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# Goal/Experiment:
To acquire photosynthetic efficiency (F<sub>v</sub>/F<sub>m</sub>) and quantum yield of photosystem II (Y(II)) of Microcystis aeruginosa cultures using far-red acclimation with the PHYO-PAM-II Compact Version.

# Application of PHYTO-PAM-II (Compact Version) For Running Rapid Light Curves on Cyanobacterial Samples

**Forked from**: [Application of PHYTO-PAM-II (Compact Version) on Aureococcus anophagefferens cultures for photosynthetic efficiency and quantum yield of PSII](https://dx.doi.org/10.17504/protocols.io.eq2ly7jqelx9/v1)

### Citation:
Gwen Stark, Emily E. Chase, Steven W. Wilhelm 2022. Application of PHYTO-PAM-II (Compact Version) For Running Rapid light curves on Cyanobacterial samples. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7jqelx9/v1

### Contributors:
Gwen Stark<sup>1</sup>, Emily E. Chase<sup>2</sup>, Steven W. Wilhelm<sup>1</sup>
1. The University of Tennessee, Knoxville
2. University of Tennessee, Knoxville

---

## Abstract
A protocol to acquire photosynthetic efficiency (F<sub>v</sub>/F<sub>m</sub>) and quantum yield of photosystem II (Y(II)) of Microcystis aeruginosa cultures using far-red acclimation.

## Keywords
- Photosynthetic efficiency
- Quantum yield
- F<sub>v</sub>/F<sub>m</sub>
- HAB
- Chlorophyll fluorescence
- Cyanobacteria

## License
This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

---

## Guidelines
The collection of quantum yield and photosynthetic efficiency is highly sensitive to modifications in sampling protocol.

## Materials
- PHYTO-PAM-II Compact Version and components
- Laptop with a USB port, and PhytoWin_3 installed
- Measurement cuvettes
- At least 3 mL of culture material for each sample
- 70% Ethanol
- KimWipes
- Necessary materials and setup for both dark adapting cultures, and a no light or low light environment for sampling

## Before Starting
Familiarize yourself with the PHYTO-PAM-II equipment, manufacturer’s provided manual, and the basics of chlorophyll fluorescence parameters for full use of data collected and accuracy of results.

---

## Equipment Preparation
1. **Setting Up the PHYTO-PAM-II Compact Version:**
    - Toggle the power and connect to a laptop via USB.
    - Open the PhytoWin_3 program and select "Phyto Compact Unit."
    - Ensure the program is in "MEASURE" mode, and that the "ML" light is green (indicating it is ready).
    - Ensure that "AL" and "FR" lights are not selected.

![Equipment Setup](image1.png)

2. **Adjusting Settings:**
    - Modify Phyto-Pam settings based on the species being tested.
    - Use the "View Pulse" check box to observe the saturation pulse kinetics, ensuring a distinct plateau is seen.
  
---

## Sample Preparation
3. **Sample Transfer:**
    - Transfer 3 mL of culture to a quartz cuvette and minimize exposure time.
    - Place the cuvette in the optical port and cover with the darkening hood.

4. **Auto-Adjust Gain:**
    - Perform a zero-offset function using Zoff-determination to blank the PHYTO-PAM-II before runs.
    
5. **Far-Red Acclimation:**
    - Apply far-red light acclimation to induce photosystem I and oxidize the PQ pool.
    - Dark acclimation is not recommended for cyanobacteria.

    > **Note:** Evaluate the necessity between no acclimation and weak far-red acclimation for consistent results.

---

## Measurement Acquisition
6. **Experimental Measurements:**
    - Measurements can now be collected.

7. **Running Far-Red Light Acclimation:**
    - Transfer 3 mL of culture into the cuvette, auto-adjust gain, and run acclimation.
    - Turn on the "ML" button and wait for the light to turn green.

8. **Taking Measurements:**
    - Choose F<sub>v</sub>/F<sub>m</sub> measurements or run a rapid light curve.
    - Adjust PAR and exposure times appropriately.
    - Aim for the ETR curve to hit a maximum peak and level off.

9. **Recording Data:**
    - Data will be recorded automatically.
    - Use 70% ethanol to clean the cuvette between different treatments.

10. **Data Storage:**
    - Save and export data as a .csv file.
    - Unplug the equipment and charge fully for long-term storage.

> **Recommendation:** Regularly charge the equipment if stored unused for extended periods.

---

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