Autofluorescence Imaging to Evaluate Red Algae Physiology

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 manipulation^3,4.

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 trapping^5,6,7,8.

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 occasions^11,12. 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'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 cyanobacteria^13,14,16. Spectral confocal fluorescence microscopy is a superb tool for in vivo studies to evaluate photosynthetic specimens' physiology at the single-cell level^{10,17,18,19,20}, 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 used^3,4,10,17, 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 species^20,21,22.

Pigments	λflmax (nm)	λ exc (nm)
		351	364	458	476	488	514	543	633
Chl a	660.9-678.1	43.4 ± 1.8	11.2 ± 0.2	1.8 ± 0.05	2.0 ± 0.08	12.2 ± 0.7	6.0 ± 0.3	4.2 ± 0.16	80.7 ± 1.5
R-PE	569.2-583.3	5.9 ± 0.6	5.9 ± 0.16	11.1 ± 0.04	42.2 ± 0.3	100.0 ± 0	90.0 ± 0.3	99.2 ± 0.08	-
	652.1-668.6	-	-	1.5 ± 0.01	3.7 ± 0.04	26.7 ± 0.5	8.7 ± 0.16	11.1 ± 0.16	11.3 ± 0.2
C-PC	636.2-676.4	2.3 ± 0.04	1.0 ± 0.01	0.6 ± 0.004	0.7 ± 0.008	2.0 ± 0.08	2.0 ± 0.04	3.3 ± 0.16	33.6 ± 0.9
APC-XL	667.3-683.8	15.1 ± 1.5	9.6 ± 0.98	1.0 ± 0.04	1.2 ± 0.08	5.9 ± 0.7	4.1 ± 0.5	23.2 ± 3.5	91.4 ± 2.3

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 ± SE (mean ± standard error from the mean). Pure pigments were used for calibrating the confocal scanning laser microscope as follows^1,2,10. 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 µL 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.

Protokół

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.

Figure 1: Overview of the study. Chroothece mobilis is incubated under extreme habitat conditions, such as different monochromatic lights, for 2 weeks. The effect on Chroothece's physiology is evaluated by autofluorescence of the proteins contained in phycobilisome and photosystems using a confocal laser scanning microscope. Please click here to view a larger version of this figure.

1. Sample preparation

1. Prepare the inoculum of Chroothece mobilis from the agar culture of the collection by transferring it to the SWES liquid medium (Table 2).
   NOTE: SWES "Seewasser + Erddekokt + Salze" = seawater medium²³.
2. Maintain all the cultures for 2 weeks with a 16:8 light/dark photoperiod at 20 °C under low white light intensity (LL: 80 µM/m²/s) conditions and without shaking, until the desired cell density is obtained (see step 1.3).
   NOTE: The growth light conditions are 80 µM/m²/s light intensity (active photosynthetic radiation, PAR). These conditions are used as the low light conditions (control). The composition of the SWES medium is provided in Table 2.
3. Carry out inoculations for the different experiments in the exponential culture phase with a cell density of 5 x 10³ cells/mL in a 24-well plate, using 1 mL per well.
   NOTE: Perform cell counting with a Neubauer chamber²⁴, and dilute the culture with SWES whenever necessary.

SWES medium composition
Component	Concentration
KNO3	1.98 mM
K2HPO4	115 µM
MgSO4	81 µM
ZnSO4, 7H2O	17 nM
MnSO4, 7H2O	45 Nm
H3BO3, 4H2O	3.1 mM
Co(NO3)2	17 mM
Na2MoO4, 6H2O	21 nM
CuSO4, 2H2O	0.1 nM
FeSO4, 5H2O	13 µM
EDTA, 7H2O	11 µM
Vit B12	5 µg
Soil extract	30 mL
Filtered river water	455 mL

Table 2: SWES medium composition.

2. Reproducing extreme algae habitat conditions: green and red monochromatic light effect

1. Add 1 mL of the cell culture from the stock culture prepared at the aforementioned cell density (step 1.2) to inoculate each well of a 24-well plate.
2. Keep the cell culture covered for 2 weeks to reproduce the monochromatic light effect.
   1. Use the green filter that allows green light to pass through from 470 to 570 nm with a peak at the 506 nm wavelength (according to the manufacturer's specifications; see Table of Materials) and expose it to the culture²⁵.
   2. Use the red filter that allows red light between 590 and 720 nm and peaks at 678 nm (according to the manufacturer's specifications; see Table of Materials) to expose it to the culture²⁵.
      NOTE: The low white light intensity (LL: 80 µM/m²/s; see Table of Materials) condition is used as the light intensity control to compare the obtained effects. The employed light intensity units are micromole per second and square meter (µmol m^-2 s^-1) or photosynthetic photon flux density (PPFD).

3. Autofluorescence imaging

NOTE: The setup of the imaging software (see Table of Materials) is illustrated in Figure 2.

1. Turn on all the components of the inverted confocal laser scanning microscope (CLSM; see Table of materials), including the laser.
2. Mount the cells from each experimental well of the 24-well plate in SWES growth medium to a 35 mm glass bottom dish (see Table of Materials) for imaging.
3. Choose the 63x/1.30 NA glycerol immersion objective and place glycerol over the lens (see Table of Materials).
4. Place the well plate on the microscope stage and ensure the specimen does not move during image acquisition.
5. Center the specimen in the light path and focus on the center of the cell by selecting the plane with the highest fluorescence intensity.
6. Open the image acquisition software and choose xyλ from the drop-down list in the acquisition mode²⁶.
7. Select the excitation line of the laser to 561 nm DPSS, 8 bits of dynamic range, and 1024 x 1024 pixels.
   NOTE: Ensure the confocal microscope has a 561 nm DPSS laser or a white light laser (WLL).
8. Collect the fluorescence emission spectra in the 10 nm bandwidth and the lambda step size of 4 nm within the 570-760 nm range.
9. Set the pinhole at 1 Airy unit and run the lambda scan acquisition.
10. Repeat this process as many times as possible in different fields of view to collect an acceptable amount of data for statistical analysis (usually a standard deviation lower than 10%²²).
11. Repeat the last step under the different conditions (under red and green lights) and save the data.
    NOTE: Use the same acquisition settings for the different samples and conditions to compare and conduct the statistical analysis.

Figure 2: Software setup. Imaging software user interface to set up the lambda scan parameters. (A) From left to right, to select the acquisition mode xyλ from the drop-down list, that corresponds to step 3.6 in the protocol, and to select the right immersion lens type, corresponding to step 3.3 in the protocol. Ensure to remove any filter from the light path in step 3.9. (B) The panel for setting up the lambda scan parameters corresponds to step 3.8 in the protocol. (C) Run the lambda scan in step 3.10. Please click here to view a larger version of this figure.

4. Parameters to evaluate Chroothece's physiology

1. Once the lambda scan is acquired, click on the quantify window at the top of the software (as shown in Figure 3) to evaluate the collected fluorescence emission spectra. Go to the Open project window and select one xyλ file (Figure 3).
2. Select Stack profile analysis in the imaging software.
3. Define a region of interest (ROI) of 4 µm² in the center of a cell to analyze the mean fluorescence intensity (MFI). Export the data in CSV format.
   NOTE: It is important to avoid the presence of black pixels (to ensure the pixels selected contain positive values) and always maintain a same sized ROI.
4. Repeat this process with different cells under different conditions to produce enough data to perform statistical analysis.
5. Open the CSV files to select the different fluorescence emission peaks from the phycobiliproteins and chlorophylls of all the measured ROIs.
   1. Select the fluorescence data of phycoerythrin-phycocyanobilin (PE-PCB; 620 nm), C-phycocyanin (CPC; 648 nm), allophycocyanin (APC; 660 nm), and chlorophyll a (Chl a; 680 nm), respectively, in the csv file.
6. Create a new table with all the maximum fluorescence values obtained from each phycobiliprotein and chlorophyll peak and plot the data on a graph.
7. Perform statistical analysis.
   1. Analyze if the obtained data meets normality and homoscedasticity²⁷.
   2. Proceed to the t-test analysis^27,28 if the first condition is true. If not, run a Mann-Whitney U test²⁹.
   3. Consider the differences significant at p values <0.05.

Figure 3: Evaluating Chroothece's autofluorescence. To perform the autofluorescence analysis, ensure to select the quantify window and one xyλ file in the open projects window (step 4.1); select stack profile visualization (step 4.2); select a 4 µm² ROI in the center of a cell and click on the report button to export the lambda scan data in CSV format (step 4.3). Please click here to view a larger version of this figure.

Wyniki

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 (

Dyskusje

Some unicellular or colonial red algae, such as Chroothece, 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 organisms^8,10,

Materiały

Name	Company	Catalog Number	Comments
µ-Dish 35 mm, high Glass Bottom	Ibidi	81158	-
24 black well plate	Ibidi	82406	flat and clear bottom for high throughput microscopy
Algae Incubator	Panasonic	MLR-352-PE
Confocal laser scanning microscope	Leica Microsystems	SP8 TCS	-
Flask	Fisher Scientific	15380591	Can be purchased in a local convenience store or online stores.
green filter	PNTA, LEE filters	-	Can be purchased in a local convenience store or online stores.
HC PL APO 63X/1.30 GLYC CORR CS2	Leica Microsystems	506353	Glycerol immersion lens
Image acquisition software. LAS X	Leica Microsystems	SP8 TCS	-
Light source	Panasonic	FL40SSENW/37MLR-352-PE
Quantum photoradiometer	DeltaOhm	DO 9721	-
R software	R Core Team, 2020	4.0.2.	-
red filter	PNTA, LEE filters	-	Can be purchased in a local convenience store or online stores.
SWES medium	University of Murcia	-	-
Type G Immersion liquid	Leica Microsystems	11513910	Glycerol