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Live imaging fluorescent dye for lipid droplets (LDs) LipiDye

Date:January 23 2018Web Page No:80682

Funakoshi Co.,Ltd.

LipiDyeⅡ is a highly sensitive lipid droplets staining reagent for long-term live cell imaging. In addition to its high lipid droplets specificity, LipiDyeⅡ shows low toxicity and extremely high photostability, making it useful for long-term observation, live cell imaging of lipid droplet fusion and degradation processes, and visualization of small LDs using STED super resolution microscopy.

Staining image of adipocytes with LipiDye<sup>™</sup>II

Adipocytes with LipiDye

Staining image of non-adipocytes with LipiDye<sup>™</sup> II

Non-adipocytes with LipiDye

Background

What is lipid droplet?

Lipid droplets (LDs) are organelles that are historically found in adipocytes and have a unique phospholipid monolayer and store neutral lipids such as triglycerides and sterol esters (Below figure left). LDs are thought to act as intracellular neutral lipid storage organs, and are often reported to be associated with obesity and diseases. In recent years, it has been reported that LDs are found not only in adipocytes but also in various other cell types, including hepatocytes, smooth muscle cells, and glial cells, and have various functions, such as metabolic control and regulation of gene expression, as well as their role as storage organs for neutral lipids, as classically known. LDs have been observed in a variety of cells, and it is known that LDs in non-adipocytes are less than 1 μm in diameter and much smaller than those in adipocytes (10-100 μm). The imaging reagent for the observation of small LDs in non-adipocyte has been expected, since existing reagents such as Nile Red stain other than LDs (low signal-to-noise ratio) and are unsuitable for live cell imaging and the observation method of small LDs has been limited to electron microscopy.

LipiDye

Funakoshi provides a green fluorescent dye LipiDye(#FDV-0010), which shows high sensitivity and selectivity for LDs and can detect approximately 1 μm sizes of LDs. Although LipiDye is a powerful tool to monitor small LDs in non-adipocytes, it isn’t suitable for long-term live cell imaging due to its photostability. LipiDyeⅡ is a novel lipid droplet detection reagent developed to overcome these issues. LipiDyeⅡ exhibits extremely high photostability and enables stable live cell imaging for a long time with low toxicity.

Schematic diagram of lipid droplet

Schematic diagram of lipid droplet (LD)

Lipid droplets of Adipocyte

Lipid droplets (LDs) in Adipocyte

Lipid droplet of Adipocyte

LD of Adipocyte

Lipid droplet of non-adipocytes

LD of non-adipocytes


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Features

Fluorescence wavelengths (excitation and detection wavelengths)

  • Absorption maxima are 410 to 420 nm, but excitation is also possible with light sources in the 450 to 500 nm region. For details, please refer to excitation and fluorescence spectrum.
  • Two-photon excitation with an 800 nm laser is available. For details, please refer to observation of microglia using two-photon microscopy.
  • Multiple staining is possible, but wavelengths should be selected carefully. A dye with an excitation light of 500 nm or higher is recommended; if a common blue fluorescent dye that excites at 450 nm or lower is used, LipiDyeⅡ may be excited at the same time.

Light source example

  • Blue excitation lasers: 405, 445, 458, 473 and 488 nm lasers
  • Light source (Xenon lamp or LED) + filters: Common FITC and GFP filters are available
  • STED super resolution microscopy: Recommended excitation light: 473 nm laser, STED light: 660 nm laser
488 nm 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.

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Superiority

Name Color Excitation light Staining for
fixed cells
Staining for
live cells
Multicolor imaging S / N ratio Photo-stability Time-lapse imaging
LipiDye Green fluorecence 400-500 nm HighExtremely High
LipiDye Green fluorecence 400-470 nm High High
Fluorecent dye B Green fluorecence ≦ 480 nm Middle Low
Nile RedRed fluorecence ≦ 510 nm Low Low
LDs staining dye A RedGreen fluorecence- High -
Oil Red ORed fluorecence - - Low -

Reference Data

Excitation / Fluorescence spectrum

Excitation spectra of LipiDye<sup>™</sup> Ⅱ

Excitation spectra of LipiDye
LipiDyeⅡ is a solvatochromic dye and shows a different spectrum in each solvent. The absorption spectrum is almost insensitive to the solvent, with absorption in the 380-500 nm range.

  • Recommended excitation light:405 nm, 445 nm, 458 nm, 473 nm laser
  • Available excitation light:488 nm laser(Due to the weak fluorescence intensity, optimization of condition including concentration is recommended in each experiment.)

Emission spectrum in various solvents

Emission spectrum in various solvents
LipiDyeⅡ is a solvatochromic dye and shows a different spectrum in each solvent. Under low polaric solvents like toluene and dichloromethane, LipiDyeⅡ emits from blue to green fluorescence with high quantum yield. On the other hand, under high polaric solvents like acetonitrile, DMSO and water LipiDyeⅡ exhibits a weak fluorescence intensity with a red shift fluorescence. This property allows the observation of hydrophobic environment-specific green fluorescence in fat droplets.

Fluorescence of the LDs

The spectrum of LDs in the cells stained with LipiDyeⅡ were scanned with a microscope to evaluate the fluorescence of the LDs, and a fluorescence spectrum with a maximum around 500 nm was observed.

Photostability of LipiDyeⅡ in the cell

Photostability of LipiDye<sup>™</sup>Ⅱ 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 2 um 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). This reveals that LipiDyeⅡ exhibits extremely high photostability and is useful for long-term time-lapse imaging (including Z-stack imaging).


Cytotoxicity of LipiDye

Cytotoxicity of LipiDye<sup>™</sup>Ⅱ

3T3-L1 adipocytes were treated with various concentrations of LipiDyeⅡ for 24 hours. After incubation, cell viability was evaluated by MTT assay. The recommended concentration of LipiDyeⅡ for LD-staining is 0.1-1 uM. At least 5 uM showed little cytotoxicity on adipocytes and cytotoxicity was observed above 10 uM.


Comparison of the fluorescence intensity under live cell and after PFA fixation

Comparison of the fluorescence intensity under live cell and after PFA fixation

3T3-L1 adipocytes stained with LipiDyeⅡ 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. Fixation has little effect on the fluorescent intensity of LipiDyeⅡ. LipiDyeⅡ is compatible with any immunocytochemical experiments after the live cell imaging experiments.


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Application Data

Staining of various cells

3T3-L1, HepG2, COS-7 and HeLa cells were stained with LipiDyeⅡ (1 µM) and observed by confocal microscopy (Ex. 473 nm/Em 490-540 nm). In the case of HepG2, cells were pretreated with fatty acids for 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).

3T3-L1 HepG2 Cos7 HeLa
Fluorescence imaging of 3T3-L1 cells Fluorescence imaging of HepG2 cells Fluorescence imaging of COS7 cells Fluorescence imaging of HeLa cells Fluorescence imaging of HeLa cells (enlarged)
Bright field image of 3T3-L1 Bright field image of HepG2 Bright field image of COS7 Bright field image of HeLa

Multicolor imaging with ER marker

COS7 cells expressing ER-resident red fluorescent protein (ER-mKO1) were stained with LipiDyeⅡ(1 uM) and observed by confocal microscopy (LipiDyeⅡ; Ex 473 nm/Em 490-540 nm, ER-mKO1; Ex 635 nm/Em 660-710 nm). Small LDs less than 1 um were frequently observed in the network structure of ER. (Scale bar: 20 um, 5 um and 1 um). This result correlates well with the fact that LDs are biosynthesized from the ER.

LipiDye ER Merge
LipiDye Ⅱ fluorescence image Fluorescence image of ER-resident red fluorescent protein (ER-mKO1) expressing cells Merge
LipiDye Ⅱ fluorescence image Fluorescence image of ER-resident red fluorescent protein (ER-mKO1) expressing cells Merge Merge

Long-term staining during adipocyte differentiation and maturation

Lipid droplet maturation of 3T3-L1 preadipocytes induced to differentiate into adipocytes was observed by confocal microscopy using LipiDyeⅡ (1 uM) for 8 days (Ex 473 nm / Em 490-540 nm). 3T3-L1 cells were cultured in differentiation medium containing LipiDyeⅡ (1 uM) for 2 days and then the medium was replaced with maintenance medium containing LipiDyeⅡ (1 uM) every 2 days. The adipocyte maturation process could be observed even after long-term treatment with LipiDyeⅡ without any cytotoxicity or effect on adipocyte differentiation.

Prolonged observation of adipocyte differentiation-0 day Prolonged observation of adipocyte differentiation-2 days Prolonged observation of adipocyte differentiation-4 days Prolonged observation of adipocyte differentiation-6 days Prolonged observation of adipocyte differentiation-8 days
Prolonged observation of adipocyte differentiation
1 μM LipiDye
in differentiation medium
1 μM LipiDye
in maintain medium
1 μM LipiDye
in maintain medium
1 μM LipiDye
in maintain medium

Dynamic behavior analysis of newly formed LDs by live cell imaging

Newly formed LDs of 3T3-L1 preadipocytes during induction of differentiation into adipocytes were observed by long time-lapse imaging (Z-stack imaging) for about 24 hours (confocal microscopy: excitation 473 nm / fluorescence 490-540 nm, 20 z-images/10 min). Time-lapse imaging was started immediately after the addition of differentiation medium (containing LipiDyeⅡ) to 3T3-L1 previously stained with LipiDyeⅡ. After ~10 hours differentiation, small LDs were observed (650 min, ) and some LDs were docking with other LDs during adipogenesis (950-1450 min, ). (Scale bar; 1 um).

Long-term staining during adipocyte differentiation and maturation (450 min) Long-term staining during adipocyte differentiation and maturation (650 min) Long-term staining during adipocyte differentiation and maturation (950 min) Long-term staining during adipocyte differentiation and maturation (1,050 min)
Long-term staining during adipocyte differentiation and maturation (1,150 min) Long-term staining during adipocyte differentiation and maturation (1,250 min) Long-term staining during adipocyte differentiation and maturation (1,350 min) Long-term staining during adipocyte differentiation and maturation (1,450 min)

Time-lapse Z-stack imaging of lipolysis and lipogenesis

After the differentiated 3T3-L1 cells were treated with forskolin (10 μM), an activator of adenylyl cyclases, and IBMX (100 nM), an inhibitor of phosphodiesterases, time-lapse Z-stack imaging (15 z-images/4 min, for 800 min) were performed by confocal microscopy (Ex. 473 nm/Em 490-540 nm) to observe the lipid droplet shrinkage process associated with triacylglycerol degradation. Some large LDs clearly contracted () and numerous newly formed small LDs were observed () in a time-dependent manner. Since the high photostability and high S/N ratio of the LipiDyeⅡ allows us to observe even the smallest newly formed LDs, the increase and decrease of LDs can be quantitatively observed.

Forskolin + IBMX
0 min 160 min 320 min 480 min
Time course observation of lipid droplet degradation and neogenesis process-0 min Time course observation of lipid droplet degradation and neogenesis process-160 min Time course observation of lipid droplet degradation and neogenesis process-320 min Time course observation of lipid droplet degradation and neogenesis process-480 min

Semi-quantitative analysis of cellular lipid droplets by fluorescent plate reader

Three cell lines (human renal cancer cell line 786-O, mouse neuroblastoma cell line Neuro2a, and Chinese hamster ovarian cell line CHO) were seeded in 96 well plate at 1 x 104 cell/well and cultured in DMEM containing 10% FBS (10% FBS/DMEM). After 24 hours, cells were treated with 10-200 uM oleic acid in 10% FBS/DMEM for further 24 hours to promote growth of lipid droplets. After washing cells, the cells were stained by 5 uM LipiDyeⅡ-containing 2% FBS/DMEM for 2 hours. Just before fluorescent plate leader measurement, cells were washed by PBS twice and fluorescent intensity (Ex 420 ±5 nm/Em 503 ±10 nm) of each well was measured. In all cell lines, oleic acid dose-dependency was observed.

Semi-quantitative analysis of cellular lipid droplets by fluorescent plate reader


Live-cell STED super resolution microscopy imaging of small LDs

HeLa cells were treated with 1 uM LipiDyeⅡ and small LDs were imaged by confocal 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 original paper below.

Live-cell STED super resolution microscopy imaging of small LDs (Confocal laser microscope) Live-cell STED super resolution microscopy imaging of small LDs (Confocal laser microscope)
Live-cell STED super resolution microscopy imaging of small LDs (STED microscope) Live-cell STED super resolution microscopy imaging of small LDs (STED microscope)

Observation of microglia using two-photon microscopy

LipiDyeⅡ (1 uM) was added to primary cultured rat microglia, stained overnight, fixed with 4% PFA, and observed with two-photon microscopy (Ex 800 nm /Em 510-560 nm). LipiDyeⅡ can also be excited by two-photon excitation method and LDs of various sizes in primary cultured microglia could be detected.

Observation Observation of microglia using two-photon microscopy (bright field-enlarged)
Observation of microglia using two-photon microscopy (fluorescence-enlarged)

*Data courtesy of Dr. Hyun Beom Choi and Dr. Brian MacVicar, The University of British Columbia.


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Original Paper

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

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Product Information

[Date : June 05 2025 00:07]

Detail Product Name Product Code Supplier Size Price
LipiDye II, Lipid Droplet Live Imaging
DatasheetThis may not be the latest data sheet.
FDV-0027 FNAFunakoshi Co.,Ltd. 0.1 mg $350

Description LipiDye™ II is a highly sensitive lipid droplets staining reagent for long-term live cell imaging. In addition to its high lipid droplets specificity, LipiDye™ II shows low toxicity and extremely high photostability, making it useful for long-term observation over several days, live cell imaging of lipid droplet fusion and degradation processes, and visualization of small LDs using STED super resolution microscopy.
Storage RT CAS
Link

Lipid Metabolism Detection Tools for Cancer Research

[Date : June 05 2025 00:07]

LipiDye II, Lipid Droplet Live Imaging


  • Product Code: FDV-0027
  • Supplier: FNA
  • Size: 0.1mg
  • Price: $350

Description LipiDye™ II is a highly sensitive lipid droplets staining reagent for long-term live cell imaging. In addition to its high lipid droplets specificity, LipiDye™ II shows low toxicity and extremely high photostability, making it useful for long-term observation over several days, live cell imaging of lipid droplet fusion and degradation processes, and visualization of small LDs using STED super resolution microscopy.
Storage RT CAS
Link

Lipid Metabolism Detection Tools for Cancer Research



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