maximum is reached within a minute [10]. BODIPY has the advantage
that it is less sensitive to light and oxidation and does not bind to
cytoplasmic compartments other than lipid bodies, and they are
stained green that can be easily distinguished from red fluorescence
of chloroplasts [3, 5, 12]. Also, this dye has a narrower emission
spectrum than NR, making it potentially more useful for confocal
imaging [12]. BODIPY is able to stain a wide range of algae groups
without the need of using high concentrations of dimethylsulfoxide
(DMSO) or acetone as a dye carrier; therefore, it can be key to wider
and faster cell recovery and scale-up [12]. Dye staining is
nondestructive allowing the use of algae for future analysis after
lipid determination and supports the establishment of lipid-rich
microalgal cultures [5, 12, 14]. According to Rumin and
co-authors [10], background fluorescence of the dye in the medium
and the failure to quantify neutral lipids between rich and low oil
strains are among the disadvantages of BODIPY 505/515 dyes.
The BODIPY staining method is rapid, easy, and reliable and has
the potential to be an effective screening tool for the semiquantification of neutral lipids in various microalgal taxa (even with thick
and robust cell wall) grown in different conditions [10, 12]. A good
correlation was observed between BODIPY 505/515 lipid fluorescence and gravimetric measurements for Tetraselmis subcordiformis
[9]. However, the other researchers noted that the staining protocol of BODIPY is species specific and a given fluorescence value
cannot be associated with the same amount of lipids in different
species [15]. Indeed, a reliable estimate of the neutral lipid content
in microalgae with dye fluorescence depends upon a variety of
staining and measuring conditions; therefore, the use of nonoptimal or inconstant conditions often results in poor lipid content
assessment [10]. Therefore, before the application of BODIPY to
measure neutral lipids, the instrument, organic solvents, and staining dye protocol should be calibrated and strictly followed because
of the nonlinear intensity emission with respect to time. Rumin and
co-authors [10] firstly proposed the general framework and guidelines of protocol for lipid staining with fluorescent dyes.
2 Materials
1. Spectrofluorometer (488 nm excitation, 510 nm emission) or
epifluorescence microscope or flow cytometer (depending on
the chosen oil evaluation method).
2. BODIPY 505/515 dye (4,4-difluoro-1,3,5,7-tetramethyl-4-bora3a,4a-diaza-s-indacene).
3. Dimethylsulfoxide (DMSO).
4. Microalgae culture.
48
Judita Koreiviene ˙
that it is less sensitive to light and oxidation and does not bind to
cytoplasmic compartments other than lipid bodies, and they are
stained green that can be easily distinguished from red fluorescence
of chloroplasts [3, 5, 12]. Also, this dye has a narrower emission
spectrum than NR, making it potentially more useful for confocal
imaging [12]. BODIPY is able to stain a wide range of algae groups
without the need of using high concentrations of dimethylsulfoxide
(DMSO) or acetone as a dye carrier; therefore, it can be key to wider
and faster cell recovery and scale-up [12]. Dye staining is
nondestructive allowing the use of algae for future analysis after
lipid determination and supports the establishment of lipid-rich
microalgal cultures [5, 12, 14]. According to Rumin and
co-authors [10], background fluorescence of the dye in the medium
and the failure to quantify neutral lipids between rich and low oil
strains are among the disadvantages of BODIPY 505/515 dyes.
The BODIPY staining method is rapid, easy, and reliable and has
the potential to be an effective screening tool for the semiquantification of neutral lipids in various microalgal taxa (even with thick
and robust cell wall) grown in different conditions [10, 12]. A good
correlation was observed between BODIPY 505/515 lipid fluorescence and gravimetric measurements for Tetraselmis subcordiformis
[9]. However, the other researchers noted that the staining protocol of BODIPY is species specific and a given fluorescence value
cannot be associated with the same amount of lipids in different
species [15]. Indeed, a reliable estimate of the neutral lipid content
in microalgae with dye fluorescence depends upon a variety of
staining and measuring conditions; therefore, the use of nonoptimal or inconstant conditions often results in poor lipid content
assessment [10]. Therefore, before the application of BODIPY to
measure neutral lipids, the instrument, organic solvents, and staining dye protocol should be calibrated and strictly followed because
of the nonlinear intensity emission with respect to time. Rumin and
co-authors [10] firstly proposed the general framework and guidelines of protocol for lipid staining with fluorescent dyes.
2 Materials
1. Spectrofluorometer (488 nm excitation, 510 nm emission) or
epifluorescence microscope or flow cytometer (depending on
the chosen oil evaluation method).
2. BODIPY 505/515 dye (4,4-difluoro-1,3,5,7-tetramethyl-4-bora3a,4a-diaza-s-indacene).
3. Dimethylsulfoxide (DMSO).
4. Microalgae culture.
48
Judita Koreiviene ˙