Lasering in on Metabolism; Utilizing Fluorescence Spectroscopy to Quantify Metabolic Responses to Heat in Yeast Cells.

The yeast cell cultivation followed a four-step protocol designed to standardize metabolic conditions prior to data collection. Initially, yeast cultures were activated at a constant temperature of 40°C to initiate metabolic activity. Following activation, the yeast was plated onto agar growth media to establish healthy colonies. These colonies were then used to inoculate liquid cultures, which were maintained for a duration of 2 to 3 days to ensure the samples reached a stable growth phase. A plating and inoculation cycle was utilized as necessary to maintain culture viability. Finally, the yeast cells were harvested and underwent final sample preparation in anticipation of UV-excited fluorescence spectroscopy and subsequent spectral phasor analysis (Fig. 3).

Samples of Baker’s Yeast were taken 2-3 days after cultivation on an Agar tray. Prior to the yeast addiction, a centrifuge tube was filled with 1mL of phosphate buffer solution (PBS), and massed on a analytical balance, where then samples of yeast were qualitatively transferred to the centrifuge and re-massed to an ideal mass of 0.05g (with precision of ±0.01g). Afterwards, the PBS was then triple washed and prepared in a cuvette according to standard protocols [3]. In order to properly test the effects of heat on the fluorescence spectra of yeast. Target groups of room temperature (22℃), 37℃, and 52℃ were established, and tested against room temperature samples of PBS media, the washed media, and PBS media with Agar to eliminate background fluorescence. A sample with 0.15mL of 95% ethanol added to it was also tested to validate our methods [4].

Fig. 4 - Sample Preparation Roadmap [3], [4].

Sample Preparation

Samples of Baker’s Yeast were taken 2-3 days after cultivation on an Agar tray. Prior to the yeast addiction, a centrifuge tube was filled with 1mL of phosphate buffer solution (PBS), and massed on a analytical balance, where then samples of yeast were qualitatively transferred to the centrifuge and re-massed to an ideal mass of 0.05g (with precision of ±0.01g). Afterwards, the PBS was then triple washed and prepared in a cuvette according to standard protocols [3].

In order to properly test the effects of heat on the fluorescence spectra of yeast. Target groups of room temperature (22℃), 37℃, and 52℃ were established, and tested against room temperature samples of PBS media, the washed media, and PBS media with Agar to eliminate background fluorescence. A sample with 0.15mL of 95% ethanol added to it was also tested to validate our methods [4].

The spectrophotometer system consists of a 340 nm LED excitation bulb, a sample loading site, and a receiving tube to deliver the emission spectra from the sample to the spectrometer (Fig. 5).

Prior to entering the receiving tube, the emission spectra of NAD(P)H entered a long pass filter, which filtered out light from <385 nm to eliminate the signal from the 340 nm LED excitation bulb, which was then integrated in the spectrometer and graphed at the computer with an integration time of 5000 ms (Fig. 6), where the average fluorescence intensity from a band width of 450-480 nm was averaged for each sample and compared the groups using an ANOVA test.

Fig. 5 & 6 - Spectrophotometer layout and schematics.

Spectrophotometer Layout/Analysis

The spectrophotometer system consists of a 340 nm LED excitation bulb, a sample loading site, and a receiving tube to deliver the emission spectra from the sample to the spectrometer (Fig. 5). Prior to entering the receiving tube, the emission spectra of NAD(P)H entered a long pass filter, which filtered out light from <385 nm to eliminate the signal from the 340 nm LED excitation bulb, which was then integrated in the spectrometer and graphed at the computer with an integration time of 5000 ms (Fig. 6), where the average fluorescence intensity from a band width of 450-480 nm over the 5 minutes durations was corrected using previous data the Urayama's lab took on the yeast fluorescence signal, and combined averaged for every group. These groups were then all compared using an ANOVA test to demonstrate statistical significance.

Results/Finding and Discussion

Fig. 7 - Graph showing the emission spectra received from the 22C (red), 37C (green), 52C (blue), Ethanol (black), and PBS with Agar (pink) groups. Previous data of the NADH emission spectra was laid over as well (yellow) to show which parts of the emission spectra seen can be attributed to NADH excitation.

From the band width of 450-480 nm, data from all heat trials tended to be relatively equal in intensity, while for the ethanol trial seemed to be slightly lower than the rest (Fig. 7). The results from the ANOVA test showed no statistical significance (p > 0.05) between any of the corrected average intensity readings from the band width of 430-480 nm for all samples. It should be noted that the highest peaks of all intensity functions for all groups was around the 500 nm range, showing that there was some background fluorescence between each trial that did scale with temperature. We believe that this background fluorescence could've been the result of any byproducts of metabolism unaccounted for, as the agar plates we used were expired.

Conclusions and Future Study

Metabolism is not just due to increasing NADH production, but instead also dependent on the enzyme activity and how fast the reactions within cellular respiration can take place. Thus, our experiment provides evidence that in the short term, the increase in metabolism due to heat is dominated not by increasing NADH sytnthesis, but that the heat provides a more favorable envionment for enzyme activity and increases the rates of the reaction chains within cellular respiration. Future research could be directed towards conforming this in the long term to see if this relationship changes over time.

As for the lab, our next steps will be to design a diffuse fluorescence fiber optics system to quantify both the NADH and FAD emission spectra In Vivo. Fluorescene Spectroscopy in it's most sophisticated forms allows the the quantification of aerobic vs. anaerobic metabolism, and when combined with diffuse reflectance spectroscopy, can be corrected to investigate things such as the fight or flight response, presence of tumors and quantification of their access to blood or not, and in noninvasive evaluation of a transplant tissue's viability.

Acknowledgements

We would like to thank Dr. Urayama and his lab for allowing us to use their spectrophotometer and for their support throughout the project.

References

[1] I. Georgakoudi et al., Journal of Biomedical Optics 30, S23901 (2025).

[2] B. Gaitan et al., Metabolites 12, 1097 (2022).

[3] A. H. Short et al., RSC Advances 11, 18757 (2021).

[4] S. M. Bailey, E. C. Pietsch, and C. C. Cunningham, Free Radical Biology and Medicine 27, 891 (1999).

NACE Career Readiness Competencies

Critical Thinking

At the beginning of the project, we had a vision vastly different than where we ended up at. Originally, we had wanted to create a complicated model combining both diffuse reflectance and fluorescene scpectroscopy. As we learned more about the spectrophotometer and yeast sample cultivation and preparation, many questions arose in our minds and our vision became clouded, resulting in our original direction being lost. When looking retrospectively at all of our goals again, we decided to priotitize our overarching goal of presenting at Undergraduate Research Forum. After rethinking the scope of the project, we decided to simplify and focus in on only possible affects of heat in the NADH emission spectra in yeast cells, and only use diffuse fluorescene spectroscopy to record and quantify our data. This new approach culminated in our ability to successfully run and analyze our new experiment, along with the ability to have a complete, finished product to present to our peers at the 2026 Miami University Undergraduate Research Forum. These bumps in our experiment, and our triumph over them, helped to develop the critical thinking skills necessary to navigate future challenges as we develop research projects and tackle professional challenges.

Teamwork

Throughout this research project, a necessity for collaboration not just between ourselves but also with other labs arose. In the beginning portions of our project, progress towards our goal was made slowly, and as deadlines arose, so did a necessity for innovation. We developed a systematic approach to data collection in collaboration with the Urayama lab, which allowed us to finish all of our data collection within a single day. Along with that, we no longer allowed scheduling conflicts to prevent us from learning new lab techniques or receiving input from others about our project, instead deciding to have some members go to meetings and relay any new information learned to the rest of the group. From these experiences, we learned not just to rely on ourselves, but on each other and the people and resources around us. Because of these changes, we were able to finish the project with plenty of time to spare. Overall, these learning moments and experiences developed the teamwork skills necessary for our lab of strictly first-year students to work as one coherent group, an attribute that will follow us all as we pursue different academic interest and projects, and in our transition from college life to our careers.

Communication

Our research project involved collaboration of multiple peers and mentors, communicating as a team was key to be able to keep everyone on the same page. We worked hard to actively listen to each other, compromise, and utilize clear explanations for our thought processes. For example, while discussing ideas for our project, we combined our ideas of using ethanol and temperature to change the state of yeast's metabolism. In the end, finding a compromise through utilizing multiple ideas elevated our research and further developed our project. Without our communication and meetings with Dr. Urayama and upperclassman Dylan Aubel and Anna Lagona we wouldn't have been able to successfully complete our research. By sending professional emails and discussing methods in person we were not only able to learn and grow our knowledge about yeast, but also practice the proper way to respectfully address, communicate and present ourselves to authoritative figures. Through our time talking with mentors we also learned that it is important to ask the right questions and provide meaningful details and information. With thorough communication we ensured that we were prepared for the future and that whomever we were speaking with understood what we needed to be successful. Maintaining proper communication has prepared us to tackle professional challenges with collaboration and clarity.

Abstract/Introduction

Abstract

Mitochondrial metabolic processes are highly sensitive to thermal fluctuations. To evaluate this relationship, we employed fluorescence spectroscopy to measure NADH autofluorescence in yeast cell cultures subjected to incremental heat stress. Readings were taken from yeast suspensions from a range of temperatures, along with the media without yeast and yeast with 0.15mL added ethanol. We found that increasing the temperature did not increase the NADH emission spectra on a statistically significant level, concluding that in the short term, increasing metabolic responses are due to increasing enzyme activity rather than increasing NADH biosynthesis.

Introduction

In biomedical sensing there is a distinct lack of standardized medical technologies that provide minimal to no interference, or potential harm to a patient in some fashion. The various biological properties of tissues, as they relate in the field of NIR (near-infrared) spectroscopy, have the potential to provide a method by which future medical technology can quantify and monitor tissues In Vivo, label-free, and noninvasively. Currently, many near-infrared sensing techniques have been applied to biological tissues, one of the most popular being fluorescence spectroscopy.

Fluorescence spectroscopy, often also referred to as fluorescence diffuse optical tomography (FDOT) or diffuse fluorescence imaging (DFI), is a spectroscopy technique which uses a laser input to quantify the metabolism of tissues. FAD+ and NAD(P)H, both oxidized and reduced molecules in cellular respiration respectfully (Fig. 1), have the intrinsic property of being naturally fluorescent [1]. By using a laser input at specific wavelengths to excite these molecules, an intensity/wavelength function can be derived (Fig. 2). Typically, the NAD(P)H emission spectra peaks from 430 nm-480 nm, and the FAD+ emission spectra peaks around 520-550 nm [2]. Through the intensity/wavelength functions of both NAD(P)H and FAD+, we’re able to quantify metabolic states in surveyed tissues.

Baker’s yeast (Saccharomyces Cerevisiae) is valuable in metabolic sensing studies due to its capabilities for cellular respiration and general ease of care, providing a method by which metabolic data can be collected. It has been generally understood that heat has the capabilities to increase cellular respiration up to the point of protein denaturation, but there is a distinct lack of literature which investigates how the emission spectra from the auto-fluorophores, specifically NAD(P)H, changes with different levels of heat.

A figure of the mitochondira showcasing glycolysis, the Citric Acid Cycle, and oxidative phosphorylation and the places where NADH can be found among each of those cycles.

Fig. 1 - Theoretics for Fluorescence Spectroscopy [1].

A fluorescence vs. wavelength graph that shows NADH emission spectra at a wavelength of 450 and an FAD emission at 550 nm.

Fig. 2 - Emission spectra of NADH and FAD+ in Yeast [2].

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