BODIPY 505/515 is mostly measured using spectrofluorometer
equipped with a 488 nm narrowband excitation and 510 nm emission filters. The following conditions were applied: excitation slit
10 nm, emission slit 10 nm, and photomultiplier 400 V [9]. Nonautomated spectrofluorometry using vials requires more time and a
larger amount of biomass (up to 4 ml) and solvent to compare with
automatic microplate method, where the 0.5 ml aliquots of stained
cell culture are placed into a 96-well plate. (b) Fluorescence microscopy
offers the possibility to calculate the volume of lipid vesicles. Place a
drop of stained microalgae culture on the objective glass, and cover it
with a coverslip. Algae are analyzed using epifluorescence microscope
equipped with digital camera. The 515 nm emission wavelength
long-pass filter is used to obtain green fluoresce of oil in the cells.
Microphotographs should be made using 100Â objectives (use
immersion oil) to have the best image quality of the stained algae
for the analysis. Images of stained algae further are analyzed using the
computer software, e.g., NIS-Elements BR software. The total volume of oil droplets in relation to cell volume was evaluated based on
mathematical formulas and statistical analysis (Fig. 1; Koreiviene ˙
et al., unpublished data). (c) Flow cytometry addresses the per-cell
fluorescence and makes it possible to distinguish between cells,
debris, and dye precipitate. Also, it allows sorting fluorescentactivated cells and establishing high lipid-accumulating cultures. In
general, the method allows to perform rapid and quantitative processing of samples and gives very good results with BODIPY 505/515
dyes [10]. However, preparation of the samples for flow cytometry is
quite time-consuming (especially in the case of a small number of
samples). The blank measurement with appropriate applied concentration of dyes should be used between culture measurements as
BODIPY 505/515 for the evaluation of background autofluorescence
Fig. 1 The analysis of oil accumulated in the Ankistrodesmus falcatus cell using epifluorescence microphotographs: (a) original image of the alga with BODIPY 505/515 stained oil droplets inside of the cell, (b) evaluation
of number and diameters of oil droplets in the cell, (c) the total volume of the oil droplets evaluated by
NIS-Elements BR software, (d) evaluation of the total cell volume using NIS-Elements BR software
Microalgae Lipid Staining with Fluorescent BODIPY Dye
51
equipped with a 488 nm narrowband excitation and 510 nm emission filters. The following conditions were applied: excitation slit
10 nm, emission slit 10 nm, and photomultiplier 400 V [9]. Nonautomated spectrofluorometry using vials requires more time and a
larger amount of biomass (up to 4 ml) and solvent to compare with
automatic microplate method, where the 0.5 ml aliquots of stained
cell culture are placed into a 96-well plate. (b) Fluorescence microscopy
offers the possibility to calculate the volume of lipid vesicles. Place a
drop of stained microalgae culture on the objective glass, and cover it
with a coverslip. Algae are analyzed using epifluorescence microscope
equipped with digital camera. The 515 nm emission wavelength
long-pass filter is used to obtain green fluoresce of oil in the cells.
Microphotographs should be made using 100Â objectives (use
immersion oil) to have the best image quality of the stained algae
for the analysis. Images of stained algae further are analyzed using the
computer software, e.g., NIS-Elements BR software. The total volume of oil droplets in relation to cell volume was evaluated based on
mathematical formulas and statistical analysis (Fig. 1; Koreiviene ˙
et al., unpublished data). (c) Flow cytometry addresses the per-cell
fluorescence and makes it possible to distinguish between cells,
debris, and dye precipitate. Also, it allows sorting fluorescentactivated cells and establishing high lipid-accumulating cultures. In
general, the method allows to perform rapid and quantitative processing of samples and gives very good results with BODIPY 505/515
dyes [10]. However, preparation of the samples for flow cytometry is
quite time-consuming (especially in the case of a small number of
samples). The blank measurement with appropriate applied concentration of dyes should be used between culture measurements as
BODIPY 505/515 for the evaluation of background autofluorescence
Fig. 1 The analysis of oil accumulated in the Ankistrodesmus falcatus cell using epifluorescence microphotographs: (a) original image of the alga with BODIPY 505/515 stained oil droplets inside of the cell, (b) evaluation
of number and diameters of oil droplets in the cell, (c) the total volume of the oil droplets evaluated by
NIS-Elements BR software, (d) evaluation of the total cell volume using NIS-Elements BR software
Microalgae Lipid Staining with Fluorescent BODIPY Dye
51