Live imaging fluorescent dye for lipid droplets (LDs) LipiDye Ⅱ | Funakoshi

Ex. 400-500 nm (maximum ~420nm)
Compatible with blue excitation lasers (ex. 405, 445, 458, 473 and 488 nm lasers, etc.), Xenon lamp or LED with commercial FITC or GFP filters.
*Note
488nm laser can excite LipiDyeⅡ but shows weak fluorescence compared with 473 nm excitation. If using 488 nm laser, please empirically optimize imaging conditions such as dye concentration etc. for your experiments.

Em. 450-650 nm (depend ent on solvents)
Maximum ~510 nm in soybean oil similar to LDs. Around 490-550 nm range is recommended.

Spectrum of LipiDyeⅡ
(A)Absorption spectrum in various solvents.
(B)Emission spectrum in various solvents. LipiDyeⅡ is a solvatochromic dye and shows a different spectrum in each solvent. Under low polaric solvents, toluene and dichloromethane, emits from blue to green fluorescence with high quantum yield. On the other hand, under high polaric solvents, acetonitrile, DMSO and water, LipiDyeⅡ exhibits a weak fluorescence intensity with a red shift fluorescence.
(C) Emission spectrum in cellular LDs measured by fluorescent microscopy.

Photostability of LipiDyeⅡ in the cell
3T3-L1 adipocytes pre-fixed in 4% formaldehyde were stained with LipiDyeⅡ , prototype LipiDye and conventional LD dye ( B ). The free dyes were removed by washing and z-stack images (z=10 with a 2μm step ) of the adipocytes in the same area were repeatedly acquired by confocal microscopy (Ex 473 nm/Em 490-540 nm). The total fluorescence was measured and normalized intensity was calculated. The fluorescent intensities from conventional dye B dramatically reduced by repeated light irradiation including LipiDye gradually decreased. LipiDyeⅡ maintained its fluorescence at least 50 z-stack images (total 500 images).

Staining of various cells
3T3-L1, HepG2, COS-7 and HeLa cells were stain e d with LipiDyeⅡ (1 µM) for 12 hours and observed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). In the case of HepG2 cells were pretreated with palmitic acid (0.33 mM) /oleic acid (0.66 mM), one day before LipiDyeⅡ staining. In HeLa cells, small LDs of approximately 1 µM were clearly observed. (Scale bar: 20 µm, HeLa cell enlarged 5 µm).

Multicolor imaging with ER marker
COS7 cells expressing ER resident fluorescent protein (mKO1) was stained with LipiDyeⅡ (1 µM) for 12 hours. After washing the cells were observed by confocal microscopy (LipiDyeⅡ; Ex. 473 nm/Em 490-540 nm, mKO1; Ex. 635 nm/Em 660-710 nm). Small LDs less than 1 µm were frequently observed in the network structure of ER (Scale bar: 20 µm, 5 µm and 1 µm)

Long term staining during adipocyte differentiation and maturation
Two days after confluence, 3T3-L1 preadipocytes were stained with LipiDyeⅡ for 12 hours. After washing with fresh medium, the cells were incubated with a differentiation medium containing 1 µM LipiDyeⅡ and the first image (0 days ) was recorded by confocal microscopy (Ex. 473 nm/Em 490-540 nm). After two days of differentiation, the medium was replaced with a maintenance medium containing 1 µM of LipiDyeⅡ. During acquisition of the images, the medium containing LipiDyeⅡ was exchanged every 2 days. (Scale bar; 20 µm)

Time-lapse Z-stack imaging of adipogenesis
3T3-L1 preadipocytes cultured in differentiation medium containing 1 µM LipiDyeⅡ and time-lapse Z-stack imaging (20 z-images/10 min, for 24 hours) was performed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). After ~10 hours differentiation, small LDs were observed (650 min, white arrows) and some LDs were docking with other LDs during adipogenesis (1050-1450 min, yellow allow). (Scale bar; 1 µm)

Time-lapse Z-stack imaging of lipolysis and lipogenesis
3T3-L1 adipocytes were incubated with 1 µM LipiDyeⅡ and washed with media to remove the free dye. After then the cells were treated with Forskolin (10 µM), an activator of adenylyl cyclases, and IBMX (100 nM), an inhibitor of phosphodiesterases. These drugs increased the intracellular concentration of cAMP and subsequently promoted the hydrolysis of triacylglycerols. Immediately after the addition of drugs, time-lapse Z-stack imaging (15 z-images/4 min, for 800 min, total 3000 images) were performed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). Some large LDs clearly contracted or disappeared caused by the drugs. After two hours, numerous newly formed small LDs were observed. ( Scale bar; 5 µm)

Live-cell STED super resolution microscopy imaging
HeLa cells were treated with 1 µM LipiDyeⅡ, washed and culture d in medium. The cells were imaged by confocal laser microscopy (Ex 473 nm/ Em 490-540 nm) and STED microscopy (Ex 473 nm/ Em 500-640 nm, depletion laser 660 nm). STED imaging detected ~120 nm (FWHM) small LD which was not clearly detected by confocal microscopy. Detailed STED imaging condition and analysis methods were described in Ref.1. ( Scale bar 1 µm)

Two-photon Imaging of Microglial LDs
Rat primary cultured microglia were treated with 1 μM LipiDyeⅡ overnight and then fixed with 4% PFA. The cells were observed by two-photon microscopy (Ex. 800 nm/Em. 510-560 nm). LipiDyeⅡ could be excited by two-photon system and detect various sizes of LDs in microglia.

*The data was provided by Dr. Hyun Beom Choi and Dr. Brian MacVicar, The University of British Columbia".

"Fused Thiophene-S,S-dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets."
Taki, M., et al., ACS Mater. Lett., 3(1), 42～49 (2021)

Fig. 1 Comparison data between LipiDye and Nile Red
Lipid droplet in Adipocyte is stained with LipiDye or Nile Red. Nile Red also stains cytoplasm, while LipiDye specifically stains lipid droplet clearly.
* General filter for green fluorescent (e.g. FITC or GFP, etc) is not compatible with LipiDye. Please use an appropriate filter.

Fig.2 Preadipocytes differentiation
Differentiation process of preadipocyte (adipose progenitor cell) : 3T3-L1 is observed with LipiDye. Along with differentiation, lipid droplet also becomes grows in size.

Fig.3 Application of LipiDye in both post-fixed and pre-fixed cells
LipiDye is applied to cultured adipocytes after (left) or before (right) paraformaldehyde fixation. Regardless of fixation timing, LipiDye detects lipid droplet highly sensitive.

* Methanol fixation is not recommeded as it affect to the structure of lipid droplets. Please use cross-linking fixatives, such as paraformaldehyde.

Yamaguchi, et al., Angew. Chem. Int. Ed., 54:4539-4543 (2015).

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