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Fluorescence from a single Symbiodinium cell

The partnership between coral and its algal symbionts, Symbiodinium, is crucial to the global environment. Yet, the regulatory process within the photosynthetic machinery of Symbiodinium is still not clearly understood. Here, we studied the influence of light stress from focussed red and blue lasers on single Symbiodinium cells. Fluorescence signals were measured to show cell response. Increasing the incident laser power or the exposure time resulted in an increase followed by a decline in fluorescence intensity. The trend of fluorescence intensity changes was associated with mechanisms of light use efficiency, non-photochemical quenching, photoinhibition, and repair of the cell. Our study provides new approaches to studying the photobiology and physiology of Symbiodinium cells.


Introduction
Corals are particularly susceptible to elevated seawater temperatures and exhibit bleaching or lose their algal endosymbionts of the genus Symbiodinium under stress. Symbiodinium belong to phylum Dinoflagellata, which is one of the largest groups of marine phytoplankton. Symbiodinium cells perform photosynthesis within the coral host cells. The photosyntheticallyproduced organic nutrients are translocated to the coral host and contribute substantially to its growth and calcification [1]. In return, the coral host provides a protective environment and a supply of nutritious metabolic waste products to the symbionts. Such partnership between the coral host and its symbionts is known to be highly sensitive to environmental stressors such as high seawater temperature and high solar irradiance [1][2][3].
Given the ecological and economic importance of corals, intensive studies have been done in an attempt to understand the photobiology and photophysiology of Symbiodinium. The fluorescence emission of chlorophyll a (chl a), the primary photosynthetic pigment of Symbiodinium, is commonly used to evaluate the photosynthetic fitness of the algal symbiont as well as the physiological condition of the coral [4][5][6][7][8]. Chlorophyll fluorescence is the re-emitted light that represents the fate of excitation energy in the photosynthetic apparatus [9].
In most coral studies, non-invasive methods such as pulse-amplitude-modulated (PAM) and fast-repetition-rate (FRR) fluorometers are capable of detecting total fluorescence of cell populations in vitro or in hospite [10,11]. Extracted chl a pigment has also been extensively studied [4,5]. However, natural variations in pigment content and thickness of the membrane layer can exist among cells of the same Symbiodinium type. So far, conventional fluorometers are not able to provide measurements for single Symbiodinium cells [4,5,12]. Only a few articles have reported fluorescence intensity studies for single phytoplankton cells [13,14].
In this study, we performed optical stimulations to single Symbiodinium cells using a focussed laser beam. Dependences of the fluorescence intensity on incident laser power and exposure time were examined. Our findings may provide new insights into the photobiology and physiology of a Symbiodinium cell under light stress.

Methods
Two strains of cultured Symbiodinium, Symbiodinium sp. (type A3, cell size=10-12 μm) and S. minutum (type B1, cell size=6-8 μm), were maintained at 25°C as previously reported [15]. All cultures were maintained in artificial seawater (Guillard's (F/2) Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. marine-water enrichment solution, Sigma-Aldrich). Three antibiotics, ampicillin (100 μg ml −1 ), kanamycin (50 μg ml −1 ), and streptomycin (50 μg ml −1 ) were also included. Mass culturing was performed in a 2 litre final volume by inoculating, and sampling was performed once every three weeks. The incubator was maintained on a 12 h-light/12 h-dark regime with illumination.
An optical tweezer (Thorlabs EDU-OT1), containing a red laser (λ=658 nm), was configured with a blue excitation light source (λ ex = 488 nm) for fluorescence (λ fluo ≈675 nm) experiments. The schematic of the experimental setup is shown in figure 1. Studies have reported that chl a pigment is usually located near the cell wall. Thus, the focus spots of both the red and blue laser beams were overlapped and directed near the cell's periphery. The red laser was focussed through a Zeiss 63X, 0.8NA objective. The excitation beam was collimated and directed into the same objective lens through a dichroic mirror. Based on the incident laser beam wavelength (658 nm) and the objective lens, the diameter of the incident beam was approximately 1 μm based on the diffraction limit; this is a few times smaller than the average diameter of the Symbiodinium cells. A localised spot on the Symbiodinium cell was exposed to light stress and excited for fluorescence emission. Sample illumination was accomplished using a white LED, and a CMOS camera (Thorlabs DCC1645C) was used to observe Symbiodinium and collect the fluorescence (recording parameters: rate=83 f s −1 , exposure time=10 s). Laser powers were measured before the microscope objective lens. Time-lapse recordings were cropped using ImageJ with only the cell region included. The fluorescence intensity was obtained using a custom script in MATLAB. A longpass filter (650 nm) was placed in front of the camera to remove stray light from the red and blue laser beams.

Without red laser influence
To verify if short-term exposure to the blue laser has an effect on the fluorescence measurements, a control measurement without the red laser being present was performed. Fluorescence measurements were carried out at two-minute intervals using only the blue excitation laser (incident power 45 μW) applied for 10 s, and a video was recorded simultaneously. Two cells per Symbiodinium type were studied and the average fluorescence emissions were calculated. Within 10 s of exposure to the blue laser, the intensity of the fluorescence did not change significantly for either Symbiodinium type, as shown in figure 2. The fluorescence intensities of types A3 and B1 could be considered as constant under a natural and normal environment, without any red laser beam influence.

With red laser influence
The fluorescence intensity of a single cell was obtained following a two-minute red laser exposure with 10 s of the blue laser excitation. The duration of one series of measurements was approximately 30 minutes. During fluorescence measurements, the red laser was turned off and the excitation blue laser opened for 10 s. The average fluorescence intensities were calculated and plotted against the laser operating current for both Symbiodinium types as shown in figure 3. In this figure, laser power is plotted on the right side against laser operating current. Fluorescence intensity started to increase until 0.5 mW laser power input was applied, after which it decreased gradually with further increasing laser power. Specifically, the fluorescence intensity of type A3 started to increase after 35 mA (threshold current) of red laser. Meanwhile, an increase in type B1 fluorescence was observed at 40 mA exposure. Fluorescence intensities before and after the red laser exposure are also demonstrated. Interestingly, the fluorescence intensity returned to a value similar to pre-exposure levels when the cells were allowed to be relaxed from the exposure to the red laser beam for five minutes, after the last red laser exposure using the strongest exposure parameters (80 mA laser current, 3 mW laser power).

With blue laser influence
Although exposure to the excitation blue light under a short time (10 s in our study) did not significantly affect cell fluorescence, a study has reported that Symbiodinium cells are more sensitive to short light wavelengths (UV/purple region) [4]. Thus, we also  . Fluorescence intensities of Symbiodinium A3 and B1 cells under two-minute red laser exposure with varied laser currents (yaxis on the left side). Fluorescence intensities for both pre-exposure and after exposure relaxation are also included. The laser power for each laser current is plotted with respect to the y-axis on the right side (grey bars). exposed the cells to a blue laser beam continuously at a fixed incident power of 45 μW for up to thirty minutes and collected the fluorescence intensity for 10 s at varied time points. Similarly to our results under red laser beam influence, an initial increase in fluorescence was observed in the first 10 minutes and a decrease was observed with longer exposure time, as shown in figure 4. Fluorescence was no longer detectable after 40 minutes. For recovery, a 20-minute interval was provided after which fluorescence could not be observed again. Compared to the control experiment results, a steep slope of fluorescence increase was observed for both types of Symbiodinium cells. After reaching the peak, the fluorescence intensity decreased with further increasing the exposure time. Although the incident power from the blue laser was not as strong as the red laser, the effect from light stress was observed.

Discussion
In our study, we performed localised illumination of red and blue lasers on single Symbiodinum cells as well as localised fluorescence excitation. The change of fluorescence intensity was measured at the single-cell level. This is in contrast to many studies wherein fluorescence measurements are acquired from extracted pigments solutions, cell cultures, or coral fragments [4][5][6][7][8].
In this study, we compared the light stress responses of Symbiodinium cells A3 and B1, both of which have been previously studied under heat stress [16]. Although we observed high variations in chl a fluorescence from the same type of cells under the same excitation conditions, the trend of the fluorescence signal change was apparent for both types. It is widely known that Symbiodinium exhibits high genetic and physiological diversity within and among different types [12,17,18]. Variations in fluorescence intensity could be attributed to the morphological characters and physiological features of the cells [19], e.g. size of the cell, concentration of pigment, and the thickness of the membrane layer.
We separate the trend of fluorescence change into three phases: (i) increase to a peak, (ii) decrease after the peak, and (iii) recovery after light stress release. In both the red (power dependent) and blue (exposure time dependent) laser experiments, the fluorescence intensity increased initially until it reached a peak. Absorption of photons results in singlet-state excitation of chl a which can be relaxed to the ground state by emitting fluorescence [1], hence an increase in fluorescence was observed. With increased exposure time or light power, photosynthetic organisms perform various protective mechanisms, and the relationship among these mechanisms is not well understood so far. One of the protective mechanisms is called nonphotochemical quenching (NPQ), which dissipates excessive absorbed light through heat [4,10,20]. Because of NPQ, the slope of the fluorescence increase reduced with higher laser power or longer exposure time, and the fluorescence intensity reached a peak.
After reaching the peak, the fluorescence intensity decreased with further increased laser power or exposure time. With further redirection of the light energy via NPQ, the yield of the fluorescence could be decreased [10]. At the same time, impairment of functional Photosystem II (PSII) centres due to absorption of high light energy could also happen [3,10]. A PSII centre is composed of a multi-subunit pigment-protein complex that drives a photochemical reaction [21]. Upon light or heat stress, highly reactive intermediates and by-products, such as reactive oxygen species (ROS), will cause photoinhibition and consequently photodamage to the photosynthetic apparatus [3,4,7,10]. However, the PSII is constantly repairing itself from the damage [22,23]. Therefore, photoinhibition occurs when the rate of damage exceeds the rate of repair [3]. This could be the reason we observed a decrease in fluorescence with increased laser power or exposure time.
We measured the fluorescence after cells were released from the light stress associated with the red or blue laser, to check the recovery of these cells during the repairing process. However, the response to the blue and red lasers was different when the relaxation period was considered. After the cells were released from the red laser beam, the fluorescence intensity could return to the pre-exposure level for both types of Symbiodinium. For the blue laser exposure, fluorescence signals were no longer detectable even after long relaxation periods of time, up to 20 min, were given. This implies that complete photodamage of all the PSII reaction centres within the cell may have already occurred. It has been reported that Symbiodinium are more sensitive to photodamage under shorter light wavelengths [4].
From our study, both fluorescence increase and decrease was observed in a single Symbiodinium cell belonging to type A3 or B1. Temperature change from heating of the water solution via the applied focussed laser beams may not be the primary contributing factor to the changes in fluorescence intensity. In a study that utilized a 1040 nm laser (300 mW) to heat and measure the fluorescence of single marine phytoplankton cells, the temperature increase induced by the laser was 4.0°C [14]. In our experiments, the maximum red laser power was only 3.0 mW, hence the temperature increase is expected to be lower than 0.04°C since water has a much lower absorption coefficient (<100×) at 658 nm than at 1064 nm [24,25].

Conclusion
The fluorescence intensity of single Symbiodinium A3 and Symbiodinium B1 were studied under two laser beams (488 nm and 658 nm) separately. We detected a fluorescence rise, of about 10 000 counts, from a single cell from its initial stage while increasing the incident laser powers or exposure time. With further increasing incident power or exposure time, a decrease in fluorescence intensity was observed. To our knowledge, non-photochemical quenching and photoinhibition were observed, for the first time, in a single Symbiodinium cell. Fluorescence measurement of an intact single cell, rather than cell cultures, cells in hospite, or pigment extracts, could improve our understanding of the photobiology and physiological response of Symbiodinium. A more delicate and systematic design of the experiments could also be considered in order to study single Symbiodinium cells, e.g. using optical tweezers to suspend a single cell in solution or to trap a single motile cell and examine the fluorescence.