Autofluorescence Imaging to Evaluate Red Algae Physiology

Summary

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.

Abstract

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.

Introduction

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 sam....

Protocol

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....

Results

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 (

Discussion

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,

Materials

List of materials used in this article

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