Fluorescent dyes offer a useful method for the measurement of intracellular lipids. They are inexpensive and require simple optical measurement instrumentation, whilst simultaneously providing high throughput application. Nile Red is a hydrophobic, metachromatic dye which has been widely used for detection of intracellular lipids. However, Nile Red fluorescence depends on its concentration, microenvironment polarity, incubation time and, therefore, requires strain specific optimization. Hence, neutral lipids in
Spectral properties (excitation and emission characteristics) of Nile Red are highly dependent on the microenvironment polarity (1, 2). The peak emission of Nile Red is blue shifted as the surrounding polarity decreases, however, it shifts to red with an increase in microenvironment polarity (3, 4, 5). To enhance neutral lipid staining by Nile Red, solvents such as acetone, dimethylsulfoxide (DMSO), ethanol, isopropanol, hexane or chloroform have been used; with DMSO and acetone being the most commonly used solvents (Cooksey et al., 1987b; Chen et al., 2009; Satpati and Pal, 2015). Intracellular neutral lipids can be detected via a Nile Red fluorescence signal with a maximum emission at 570-580 nm. By choosing an excitation/emission setup of 480/570–580 nm, Nile Red was successfully used for staining neutral lipids from Chlorella vulgaris under nutrient starvation conditions (Morschett, Wiechert and Oldiges, 2016). In addition to the variability generated from hydrophobic environments, Nile Red fluorescence parameters also vary for microalgae species (6, 7, 8, 9). As the composition and structure of the microalgal cell wall varies, the time and Nile Red concentration required to generate optimal fluorescence varies too. Robust and thick cell walls (particularly in green algae and nutrient starved microalgae) act as a barrier; preventing efficient penetration of Nile Red in cells and neutral lipid staining and, therefore, increased staining time is required (10). Even after the establishment of appropriate staining conditions (e.g. dye concentration, temperature, organic solvents) for a microalgae strain, the ability to measure fluorescence intensity remains dependent on the cell concentration used during the assay. A decrease in fluorescence (or saturation) for neutral lipid staining has been reported below and above the threshold of cell concentration at fixed Nile Red concentrations. Therefore, considering all the parameters that can affect the final neutral lipid quantification using Nile Red staining, it is important to establish an assay which is robust enough to addresses these factors. Here, for quantification of neutral lipids, such a standard Nile Red assay development, and optimisation, is detailed for
Bold basal medium, with 3-fold nitrogen and vitamins (3N-BB-M+V modified) was prepared by mixing the components detailed in Table 1. The trace element solution mentioned in Table 1 was prepared by addition of chemicals outlined in Table 2 in the exact same order.
Composition of 3N-BBM+V media for the synthesis of microalgal biomass
Composition of trace element solution
Stock solutions of Vitamin B1 (Thiaminhydrochloride) and Vitamin B12 (Cyanocobalamin) were prepared as follows; 0.12g of Vitamin B1 was dissolved in 100ml of ddH2O. 1ml of this Vitamin B1 was further diluted into 99ml ddH2O. For Vitamin B12 stock; 0.1g of Vitamin B12 was dissolved in 100ml of ddH2O. Both working stocks of vitamin B1 and B12 were filter sterilized through a 0.2μm sterile filter and stored at -20°C until required for use. Cultures were grown in 3N-BBM+V media at 18°C with 16h:8h (light:dark) cycle at 120rpm. Cell growth from was checked at O.D 590nm, and fixed number of cells (1 O.D@590nm = 107 cells/ml) were transferred into nitrogen deficient media (3N-BBM+V media, without NaNO3). Cells were maintained in the same nitrogen deficient media without any further supplementation for 7days at 18°C with 16h:8h (light:dark) cycle at 120rpm. After 7days, cells were harvested and were used for neutral lipid quantification.
After 7 days of incubation in nitrogen deficient media; 10ml of culture was removed aseptically to a universal tube. The culture was centrifuged at 5,000*g for 5mins and the culture media (supernatant) was discarded. The microalgal cell pellet was later mixed thoroughly in 10ml of double distilled water. 50μl of this water dissolved pellet was used as one assay aliquot. Into a flat bottom, transparent 96-well microtiter plate, 100μl of double distilled water was added in quintuplet to which 50μl of microalgae culture prepared earlier was added. These diluted microalgae cultures were used for further assay development.
As based on Chen, and Huang and colleagues, (7 and 11) 10μg/ ml of Nile Red solution was prepared over a concentration range (10-60% v/v) of the solvents examined (DMSO and acetone). To 150μl of diluted microalgae culture, 100μl of 10% (v/v) Nile Red prepared in DMSO was added in quintuplet. The process was repeated for all the solvent concentrations (1060% v/v DMSO and acetone) containing 10μg/ml Nile red. The sample (ddH2O+microalgae+Nile Red) was incubated at room temperature in the dark for 30mins before monitoring the fluorescence intensity using a Perkin Elmer Lambda 900 UV/VIS/ NIR Spectrometer at various excitation wavelengths (485nm-530nm) and at an emission wavelength of 580nm.
To identify the concentration of Nile Red required to obtain the maximum fluorescence intensity; different concentrations of Nile Red, ranging between 1μg/ml to 50μg/ml were prepared in 20% (v/v) DMSO. 100μl of this solution was added to 150μl of diluted microalgal culture in quintuplet. The assay plate was then statically incubated, at 40°C in the dark, for 60mins before recording the fluorescence intensity using a Perkin Elmer Lambda 900 UV/VIS/NIR Spectrometer at 530nm excitation and 580nm emission wavelength.
A 10μg/ml and 5μg/ml Nile Red solution was prepared in 20% (v/v) DMSO. 100μl of 10μg/ml and 5μg/ml of Nile red was added to 150μl of diluted
Different cell concentrations (0.05-0.9 at OD590nm) of
A working range of triolein standard (between 1μg/ml to 15μg/ ml) was prepared in neat chloroform. In a 96-well plate, 100μl of ddH2O, 100μl of 10μg/ml Nile Red (in 20% v/v DMSO) and 50μl of the respective triolein concentration was mixed in quintuplicate. The assay monitored fluorescence intensity at 530nm excitation and 580nm emission wavelength after 5mins incubation. The fluorescence intensity measured using a Perkin Elmer Lambda 900 UV/VIS/NIR Spectrometer for various concentrations of triolein was used for preparing a standard curve. The intensity of the unknown microalgal strain observed after optimization of the Nile Red assay was compared to the Triolein standard curve and neutral lipid content was quantified.
This study aimed to optimise the Nile Red assay for use with two microalgae strains,
Subsequently, the optimum solvent to obtain maximum fluorescence intensity, for both
The optimum concentration of Nile Red was explored, and the maximum fluorescence intensity was observed with 10μg/ ml concentration of Nile Red in the case of
An increase in fluorescence intensity was observed with increased incubation time for both the microalgal strains, to a maximum of 60mins (
Therefore, since a minimum incubation of 60mins is required to attain maximum fluorescence intensity for
The optimum range of cell concentration is species specific and varies between 5x104 to 1x106 cell/mL, as observed with Chlorella vulgaris (7). A linear relationship was observed between fluorescence intensity and number of cells when an OD590nm between 0.2-0.7 for both
Finally, microscopic examination of microalgal cells indicated the shape of
Fluorescent dyes offer a useful method for the measurement of intracellular lipids in microalgae as they are inexpensive and require simple optical measurement instrumentation while simultaneously providing the opportunity of high throughput application. In this study, a standard and bespoke fluorescent Nile Red assay was developed and optimised for two microalgae strains;