Membrane Lipid Order Imaging Dye LipiORDER | Funakoshi

LipiORDER is a novel solvatochromic dye for membrane lipid order imaging

Fluorescent change of LipiORDER depending on lipid order of membrane (Left) and an example of ratiometric imaging on dendrites of cultured neurons.

Sensing of lipid order by using LipiORDER is based on the following two unique properties.
1) LipiORDER is a pyren-based solvatochromic fluorescent dye which changes fluorescent property in response to their solvent environment (Figure P1). In low polaric solvents such as toluene, LipiORDER shows green fluorescence. On the other hand, in highly polaric solvents such as DMSO and methanol, this dye changes color to orange or red.
2) LipiORDER is a highly hydrophobic compound and quickly accumulates in the various biological membranes. Combining the two features above, LipiORDER can sense the local environment in a lipid bilayer. Generally, Lo is a high packing lipid bilayer and shows lower polarity, whereas Ld is a sparse packing lipid bilayer and shows high polarity. Based on polarity of lipid bilayer derived from lipid order, LipiORDER will change fluorescent color, from green on Lo membrane to red on Ld membrane (Figure P2).
Ratiometric fluorescent value (F_R/F_G ) is correlated to lipid order (Lo and Ld). Actually, in sphingomyeline/cholesterol (SM/Chol) liposome, one of the model Lo, LipiORDER emits green fluorescence and in 1,2-dioleoyl-sn-glucero-3-phosphocholine (DOPC) liposome, a model Ld, shows red fluorescence. In DOPC/Chol, an intermediate model, the reagent show yellow to orange. The ratiometric values (F575/F510) clearly depend on lipid order, SM/Chol (Lo) is low and DOPC (Ld) is high (Figure P3).

Figure P1 Absorption and fluorescent spectrum of LipiORDER in various solvent

Figure P2 Graphical overview of lipid order-dependent fluorescent change of LipiORDER

Figure P3 Fluorescent spectrum of LipiORDER in model liposomes

COS7 cells were treated with 300 nM LipiORDER in HBSS for 10 min and observed by confocal laser microscopy (Ex. 405 nm, Em 470-550 nm for Green channel and >550 nm for Red channel). Ratiometric analysis was performed with ImageJ using green and red channel data and lipid order was shown by green-to-red pseudocolor (Lo■■■■■■■Ld). Plasma membrane and intramembranes are shown Lo and Ld, respectively.

Primary cultured hippocampal neurons (DIV 3 or DIV 12) from E17.5 mice were stained with 300 nM LipiORDER in HBSS for 10 min and observed by confocal laser microscopy (Ex. 405 nm, Em. 470-550 nm for Green channel and >550 nm for Red channel). Ratiometric analysis was performed with ImageJ using green and red channel data and lipid order was shown by green-to-red pseudocolor (Lo■■■■■■■Ld).

COS7 cells were treated with 15 mM beta-cyclodextrin (beta-CD), a membrane-disrupting chemical via removing endogenous cholesterol, for 4 hours. After beta-CD treatment, cells were washed and stained with 300 nM LipiORDER in HBSS for 10 min. The cells were observed by confocal laser microscopy (Ex. 405 nm, Em. 470-550 nm for Green channel and >550 nm for Red channel). Ratiometric analysis was performed with ImageJ using green and red channel data and lipid order is shown by green-to-red pseudocolor (Lo■■■■■■■Ld).
The cell structure was dramatically changed by beta-CD and at the same time, the distribution of Lo phase (■) clearly changed.

LipiORDER and Laurdan, a conventional membrane lipid order imaging dye in lipid vesicles composed of 0.2 mM DOPC in 20 mM HEPES (pH 7.4) were irradiated with Xe lamp. LipiORDER and Laurdan were excited at 405 nm and 360 nm, respectively and fluorescent intensity was measured. Laurdan was quickly photodegraded, whereas LipiORDER maintains fluorescent intensity for at least 1 hour. LipiORDER is highly stable compared to Laurdan.

1. Valanciunaite et al., Anal. Chem., 92, 6512-6520 (2020)　Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe.

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