.
It also allows researchers to map the spatial and temporal distribution of vegetation productivity on a global scale, and the changes in land use, such as deforestation or urbanization, can significantly impact vegetation dynamics and alter the amount of SIF emitted by affected areas.
The SIF can provide insights into variations in carbon uptake by vegetation.Monitoring these changes helps researchers better understand how ecosystems are responding to increasing atmospheric carbon dioxide levels and can refine climate models to predict future carbon cycle dynamics.The satellite SIF provides as a direct and measurable signal of terrestrial photosynthesis from space [14][15][16].

Chlorophyll fluorescence
Chlorophyll absorbs light mostly at red and blue wavelengths and reflects green wavelengths, but it also fluoresces at the red wavelength of 670 nm (figure 3) [6].

Methods: hands-on activities
Three experiments were conducted to demonstrate the fluorescent phenomena.The students were requested to bring olive oil from their homes as part of this activity, and it is interesting to note that only the extra virgin olive oils exhibited the red emission of light, as previously mentioned [17].

Preparation of chlorophyll solvents
Spinach leaves were used as the test material.An appropriate amount of spinach leaves was finely chopped and then mixed with ten times their volume of ethyl alcohol 75% to obtain chlorophyll ethanol extract (CEE).

Activities I
The extra virgin olive oil and the CEE were both excited by the same violet-blue laser.We observed that the red fluorescence specifically appeared close to the surface, and the fluorescent emission from CEE was less than that from the olive oil (figure 4).The phenomena are recognized as the inner-filter effect in fluorescence spectroscopy.When there are too many fluorescence molecules and not enough incident light (photons) to excite them, the fluorescence tends to be confined to the surface.As the incident beam is fully absorbed, the fluorescence diminishes rapidly because there are no more incident photons to continue exciting the molecules deeper into the material [17].The concentration of chlorophyll in CEE is likely higher than in olive oil from visual observations.A further experiment will be conducted to observe fluorescence at various solutions with different levels of chlorophyll intensity, and enhance students' understanding of inner-filter effect.

Activities II: green-light and red-light laser pointers excited chlorophyll fluorescence
The CEE and olive oil were tested by green and red laser pointers, and the red fluorescence was emitted (figure 5).The olive oil showed better red fluorescence than CEE, and both red and green laser pointers were able to excite red fluorescence at 670 nm.The red laser pointer was able to excite red fluorescence in CEE and olive oil (figures 5(C) and (D)), although our eyes are unable to distinguish between the incident red light and the red fluorescence [17].
In the second stage of the demonstration, different wavelengths of laser pointers were employed to clarify the fluorescence phenomenon; this activity demonstrated that fluorophores absorb light energy and then emit light at longer wavelengths.Next, the third experiment was set up to display the fluorescence spectrum by using a Scimage ® smartphone spectrometer (figures 6 and 7) with spectrophotometer module (figure 8(A)) [18].
This experiment utilizes the SciView smartphone spectrometer and its design adopts a vertical short optical path configuration, combined with the use of specially made high-efficiency gratings to address the issue of spectral curvature  often observed in homemade spectrometers.The construction materials uses fully sealed black acrylic to avoid light leakage (figure 7).The slit located at the front end of the spectrometer can be replaced with a spectrophotometer module with a cuvette (as shown in figure 8(A)).When the light source passes through the central slit to illuminate different test materials, by  utilizing the transmittance and absorbance values of different materials, its spectrum or absorbance can be observed and calibrated.The spectrophotometer module can also be fixed with a box (as shown in figures 8(B) and (C)), and a light source (e.g.UV light) installed above the spectrophotometer cuvette filled up with materials to observe the fluorescence spectrum.The fluorescence phenomenon generated on the surface of the tested material and its spectrum is displayed through the smartphone camera mode (as shown in figure 8(D)).

Activities III: chlorophyll fluorescence spectrum
The activity was designed to view the chlorophyll fluorescence and also to see the wavelengths of the light absorbed by chlorophyll (figure 8).
Step (1) Acquire a spectrophotometer module and pour 1 c.c. CEE into the tube (as the red arrow pointing on figure 8(A)).
Step (2) Position the spectrophotometer module in the dark box to ensure that the UV light beam accurately irradiates into the cuvette (figure 8(B)).
Step (3) Position the mobile phone lens at the observation port of the spectrometer (figure 8(C)).

Results of activities III
From these experiments, we observed chlorophyll's absorption and fluorescence spectra (figure 9).The results clearly show that most blue and red wavelengths were absorbed by chlorophyll (figure 9(B)) when white LED light flashed through the solvent; moreover, when the UV light flashed, the red spectrum was detected by the spectrometer (figure 9(C)).

Discussion
We also extracted chlorophyll from algae to learn if it would produce the same red emission as spinach chlorophyll.The two extracted chlorophyll test tubes were illuminated with UV light, and a beautiful red fluorescent phenomenon resulted as seen in figure 10.
Plants and algae contain chlorophyll, which enables them to convert sunlight into energy and absorb CO 2 during photosynthesis.Algae encompass thousands of known species, categorized into several main groups based on their characteristics: green algae (Chlorophyta), red algae (Rhodophyta), brown algae (Ochrophyta), and diatoms (Bacillariophyta).All algae contain chlorophyll a, which enables them to carry out photosynthesis and obtain nutrients.Therefore, the fluorescence phenomenon can be observed through the chlorophyll and UV light they contain.Additionally, different types of algae contain various forms and amounts of accessory pigments, such as chlorophylls (a, b, c, d), α-, β-carotene, phycocyanin, phycoerythrin, fucoxanthin, and xanthophylls.The combination and proportion of these accessory pigments give different algae their diverse colours.Algae growing in nearshore areas, like green algae, can grow by using sunlight to perform photosynthesis.Plants growing in the deep sea cannot directly receive sunlight but can absorb sunlight entering the ocean, transferring it to chlorophyll for photosynthesis.For example, red algae not only contain chlorophyll but also have the photosynthetic accessory pigment phycoerythrin, which absorbs blue-green and blue light and reflects red light, giving red algae their characteristic red appearance.This is the reason why red algae appear red [19].Cyanobacteria, often referred to as blue-green algae, are a phylum of bacteria that conduct photosynthesis, although they are not classified as algae [20,21].Chlorella (green algae) and spirulina (blue-green algae) are both the most introduced for growing at home due to their high nutrient density and relatively easy cultivation processes.They are often promoted on social media platforms for their health benefits and sustainability.
These hands-on activities were designed to be performed easily by students to help them understand the concepts of fluorescence, which is a fascinating and amazing phenomenon; the visually impressive images should enhance students' interests in physics.
An experiment to demonstrate fluorescence at various depths in solutions with different levels of chlorophyll intensity is suggested for a further discussion and better understanding about the innerfilter effect to students by visual observation.

Conclusion
The red fluorescence phenomena show how physics enters into our daily lives, and this red glow also was quantified by instrumentation on a satellite to measure CO 2 taken up by plants and algae to analyse the health of ecosystems.From NASA's red fluorescence and CO 2 monitoring analysis, we can see that the absorption of CO 2 is decreasing, which causes global warming and critical climate change; moreover, the emission maps also warn us that more trees and algae are needed.It is important to note that algae is well known as easy to grow and has multiple uses such as food, food supplements, biofuel, a source of chemicals, and a bioplastic resource.Algae can also serve as a waste management system and a means of converting solar energy into electricity [22,23].Teaching red fluorescence is a fine example of an interdisciplinary program to teach physics and climate change, which needs to be addressed to slow down global warming.Further demonstration experiments that involve growing algae at home can be designed and introduced in physics and general education classes as a way of teaching students about sustainability and the greenhouse effect.This will help to reduce our carbon footprint.Harnessing photosynthesis to produce electricity using cyanobacteria, green algae, seaweeds and plants Front.Plant Sci. 13 955843

Figure 2 .
Figure 2. The fluorophores (curcumin) absorb shorter wavelengths (the best excitation wavelength is 467 nm), and emit at longer wavelengths (the fluorescence emission peak is 550 nm).

Figure 3 .
Figure 3. Chlorophylls absorb red and blue wavelengths and reflect green; they also emit fluorescent light in the red region at 670 nm.

Figure 4 .
Figure 4. Illuminating (A) CEE and (B) olive oil with a violet-blue laser pointer.

Figure 5 .
Figure 5. Emissions of olive oil and CEE under green and red laser pointers.

Figure 7 .
Figure 7. White light spectrum shown on the smartphone at camera mode.

Figure 8 .
Figure 8.A spectrometer with spectrophotometer module was set up to observe chlorophyll fluorescence spectrum.

Figure 9 .
Figure 9. (A) White LED light spectrum.(B) White LED light through CEE.(C) UV light through chlorophyll extraction.

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