Name: Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer (Video) | JoVE Journal
Uploaded: 2021-11-12T13:00:00.000+00:00
Duration: 7 min 3 s
Description: Learn to measure photosystem II photophysiology in attached Colacium sp. algae using fast repetition rate fluorometry. This protocol enables real-time assessment of photosynthetic activity in epizoic algae on zooplankton substrates.

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5 minutes

The work was supported by the Collaborative Research Fund from Shiga Prefecture entitled &#34;Study on water quality and lake-bottom environment for the protection of the soundness of water environment&#34; under the Japanese Grant for Regional Revitalization and the Environment Research and Technology Development Fund (No. 5-1607) of the Ministry of the Environment, Japan.&#160;https://www.kantei.go.jp/jp/singi/tiiki/tiikisaisei/souseikoufukin.html. The authors would like to thank Enago (www.enago.jp) for the English language review.

1. Sampling
<ol>
	<li>Collect lake water&#160;from the surface by a bucket. To target Colacium sp. attached to S. mucronata (Figure 1A-C) filter 0.5-10 L of lake water using a 100 &#181;m nylon mesh net52. 
	NOTE: S. mucronata often densely aggregate in shallow, eutrophic, muddy water, such as among reed (phragmites) areas.</li>
	<li>Store the concentrated samples in 500 mL plastic bottles with 350 mL of lake water. Keep in dark conditions.</li>
	<li>In the laboratory, pour the sample water into a 500 mL beaker and allow it to settle for a few minutes.</li>
	<li>Filter the lake water through a 0.2 &#181;m pore-size filter.</li>
	<li>Pick up S. mucronata individuals using a pipette under an optical microscope at 100x magnification. Perform species identification according to B&#322;&#281;dzki and Rybak53.</li>
	<li>Transfer them into a drop of 0.2-&#181;m filtered lake water (FLW) placed on a glass slide. 
	NOTE: S. mucronata may swim to the surface or attach to the beaker wall.</li>
	<li>Check S. mucronata under light microscopy.</li>
	<li>Wash S. mucronata individuals using FLW (3 drops or more) to prevent contamination from other organisms (Figure 2).</li>
	<li>Keep S. mucronata at&#160;an in situ temperature in a growth chamber under 40 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1.</li>
</ol>
2. Effects of S . mucronata on baseline fluorescence
<ol>
	<li>S. mucronata cultivation
	<ol>
		<li>Pick up S. mucronata individuals using a pipette under an optical microscope at 100x magnification and wash using FLW, as in step 1.8.</li>
		<li>Aerate tap water using an electric air pump via an air stone for at least 1 week. Pour 300 mL of aerated tap water into a 350 mL glass jar.</li>
		<li>Feed Chlorella (1 mg C&#183;L&#8722;1) and maintain at 20 &#176;C under 40 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1 in a growth chamber.</li>
		<li>After approximately 14 days, pick up 5-30 individuals using a pipette under an optical microscope at 100x magnification and inoculate them into 300 mL of clean, aerated tap water to keep the medium fresh.</li>
	</ol>
	</li>
	<li>Setting up FRRf
	<ol>
		<li>Launch the Act2Run software.</li>
		<li>Click on the Options tab and select Act2 FLC (white LEDs) in Mode of Operation to set the actinic LEDs color.</li>
		<li>Click on the value of Dark at step 1 of the settings of the fluorescent-light curve in the main window, and type 30 to set the duration of the dark period (Figure 3A).</li>
		<li>Click on the LED combination B, C, and D to turn off the green and orange LEDs (Figure 3C).</li>
		<li>Click on the value of Fets and Pitch under Sat, and type 100 and 2, respectively, to set the number and pitch of flashlet in the saturation phase (Figure 3D).</li>
		<li>Click on the value of Fets and Pitch under Rel, and type 40 and 60, respectively, to set the number and pitch of flashlet in the relaxation phase (Figure 3E).</li>
		<li>Activate the water jacket pump by clicking on During FLC (Figure 3F) to control sample temperature during the measurement.</li>
		<li>Activate by clicking Auto-LED and Auto-PMT (Figure 3F).</li>
		<li>Click on Synchronize to connect FRRf-Act2.</li>
	</ol>
	</li>
	<li>FRRf measurements
	<ol>
		<li>To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult S. mucronata (body size 400-650 &#181;m) from the culture in steps 2.1.1-2.1.4 without any attached organisms.</li>
		<li>To avoid fluorescence from the gut contents, starve&#160;the individuals in FLW at 20&#176;C for at least 90 mins.</li>
		<li>Pour 1.5 mL of FLW into a cuvette. Pick up 0, 1, 5, and 10 S. mucronata individuals using a pipette under an optical microscope at 100x magnification.</li>
		<li>Transfer S. mucronata individuals into the cuvette and add FLW to bring the sample up to 2 mL.</li>
		<li>Acclimate under low light (1-10 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1) at 20 &#176;C for 15 min before FRRf measurement.</li>
		<li>Click on Act2 Run to start the measurement. Repeat the measurements &#62;3 times per sample.</li>
		<li>Read the Fo value from the result plot (Figure 4).</li>
	</ol>
	</li>
</ol>
3. Effects of substrate organism on Chl- a fluorescence
<ol>
	<li>Colacium sp. cultivation
	<ol>
		<li>Prepare the FLW and AF-6 medium54 for cultivation (Table 2).</li>
		<li>Collect Colacium sp. attached to S. mucronata as in steps 1.1 and 1.2 and, keep at in situ temperature in a growth chamber.</li>
		<li>Pick up Colacium sp. with a molted carapace (Figure 1D) using a pipette under an optical microscope at 100x magnification. Wash them with FLW, as in step 1.8.</li>
		<li>Aseptically inoculate Colacium sp. and AF-6 medium in&#160;a 10 mL glass tube on a clean bench.</li>
		<li>Maintain the culture at in situ temperature under 200 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1 in a growth chamber. Shake the glass tube gently by hand at least once per day to prevent cell settlement. 
		NOTE: To keep the attenuation effect of aggregated colonies as low as possible, check the colonies under a microscope prior to FRRf measurement. Cell aggregation may cause dense colonies and affect algal photophysiology55.</li>
	</ol>
	</li>
	<li>FRRf measurements
	<ol>
		<li>To examine the effects of zooplankton individuals on Chl-a fluorescence from Colacium sp., prepare adult S. mucronata (body size 400-650 &#181;m) without any attached organisms.</li>
		<li>To avoid fluorescence from the gut contents, starve the&#160;individuals in FLW for at least 90 min.</li>
		<li>Set up a cuvette-type fast repetition rate fluorometer (FRRf).</li>
		<li>Pour a 1.5 mL subsample of precultured Colacium sp. into a cuvette. Transfer 0, 5, 10, and 15 S. mucronata individuals into these cuvettes and add 2 &#181;m of filtered medium to bring the sample up to 2 mL.</li>
		<li>Acclimate under low light (1-10 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1) at 20 &#176;C for 15 min before taking the FRRf measurement. 
		NOTE: Maintain the samples at incubation temperature during measurements</li>
		<li>Click on Act2 Run to start the measurement. Repeat the measurements &#62;3 times per sample.</li>
		<li>Read the FO&#160;and Fm values from the result plot (Figure 4). 
		NOTE: Check the R&#963;PSII value (Table 1), which shows whether the LED power is within the optimal range to estimate the PSII parameters correctly. When the Auto-LED is activated, Act2run system controls the LED power to achieve an optimal R&#963;PSII range (0.042-0.064). The experimental R&#963;PSII cut-off value was defined at 0.03 and 0.08 in a previous study48.</li>
		<li>To correct the baseline fluorescence22, filter the culture medium using a 0.2-&#181;m pore-size filter and measure the fluorescence. Subtract FO&#160;of the baseline sample from FO and Fm of Colacium sp., or modify the Blank correction value in the Settings in the Options tab.</li>
	</ol>
	</li>
</ol>
4. Photophysiology of Colacium sp. (attached stage)
<ol>
	<li>Isolate S. mucronata individuals with Colacium sp. using a pipette under an optical microscope.</li>
	<li>Wash S. mucronata using FLW, as in step 1.8.</li>
	<li>Transfer S. mucronata into a 100 mL of FLW. For starvation, keep under dark conditions at in situ temperature for 90 min.</li>
	<li>Pour 1.5 mL of FLW into a cuvette.</li>
	<li>Transfer ~10 S. mucronata individuals with Colacium sp. into a cuvette. For measurements, more than 100 Colacium cells per 2 mL are needed. Add FLW to bring the sample up to 2 mL.</li>
	<li>Acclimate under low light (1-10 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1) at in situ temperature for 15 min. Measure Chl-a fluorescence as in steps 3.2.6-3.2.8.</li>
	<li>To enumerate the number of attached cells, fix the sample with glutaraldehyde (2% final volume) after taking the FRRf measurement. Take pictures at several focal depths and positions of S. mucronata under a light microscope.</li>
</ol>
5. Photophysiology of Colacium sp. (planktonic stage)
<ol>
	<li>Cultivate sampled Colacium sp. in AF-6 medium at in situ temperature as in steps 3.1.1-3.1.5.</li>
	<li>For the stationary phase, take 2 mL of cultured Colacium sp. and pour into a cuvette.</li>
	<li>Acclimate under low light (1-10 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1) at in situ temperature for 15 min. Measure Chl-a fluorescence as in steps 3.2.6-3.2.8.</li>
</ol>
6. Effects of Ca and Mn addition on photophysiology of Colacium sp.
<ol>
	<li>Effects on attached stage
	<ol>
		<li>Isolate S. mucronata individuals with Colacium sp. using a pipette under an optical microscope. Wash using FLW, as in step 1.8.</li>
		<li>Transfer six individuals each into 12 glass beakers with 30 mL of FLW. Ensure that each beaker contains &#62;100 Colacium sp. cells.</li>
		<li>Add 200 &#181;mol&#183;L&#8722;1 CaCl2&#183;H2O (Ca treatment), 40 &#181;mol&#183;L&#8722;1 MnCl4 (Mn treatment), or ultrapure water (control) to each beaker. Incubate the samples under 200 &#181;mol photon&#183;m&#8722;2 &#183;s&#8722;1 at in situ temperature in a growth chamber.</li>
		<li>At 3 h and 21 h, transfer all individuals and molted skins into a cuvette with 2 mL of the medium.</li>
		<li>To examine the rapid response to increasing light, click on Up of the periods of 8 actinic light steps and type 20 (Figure 3A) to set the duration of each step in 20 s. To set the stepwise actinic light as 0, 11, 25, 44, 68, 101, 144, and 200 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1, click on High E and Step Up and change the values to 200 and 34, respectively (Figure 3B).</li>
		<li>After 15 min of dark acclimation, measure Chl-a fluorescence of each sample similar to steps 3.2.6-3.2.8. 
		NOTE: Verify that the R&#963;PSII and R&#963;PSII&#39; values are within the optimal range (0.03-0.08)48.</li>
	</ol>
	</li>
	<li>Effects on planktonic stage
	<ol>
		<li>Cultivate sampled Colacium sp. in AF-6 medium at in situ temperature as in steps 3.1.1-3.1.5.</li>
		<li>Transfer the cultured Colacium sp. into FLW and acclimate at in situ temperature less than 200 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1 for 3 days.</li>
		<li>Transfer 1 mL of the acclimated samples into three glass vials with 10 mL of FLW.</li>
		<li>Add 200 &#181;mol&#183;L&#8722;1 CaCl2&#183;H2O (Ca treatment), 40 &#181;mol&#183;L&#8722;1 MnCl3&#160;(Mn treatment), or ultrapure water (control) to vials. Incubate the samples under 200 &#181;mol photon&#183;m&#8722;2&#183;s&#8722;1 at in situ temperature in a growth chamber.</li>
		<li>At 3 h and 21 h, measure Chl-a fluorescence of each sample as in steps 3.2.6-3.2.8.</li>
	</ol>
	</li>
</ol>

The authors have no disclosures to declare.

This protocol demonstrated for the first time that photophysiology of Colacium sp. during the attached stage in a natural environment is comparable to its planktonic stage in AF-6 medium. Additionally, gut contents of starved S. mucronata did not affect baseline and Chl-a fluorescence when density was &#8804;5 inds&#183;mL&#8722;1 (Figure 5 and Figure 6). These results suggest this protocol can measure photophysiology of <em...

Chlorophyll variable fluorescence is a useful tool for measuring algal photosystem II (PSII) photophysiology. Algae respond to various environmental stresses, such as excess light and nutrient deficiency, by altering their PSII photophysiology. Fast repetition rate fluorometer (FRRf) is a common method for measuring PSII photophysiology1,2 and estimating primary productivity1,3,4, which enables monitoring phytoplankton PSII photophysiology, as well as primary productivity across wide spatial and temporal scales5,6,7. FRRf can simultaneously measure PSII (&#963;PSII) absorption cross section, reaction center ([RCII]) concentration, maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) (Table 1). In general, Fv/Fm and Fq&#8242;/Fm&#8242; are defined as PSII activity8, while NPQNSV&#160;is defined as relative heat-dissipated energy9.
Importantly, single turnover (ST) flashes of FRRf fully reduce the primary quinone electron acceptor, QA, but not the plastoquinone pool. Conversely, multiple turnover (MT) flashes from a pulse amplitude modulation (PAM) fluorometer can reduce both. The ST method has a clear advantage over the MT method when identifying the possible origins of NPQNSV&#160;by simultaneously measuring recovery kinetics of Fv/Fm, Fq&#8242;/Fm&#8242;, NPQNSV, and &#963;PSII10. To date, several types of FRRf instruments, such as submersible-type, cuvette-type, and flow-through-type, are commercially available. The submersible-type FRRf enables in situ measurements in oceans and lakes, while the cuvette-type FRRf is suitable for measuring small sample volumes. The flow-through type is commonly used to continuously measure the photophysiology of phytoplankton in surface waters.
Given the development of PAM fluorometers, including the cuvette-type, for a broad range of subjects11, PAM fluorometers are still more common than FRRfs in algal photophysiology research12. For example, although the sample chamber structure and cuvette capacity between these tools only differs slightly, the cuvette-type PAM has been applied to phytoplanktons13,14,15, benthic microalgae16,17,18, ice algae19, and epizoic algae20, while the cuvette-type FRRf has been applied primarily to phytoplanktons21,22,23 and a limited number of ice algal communities24,25. Given its effectiveness, cuvette-type FRRf is equally applicable to benthic and epizoic algae. Therefore, expanding its application will provide considerable insight into PSII photophysiology, particularly for lesser-known epizoic algal photophysiology.
Epizoic algae have received little attention, with few studies examining their PSII photophysiology20,26, most likely due to their minor roles in aquatic food webs27,28. However, epibionts, including epizoic algae, can positively influence zooplankton community dynamics, such as increasing reproduction and survival rates29,30, as well as negatively impact processes, such as increasing sinking rate29,31 and vulnerability to visual predators32,33,34,35,36. Therefore, exploring the environmental and biological factors controlling epibiont dynamics in zooplankton communities is crucial.
Among epizoic algae, Colacium Ehrenberg 1834 (Euglenophyta) is a common, freshwater, algal group32,37,38,39 with various life stages, including attached (Figure 1A-D), non-motile planktonic (Figure 1E,F), and motile planktonic stages40,41. During the non-motile planktonic stage, cells live as single-cell planktons, aggregated colonies, or one-layer sheet colonies, covered by mucilage42. In the attached stage, Colacium sp. uses mucilage excreted from the anterior end of the cell37,39,41 to attach to substrate organisms (basibionts), particularly microcrustaceans41,43. Their life cycle also involves detaching from the molted exoskeleton or dead basibiont and swimming with their flagella to find another substrate organism39. Both planktonic and attached stages can increase their population size by mitosis40. Although their attached stage is hypothesized to be an evolutionary trait for gathering resources, such as light44 and trace elements41,45,46, or as a dispersion strategy27, little experimental evidence is available about these aspects37,41,44 and the key attachment mechanisms are largely unknown. For example, Rosowski and Kugrens expected that Colacium obtains manganese (Mn) from substrate copepods41, concentrated in the exoskeleton47.
Here, we describe how to measure PSII photophysiology of planktonic algae and the related application method for targeting attached algae (attaching to zooplankton) with Colacium sp. cells using the cuvette-type FRRf. We use the Act2 system equipped with three light-emitting diodes (LEDs) that provide flash excitation energy centered at 444 nm, 512 nm, and 633 nm48. Here, 444 nm (blue) corresponds to the absorption peak of chrophyll a (Chl-a), while 512 nm (green) and 633 nm (orange) correspond to the absorption peaks of phycoerythrin and phycocyanin, respectively. The fluorescent signal detection peak is 682 nm with 30 nm half bandwidth. Since it is difficult to find the planktonic stage of Colacium sp. in natural environments, their attached stage was collected for the experiments. Among the numerous substrate organisms,Scapholeberis mucronata O.F. M&#252;ller 1776 (Branchiopoda, Daphniidae; Figure 1A,B,G) is one of the simplest to handle due to their slow swimming speed, large body size (400-650 &#181;m), and unique behavior (hanging upside down on the water surface). Therefore, this protocol uses Colacium sp. attached on S. mucronata as a case study of the Colacium-basibiont system. To avoid fluorescence derived from the gut contents, S. mucronata was starved. As a previous study reported that the fluorescence signal from gut contents (ingested algae) displays a five-fold decrease after 40 min49, we expected that 90 min starvation would be enough to minimize the possibility of gut content fluorescence affecting the FRRf measurement with minimum effects of experimental stress to Colacium sp., such as nutrient deficiency. Furthermore, this protocol was applied to clarify the attaching mechanism of Colacium sp. and determine how two metals, calcium (Ca) and manganese (Mn) affect the photophysiology of both planktonic and attached stages. Calcium plays key roles in the photosynthetic pathways50 in multiple ways, and both metals are required to construct the oxygen-evolving complexes of the PSII51. As calcium and manganese are highly concentrated in the carapace of crustacean zooplankton47, we hypothesize that Colacium sp. photophysiology might respond more prominently to Ca and Mn enrichment during the planktonic stage if this life stage obtains these elements from S. mucronata during the attached stage.

<table><tbody><tr><td>Acrodisc syringe filter</td><td>Pall Corporation, Ann Arbor, MI, USA</td><td></td><td>0.2 &amp;mu;m pore size</td></tr><tr><td>Act2Run</td><td>CTG Ltd., West Molesey, UK</td><td></td><td></td></tr><tr><td>Biotin</td><td>Wako</td><td>023-08711</td><td>AF-6 medium</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>031-25031</td><td>AF-6 medium</td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>036-00382</td><td>AF-6 medium</td></tr><tr><td>Citric acid</td><td>Wako</td><td>036-05522</td><td>AF-6 medium</td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>036-03682</td><td>AF-6 medium</td></tr><tr><td>Concentrated Chlorella</td><td>Recenttec, Tokyo, Japan</td><td></td><td>20 mg C&amp;middot;mL&lt;sup&gt;&amp;minus;&lt;/sup&gt;&lt;sup&gt;1&lt;/sup&gt; ; store at 4 &amp;deg;C</td></tr><tr><td>FastOcean Act2</td><td>CTG Ltd., West Molesey, UK</td><td></td><td></td></tr><tr><td>Fe-citrate</td><td>Wako</td><td>093-00952</td><td>AF-6 medium</td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>091-00872</td><td>AF-6 medium</td></tr><tr><td>HCLP-880PF</td><td>Nippon Medical and Chemical Instruments&lt;br/&gt; &amp;nbsp;Co., Ltd., Osaka, Japan</td><td></td><td>With LED light bulbs</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Wako</td><td>160-04292</td><td>AF-6 medium</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Wako</td><td>167-04241</td><td>AF-6 medium</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>137-00402</td><td>AF-6 medium</td></tr><tr><td>MnCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>139-00722</td><td>AF-6 medium</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>Wako</td><td>343-01861</td><td>AF-6 medium</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;</td><td>Wako</td><td>196-02472</td><td>AF-6 medium</td></tr><tr><td>NaNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>191-02542</td><td>AF-6 medium</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>015-03231</td><td>AF-6 medium</td></tr><tr><td>Plankton Counter</td><td>Matsunami Glass, Osaka, Japan</td><td>S6300</td><td></td></tr><tr><td>Pylex test tube</td><td>CTG Ltd., West Molesey, UK</td><td></td><td>With rim, 16 x 100 mm</td></tr><tr><td>Vit. B&lt;sub&gt;1&lt;/sub&gt;</td><td>Wako</td><td>203-00851</td><td>AF-6 medium</td></tr><tr><td>Vit. B&lt;sub&gt;12&lt;/sub&gt;</td><td>Wako</td><td>226-00343</td><td>AF-6 medium</td></tr><tr><td>Vit. B&lt;sub&gt;6&lt;/sub&gt;</td><td>Wako</td><td>165-05401</td><td>AF-6 medium</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>264-00402</td><td>AF-6 medium</td></tr></tbody></table>

measuring photophysiology of attached stage of colacium sp. by a cuvette-type fast repetition rate fluorometer

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

There was no significant effect of baseline fluorescence (Figure 5) or Chl-a fluorescence (Figure 6) by S. mucronata up to 5 individuals (inds.) mL−1. However, Fv/Fm and NPQNSV were significantly affected when S. mucronata was 7.5 inds·mL−1. Therefore, for measuring the photophysiology of Colacium sp. during the attached stage, we chose S. mu...

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

measuring-photophysiology-attached-stage-colacium-sp-cuvette-type

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Source: Kulkarni, M. et al. Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina. J. Vis. Exp. (2015)This video demonstrates the use of two-photon microscopy to monitor calcium dynamics in cone photoreceptors of a transgenic mouse retina. It outlines the steps for preparing retinal slices, imaging FRET-based calcium biosensors, and analyzing fluorescence changes to quantify calcium levels under background illumination and light stimulation.

Two-Photon Microscopy for Monitoring Calcium Dynamics in Mouse Retinal Cone Photoreceptors

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

This article presents a method for spatiotemporal analysis of mobile, single-molecule Förster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

Autofluorescence Imaging to Evaluate Red Algae Physiology

Learn to measure photosystem II photophysiology in attached Colacium sp. algae using fast repetition rate fluorometry. This protocol enables real-time assessment of photosynthetic activity in epizoic algae on zooplankton substrates.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

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