This research was carried out as part of Projects TIN2015-68454-R and 20961/PI/18, financed by the Spanish Ministry of Economy and Competitiveness and the S&#233;neca Foundation of the Murcia Region. Irene Hern&#225;ndez Mart&#237;nez and Francisco Javier Ib&#225;&#241;ez L&#243;pez from the Statistical Support Section of the Scientific and Research Area of Murcia University (Secci&#243;n de Apoyo Estad&#237;stico (SAE), &#193;rea Cient&#237;fica y de Investigaci&#243;n (ACTI), Universidad de Murcia, (Figure 1 were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed with a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)

The algal species Chroothece mobilis was used for the present study. The species was obtained from the Microalgae Edaphic SE Spain, MAESE 20.29 culture collection. An overview of the protocol is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64533/64533fig01.jpg" /> 
Figure 1: Overview of the study. Chroothece mobilis is i...

<ol>
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Some unicellular or colonial red algae, such as Chroothece,&#160;grow slowly in vitro, but contain multiple autofluorescent compounds that can be analyzed by spectral analysis under a confocal microscope, where differences in pigment emission peaks can be detected. Spectral confocal fluorescence microscopy has allowed us to conduct in vivo studies to evaluate the adaptation or acclimation of photosynthetic organisms8,10,<sup ...

Red algae, such as the genus Chroothece, can grow in extreme habitats, where they frequently have to cope with marked environmental changes1. Floods and droughts are frequent in semi-arid regions where this genus can be found, and some species have been reported in creeks, cliffs, caves, or even thermal waters2. However, most of the time, biological variables, such as competition or grazing, relegate species to non-optimal conditions for their growth. As these organisms are often difficult to culture and either do not grow or grow very slowly under laboratory conditions, one major limitation is the available sample size. Therefore, it is very important to follow non-destructive methods or methods that involve minimal sample manipulation3,4.
The physiological skills needed to survive in these harsh environments can be monitored by following changes in their photosynthetic systems. Metabolic mechanisms, photosynthetic efficiency, and sensitivity to light or culture conditions can be revealed by pigment fluorescence emission profiles, due to accurate changes in their energy transfer or trapping5,6,7,8.
Autofluorescence of cellular compounds can be used as a marker for cytodiagnosis or as a natural indicator of cellular state or metabolism in response to external and internal signals through changes in emission9. It can also be used to discriminate taxonomically different groups of photosynthetic organisms10. Depending on the phylogenetic position of phototrophic microorganisms, one can find different in vivo fluorescence features. Therefore, a taxonomic identification based on the in vivo characteristics of phototrophic fluorescence (including fluorescence absorption and emission spectra) has been attempted on several occasions11,12. Because of the diversity in accessory pigments among phytoplankton taxa, differences in the wavelengths at which chlorophyll a (Chl a) fluorescence is stimulated, or differences in emission spectra, can be used to infer taxonomy13. The in vivo fluorescence excitation and emission spectra of these specimens rely not only on the phyla of algae, but also on photosystem adaptation14. The efficiency of energy transfer to Chl a, or the ratio of Chl a to accessory pigments, and the cellular pigment content are sensitive to growth conditions5.
Red algae, particularly Chroothece, have several accessory fluorescent pigments-phycobiliproteins and carotenoids; the former concentrate in phycobilisomes attached to the thylakoids of chloroplasts. Phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) can capture light at different wavelengths and transmit it to Chl a, which makes photosynthesis in very different light and culture conditions possible15. For instance, Chroothece species can grow inside caves or almost emerge in slightly saline calcareous streams2.
Monochromatic lights affect the growth and pigment composition of photosynthetic organisms, and have been studied to prevent or control the growth of photosynthetic organisms in caves. Mulec et al. showed that red enriched lighting promotes the growth of cyanobacteria, algae, and plants16. Previous studies have also reported that green light affects the pigment composition of cyanobacteria17, while others have revealed that green light prevents the growth of most photosynthetic organisms and some cyanobacteria exhibit a reduction in thylakoids and weaker mean fluorescence intensity18.
To understand the ability of Chroothece as a model organism to overcome harsh conditions, cultured cells have been exposed to increasing light intensities and monochromatic light (green or red)15, to see how it copes with the dim conditions of caves (where red light predominates). The protocol presented herein reproduces the effect of the abovementioned variables on Chroothece&#39;s phycobiliproteins at the cellular level using its own autofluorescence.
Nowadays, fluorescence is commonly used as a tool to study the physiological responses of vascular plants, microalgae, macroalgae, and cyanobacteria13,14,16. Spectral confocal fluorescence microscopy is a superb tool for in vivo studies to evaluate photosynthetic specimens&#39; physiology at the single-cell level10,17,18,19,20, by avoiding problems associated with the low growth rate in the laboratory and the difficulties with obtaining enough biomass for the associated extraction and biochemical methods8. Once cells are treated under different culture conditions for 2 weeks, the lambda scan profile can be measured in vivo. Although there are several publications in which different wavelengths of excitation by confocal imaging have been used3,4,10,17, most phycobiliproteins and Chl a can be detected using a 561 nm wavelength excitation line, and the detected emission ranges from 570 to 760 nm wavelength. These criteria have been based on an analysis previously performed10 with commercial pure pigments (Table 1) by confocal imaging and the obtained results in different algae species20,21,22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Pigments</td>
			<td rowspan="2">&#955;flmax (nm)</td>
			<td colspan="8">&#955; exc (nm)</td>
		</tr>
		<tr>
			<td>351</td>
			<td>364</td>
			<td>458</td>
			<td>476</td>
			<td>488</td>
			<td>514</td>
			<td>543</td>
			<td>633</td>
		</tr>
		<tr>
			<td>Chl a</td>
			<td>660.9-678.1</td>
			<td>43.4 &#177; 1.8</td>
			<td>11.2 &#177; 0.2</td>
			<td>1.8 &#177; 0.05</td>
			<td>2.0 &#177; 0.08</td>
			<td>12.2 &#177; 0.7</td>
			<td>6.0 &#177; 0.3</td>
			<td>4.2 &#177; 0.16</td>
			<td>80.7 &#177; 1.5</td>
		</tr>
		<tr>
			<td rowspan="2">R-PE</td>
			<td>569.2-583.3</td>
			<td>5.9 &#177; 0.6</td>
			<td>5.9 &#177; 0.16</td>
			<td>11.1 &#177; 0.04</td>
			<td>42.2 &#177; 0.3</td>
			<td>100.0 &#177; 0</td>
			<td>90.0 &#177; 0.3</td>
			<td>99.2 &#177; 0.08</td>
			<td>-</td>
		</tr>
		<tr>
			<td>652.1-668.6</td>
			<td>-</td>
			<td>-</td>
			<td>1.5 &#177; 0.01</td>
			<td>3.7 &#177; 0.04</td>
			<td>26.7 &#177; 0.5</td>
			<td>8.7 &#177; 0.16</td>
			<td>11.1 &#177; 0.16</td>
			<td>11.3 &#177; 0.2</td>
		</tr>
		<tr>
			<td>C-PC</td>
			<td>636.2-676.4</td>
			<td>2.3 &#177; 0.04</td>
			<td>1.0 &#177; 0.01</td>
			<td>0.6 &#177; 0.004</td>
			<td>0.7 &#177; 0.008</td>
			<td>2.0 &#177; 0.08</td>
			<td>2.0 &#177; 0.04</td>
			<td>3.3 &#177; 0.16</td>
			<td>33.6 &#177; 0.9</td>
		</tr>
		<tr>
			<td>APC-XL</td>
			<td>667.3-683.8</td>
			<td>15.1 &#177; 1.5</td>
			<td>9.6 &#177; 0.98</td>
			<td>1.0 &#177; 0.04</td>
			<td>1.2 &#177; 0.08</td>
			<td>5.9 &#177; 0.7</td>
			<td>4.1 &#177; 0.5</td>
			<td>23.2 &#177; 3.5</td>
			<td>91.4 &#177; 2.3</td>
		</tr>
	</tbody>
</table>
Table 1: The pure pigment information used to run the lambda scan analysis. This table shows emission peaks and shoulders/fluorescence band maxima of different fluorochromes/pigments by confocal imaging spectrophotometry for all the excitation wavelengths, and the percentage of light emission by pigments/fluorochromes. Values were calculated by the formula: = MFI*100/255. Each value is the mean &#177; SE (mean &#177; standard error from the mean). Pure pigments were used for calibrating the confocal scanning laser microscope as follows1,2,10. Chlorophyll a was obtained from Spinacia oleracea, R-phycoerythrin (R-PE) from Porphyra tenera, and C-phycocyanin (C-PE) from Spirulina sp. All the species were dissolved in filtered distilled water. Allophycocyanin-XL (APC-XL) was obtained from Mastigocladus laminosus, which was dissolved in ammonium sulfate (60%) and potassium phosphate (pH = 7) to achieve a concentration of 38 mM. The scans were performed with 400 &#181;L of each pigment solution (concentration of 1 mg/mL) using an 8-well covered-glass bottom chamber.
The study of a single excitation wavelength is quite a useful first approximation. In this case, however, it is necessary to elucidate the relative contribution of the different complexes in the fluorescence signal, which is recommended to perform a fluorescence ratio or spectrum analysis at several wavelengths, among other methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Dish 35 mm, high Glass Bottom</td>
			<td>Ibidi&#160;</td>
			<td>81158</td>
			<td>-</td>
		</tr>
		<tr>
			<td>24 black well plate</td>
			<td>Ibidi</td>
			<td>82406</td>
			<td>&#160;flat and clear bottom for high throughput microscopy</td>
		</tr>
		<tr>
			<td>Algae Incubator</td>
			<td>Panasonic</td>
			<td>MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Fisher Scientific</td>
			<td>15380591</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>green filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>HC PL APO 63X/1.30 GLYC CORR CS2</td>
			<td>Leica Microsystems</td>
			<td>506353</td>
			<td>Glycerol immersion lens</td>
		</tr>
		<tr>
			<td>Image acquisition software. LAS X</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Panasonic</td>
			<td>FL40SSENW/37MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum photoradiometer</td>
			<td>DeltaOhm&#160;</td>
			<td>DO 9721</td>
			<td>-</td>
		</tr>
		<tr>
			<td>R software</td>
			<td>R Core Team, 2020</td>
			<td>4.0.2.</td>
			<td>-</td>
		</tr>
		<tr>
			<td>red filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>SWES medium</td>
			<td>University of Murcia</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Type G Immersion liquid</td>
			<td>Leica Microsystems</td>
			<td>11513910</td>
			<td>Glycerol&#160;</td>
		</tr>
	</tbody>
</table>

autofluorescence imaging to evaluate red algae physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

Chlorophyll a generally absorbs blue and red wavelengths of visible light, whereas phycobiliproteins use green, yellow, and orange wavelengths7. The autofluorescence of these pigments makes the first approach to study phycobiliproteins and chlorophyll behavior under experimental and field conditions possible.
By comparing the obtained data and plotting on different graphs, significant changes in mean fluorescence intensity (MFI) can be distinguished (<strong class="xfig...

Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Red Algae Physiology at JoVE.com

Autofluorescence Imaging to Evaluate Red Algae Physiology

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.

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...

This confocal microscopic protocol will permit the interpretation of the ecological behavior and adaptations of microalgae from extreme environments with slow growing in the laboratory using autofluorescence of their pigments. This technique is not invasive and minimizes artifacts as chemical compounds are not used. Knowing the performance of pigments of autotrophs and corresponding growth will permit the designing of the methods of control which is the greatest interest for touristic caves and architectural monuments.Begin preparing the inoculum of Chroothece mobilis by transferring it from the agar culture to the seawater medium or SWES liquid medium. Maintain all the cultures for two weeks with a 16 to 8 light dark photo period at 20 degrees Celsius under a low white light intensity and without shaking until the desired cell density is obtained. Use one milliliter of the exponential phase culture having five times 10 to the third cells per milliliter cell density to inoculate in a 24-well plate for different experiments.To reproduce the monochromatic light effect, use the green filter allowing green light to pass through from 470 to 570 nanometers with a peak at the 506 nanometer wavelength and to red light adjusted using a filter between 590 and 720 nanometers and peaks at 678 nanometers. Expose the cell cultures to this light for two weeks. Turn on all the components of the inverted confocal laser scanning microscope including the laser.Mount the cells in the SWES growth medium from each experimental well of the 24-well plate to a 35 millimeter glass bottom dish for imaging. Choose the 63X or 1.30 numerical aperture or NA glycerol immersion objective and place glycerol over the lens. Next, place the well plate on the microscope stage ensuring the specimen does not move during image acquisition.Center the specimen in the light path and focus on the center of the cell by selecting the plane with the highest fluorescence intensity. Once done, open the image acquisition software and choose XY lambda from the dropdown list in the acquisition mode. Select the excitation line of the laser 561 nanometers, eight bits of dynamic range and 1024 by 1024 pixels.Collect the fluorescence emission spectra in the 10 nanometer bandwidth and the lambda step size of four nanometers within the 570 to 760 nanometer range. Set the pinhole at one air unit and run the lambda scan acquisition. After repeating this process in different fields of view and under the different conditions of red and green lights, save all the data.Once the lambda scan is acquired, click on the quantify window at the top of the software to evaluate the collected fluorescence emission spectra. Go to the open project window and select one XY lambda file. Select stacked profile analysis in the imaging software and define a region of interest of four micrometer square in the center of a cell to analyze the mean fluorescence intensity.Export the data and CSV before repeating the process with different cells under different conditions. Next, open the CSV files to select the different fluorescence emission peaks of all the measured regions of interest by selecting the maximum fluorescence data of phycoerythrin phycocyanobilin, C.phycocyanin, allophycocyanin, and chlorophyll A respectively in the CSV file. Once done, create a new table with all the maximum fluorescence values obtained from each phycobiliprotein and chlorophyll peak and plot the data on a graph.Significant differences in mean fluorescence intensity were observed. The mean fluorescence intensity of phycoerythrin phycocyanobilin significantly decreased when the cells were exposed to monochromatic green light. In contrast, no difference was observed in red light compared to the control.The green light had the opposite effect on allophycocyanin and chlorophyll A in which the mean fluorescence intensity significantly increased due to the adaptation of antenna complexes due to increased quantity or improved connectivity of the antenna complexes among others. The red light produced a non-significant increase in allophycocyanin and chlorophyll fluorescence. To study the effect of different growth conditions on cells in culture, it's crucial to perform the measurements with exactly the same microscope settings.It's possible to analyze other environmental parameters relevant to the culture of autofluorescence organisms, as well as the fluorescence signal within the visible spectrum. This technique is a very powerful approach to a study life organisms with several autofluorescence pigments.

autofluorescence-imaging-to-evaluate-red-algae-physiology

Research

JoVE Journal

Biology

1.2K vistas.  University of Murcia. Este protocolo microscópico confocal permitirá la interpretación del comportamiento ecológico y las adaptaciones de microalgas de ambientes extremos con crecimiento lento en el laboratorio utilizando la autofluorescencia de sus pigmentos. Esta técnica no es invasiva y minimiza los artefactos ya que no se utilizan compuestos químicos. Conocer el rendimiento de los pigmentos de los autótrofos y el crecimiento correspondiente permitirá diseñar los métodos de control que son los de mayo...