Live imaging fluorescent dye for lipid droplets (LDs)
Date：January 23 2018Web Page No：80682
LipiDye II is a high sensitive, low cytotoxic and super-photostable fluorescent dye for Lipid Droplets (LDs).
LipiDye II enables to detect very small LDs (less than 1µm) and to perform long-term time-lapse imaging, including Z-stack imaging.
This novel dye succeeded in observation of dynamic biosynthesis, degradation and movement of LDs in live cells.
What is lipid droplets (LDs)?
Lipid droplets (LDs) are organelles that have unique phospholipid monolayer and store neutral lipids such as triglycerides and sterol esters (Below figure left). LDs are historically found in adipose tissue and considered as sites for energy storage or lipid turnover. Recent studies discovered that LDs are not only in adipocytes, but also found ubiquitously in cells from yeast to mammalian cells. The numbers, size and composition of LDs largely differ depending on cell types or even within the same cell. For example a dipocytes usually have large LD structures (>10μm) which can be observed by optical microscopy. On the other hand, non adipocytes have a much smaller LD structure compared with adipocytes (Below figure right). LDs are produced from the endoplasmic reticulum (ER), exported to the cytoplasm and expand via fusion of LDs or incorporation of additionally synthesized neutral lipids. LDs contact with various organelles including ER, mitochondrias, lysosomes, nucleus and shows dynamic movement inside the cells.
Problem of conventional LD dyes: stability, sensitively, selectivity etc...
To observe the dynamic movement of LDs in live cells and investigate the physiological functions of LDs, a specific LD dye compatible with long term live cell imaging is required. Conventional fluorescent dyes for LDs such as Nile Red contribute to elucidate biological functions of LDs but its sensitivity and selectivity are limited to detect relatively large LDs in adipocytes or cells treated with excess lipids. It is challenging for conventional dyes to detect small LDs often founds in non-adipocytes under live cell conditions. Funakoshi provides a green fluorescent dye LipiDye (catalog no. #FDV-0010) which shows high sensitivity and selectivity for LDs and can detect approximately 1 μm size s of LDs. Although LipiDye is a powerful tool to monitor small LDs in non-adipocytes, LipiDye requires 405 nm excitation and has insufficient photostability, not suitable for long-term live cell imaging to observe dynamic LDs synthesis, movement or degradation.
is an innovative dye for advanced LD research
Here,LipiDye II , an upgrade version of LipiDye , can be excited by less toxic 450-480 nm light and exhibits super photostability. LipiDyeII is very suitable for long-term live cell imaging including Z-stack time-lapse imaging with multiple time excitations for short term intervals. For example, LipiDye II was applied in long-term imaging for drug induced LD degradation processes for 12 hours with 3,000 image captures and visualized lipolysis of LDs and de novo synthesis of very small (<1 μm) LDs. Furthermore, LipiDye II is compatible with STED microscopy and enables detecting less than 500 nm LDs in HeLa cells.
- High S/N ratio: Low background in cytoplasm via following two features
1) Specific accumulation into LDs
2) Strong green fluorecent emission in non-polar oil phase like LDs
- High sensitivity: Can observe even very small (<1 μm) LDs
- Super photo-stability: Long-term live time lapse imaging is possible
- Low cytotoxicity (at the recommended concentration (0.1～1μM))
- Compatible with both live and fixed cell
- Applicable to STED microscopy: can detect less than 500 nm LDs in HeLa cells.
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.
488nm laser can excite LipiDye II 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.
Superiority of LipiDye
|Name||Color||Excitation light||Staining for live cells||Staining for fixed cells||Photo-stability||Time-lapse imaging||Multicolor imaging||S / N ratio|
|LipiDye Ⅱ||Green fluorecence||400~500 nm||〇||〇||Extremely High||◎||〇||High|
|LipiDye||Green fluorecence||400~470 nm||〇||〇||High||〇||〇||High|
|Nile Red||Red fluorecence||~510 nm||〇||〇||Low||△||△||Low|
|Fluorecent dye B||Green fluorecence||~480 nm||〇||〇||Low||〇||〇||Middle|
|LDs staining dye A||Red・Green fluorecence||-||✕||〇||-||✕||〇||High|
|Oil Red O||Red fluorecence||-||✕||〇||-||✕||-||Low|
Spectrum of LipiDye II
(A)Absorption spectrum in various solvents.
(B)Emission spectrum in various solvents. LipiDye II 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, LipiDye II exhibits a weak fluorescence intensity with a red shift fluorescence.
(C) Emission spectrum in cellular LDs measured by fluorescent microscopy.
Photostability of LipiDye II in the cell
3T3-L1 adipocytes pre-fixed in 4% formaldehyde were stained with LipiDye II , 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. LipiDye II maintained its fluorescence at least 50 z-stack images (total 500 images).
Cytotoxicity of LipiDye II
3T3-L1 adipocytes were treated with various concentrations of LipiDye II for 24 hours. After incubation, cell viability was evaluated by MTT assay. At least 5µM showed little cytotoxicity on adipocytes. The recommended concentration of LipiDye II for LD staining is 0.1-1µM.
Fixed / Live cells
Comparison of the fluorescence intensity under live cell and after PFA fixation
3T3-L1 adipocytes stained with LipiDye II and were observed under live cell conditions (left). After recording the live cell image, the cells were subsequently fixed with 4% PFA and observed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). Fixation has little effect on the fluorescent intensity of LipiDye II. LipiDye II is compatible with any immunocytochemical experiments after the live cell imaging experiments.
Staining of various cells
3T3-L1, HepG2, COS-7 and HeLa cells were stain e d with LipiDye II (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 LipiDye II 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 LipiDye II (1 µM) for 12 hours. After washing the cells were observed by confocal microscopy (LipiDye II; 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 LipiDye II 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 LipiDye II. During acquisition of the images, the medium containing LipiDye II 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 LipiDye II 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 LipiDye II 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 LipiDye II, 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 LipiDye II overnight and then fixed with 4% PFA. The cells were observed by two-photon microscopy (Ex. 800 nm/Em. 510-560 nm). LipiDye II 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)
[Date : August 13 2022 00:32]
[Date : August 13 2022 00:32]
Previous version: LipiDye
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.
[Date : August 13 2022 00:32]
|Detail||Product Name||Product Code||Supplier||Size||Price|
LipiDye, Lipid Droplet Green
||FDV-0010||FNAFunakoshi Co.,Ltd.||0.1 mg||$200|
[Date : August 13 2022 00:32]
LipiDye, Lipid Droplet Green
- Product Code: FDV-0010
- Supplier: FNA
- Size: 0.1mg
- Price: $200
LipiDye is a highly sensitive, new fluorescent dye for lipid droplet. Compared to conventional lipid droplet detection dye (Nile Red or boron-dipyrromethene), LipidDye has higher S/N ratio and photo-stability. LipiDye can be used for both live and fixed cell.
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