Imaging the accumulated intracellular microalgal lipids as a response to temperature stress

Abstract

Over the last few decades, many scientists considered microalgae as promising actors for future biofuels because of the high lipid productivity inside their cells. Moreover, much attention has been paid to algal lipids as they can be used in biodiesel production. In this study, we optimized the different suitable conditions such as incubation time, incubation temperature, Dimethylesulfoxide and Nile red concentrations of the lipophilic fluorescence dye Nile red as an excellent and fast vital stain to detect and quantify intracellular lipids. This was achieved using the green alga Nannochloropsis salina. In addition, investigating the accumulation of lipid vesicles inside different isolated microalgal species as a response to temperature stress. Furthermore, the confocal laser scanning microscopy (LS510) for imaging and measuring the size and volume of the accumulated lipid vesicles was used.

Keywords: Fluorescence dye; Microalgae; Neutral lipids; Nile red (NR); Scanning microscopy.

Fig. 1

Effect of different parameters on Nile red assay a NR concentration µg ml⁻¹(w/v), b DMSO concentration % (v/v), c Incubation time (min) and d Incubation temperature (°C) on the fluorescence intensity (a.u.) of the green alga N. salina as a model organism. The excitation and emission wavelengths used were 530 and 580 nm, respectively, the OD₇₀₀ of the algal suspension was 1. All the tested samples expressed as mean values with standard deviations of three replicates

Fig. 2

Visualization of lipid vesicles under temperature stress. Confocal fluorescence microscopy study of lipid content in different algal species: a—Micractinium sp. YACCYB33, b—Synechocystis sp. PAK12, c—Pedinomonas noctilucae C34, d—Chlorella sorokiniana KLL-G018, e—Synechocystis sp. ElfSCS31, f—Chlorella variabilis NC64A, g—Synechocystis sp. PCC 6803, h—Desmodesmus pannonicus GM4n, i—Desmodesmus intermedius CCAP 258/36. Lipophilic fluorescent dye Nile red is used as a vital stain for visualization of the oil-containing lipid bodies. The overlay shows auto-fluorescence of chlorophyll in chloroplasts (red) and Nile red stained lipid bodies (green) within algal cells. In some cases, lipid bodies appear yellow due to their spatial overlap with chloroplasts. Excitation of Nile Red was at 488 nm, emission was collected using a 530–600 nm band-pass filter. Excitation of chlorophyll fluorescence was at 633 nm, emission was imaged through a 650 nm long-pass filter