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Enabling High-Sensitivity Red Fluorescent Imaging of Lipid Droplets & Lipid Composition Analysis LipiDye™ RED

Date：March 18 2026Web Page No：95044

Funakoshi Co.,Ltd.

LipiDye™ RED is a reagent that enables high-sensitivity red-fluorescent imaging of lipid droplets in live cells. In addition to its high specificity for lipid droplets, it exhibits low cytotoxicity and exceptional photostability, making it well suited for long-term imaging and dynamic analysis of lipid droplets. Furthermore, by using fluorescence lifetime imaging, it allows analysis of the lipid composition of individual lipid droplets and the progression of lipid hydrolysis.

LipiDyeR REDの蛍光寿命イメージング

About lipid droplet and LipiDye™ series

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. 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™ series(LipiDye™ Ⅱ, LipiDye™ RED)

LipiDye™ Ⅱ (#FDV-0027, Green fluorescence) and LipiDye™ RED (#FDV-0057, Red fluorescence) are novel lipid droplet probes developed to overcome these challenges by Prof. Yamaguchi at the Institute of Transformative Bio-Molecules (ITbM), Nagoya University, and Prof. Taki at the Institute for Glyco-core Research, Gifu University (original probe names: LAQ1 and LipiPB Red). Owing to their high signal-to-noise (S/N) ratio, these probes enable detection of small lipid droplets below 1 μm in diameter. In addition, their exceptionally high photostability allows stable, low-toxicity live-cell imaging over extended periods.


Analysis of lipid composition dynamics in lipid droplets using LipiDye™ RED

As described above, lipid droplets are mainly composed of triacylglycerols (TAGs) and sterol esters. TAGs are hydrolyzed by lipases into fatty acids and diacylglycerols (DAGs), and DAGs are further sequentially degraded into monoacylglycerols (MAGs) and glycerol (lipolysis). During this process, the lipid composition of lipid droplets dynamically changes from a TAG-dominant state to one with a higher proportion of DAG. In addition, a metabolic process known as "lipophagy", in which lipid droplets are degraded through autophagy, has also been reported.
These lipid droplet degradation mechanisms are regulated according to the energy demands of cells and organisms. Fatty acids released through lipid droplet breakdown undergo β-oxidation in mitochondria and are converted into the chemical energy molecule ATP. Notably, one molecule of TAG releases three molecules of fatty acids upon hydrolysis. Because disruption of lipid droplet degradation is known to cause various metabolic diseases, analyzing the progression of lipid droplet degradation and the lipid composition of lipid droplets is of great importance.
Traditionally, lipid composition has been analyzed by extracting lipids from cells or tissues followed by analysis using techniques such as LC/MS/MS. However, this approach is labor-intensive and results in the loss of spatial information regarding lipid droplet composition. In addition, while conventional lipid droplet staining probes allow observation of the spatial distribution and size of lipid droplets, they do not provide information on the lipid composition or degradation state of individual lipid droplets.

脂質組成動態解析

LipiDye™ RED can be used not only as a red-fluorescent lipid droplet staining probe but also for analyzing the lipid composition of lipid droplets when combined with fluorescence lifetime imaging microscopy (FLIM). In general, lipid droplets exist in a highly nonpolar environment; however, their polarity gradually increases as lipid hydrolysis alters their lipid composition. Because LipiDye™ RED exhibits changes in fluorescence lifetime depending on the polarity of the surrounding environment, its fluorescence lifetime varies according to the extent of lipid droplet hydrolysis.
Specifically, lipid droplets in a low-polarity environment with a high proportion of TAG exhibit a longer fluorescence lifetime. In contrast, lipid droplets in a relatively higher-polarity environment, where the proportion of DAG increases due to hydrolysis, show a shorter fluorescence lifetime. By taking advantage of this property, cells stained with LipiDye™ RED can be observed using fluorescence lifetime imaging microscopy, allowing the lipid composition of individual lipid droplets to be visualized as differences in fluorescence lifetime.
Furthermore, LipiDye™ RED has high photostability and strong intracellular retention, enabling long-term imaging. This makes it possible to analyze the progression of lipid droplet hydrolysis both spatially and temporally.

蛍光寿命イメージング顕微鏡法

Comparison of the LipiDye™ series with conventional reagents

Name Excitation Wavelength

Fluorescence Wavelength


(Fluorescence color)

 Staining Multicolor imaging S/N ratio Photo-stability Time-lapse imaging

Analysis of lipid composition dynamics
Fixed cells Live cells
LipiDye™ RED 470-560 nm 550-700 nm
(Red)		 Yes Yes Yes

(Wavelength Selection Warning)

 High Extremely High Extremely Long Time

Fluorescence lifetime imaging microscopy (FLIM)
LipiDye™ Ⅱ 400-500 nm 490-600 nm
(Green)		 Yes Yes Yes

(Wavelength Selection Warning)

 High Extremely High Extremely Long Time No
Fluorescence B -490 nm 510 nm
(Green)		 Yes Yes Yes Middle Low Yes No
Nile Red -510 nm 631 nm
(Red)		 Yes Yes Not suitable Low Low Not suitable No
LDs staining dye A - Red/Green Yes Yes Yes High - No No
Oil Red O - Red dye No Yes - Low - No No

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Features

  • In addition to the selective enrichment to LDs, this probe emits light in response to a hydrophobic environment, thus suppressing emission in the cytoplasm, etc. and showing a high signal-to-noise ratio for LDs.
  • Capable of detecting small LDs (<1 μm) in non-adipocytes.
  • It exhibits extremely high photostability and is excellent for long-time live cell imaging.
  • At the recommended use concentration (0.1-5 μM), it shows almost no cytotoxicity.
  • Can be used for both live cells and fixed cells. Fixation treatment after staining of living cells is also possible.
  • Applicable to STED super resolution microscopy.
  • Excitation/Fluorescence wavelengths: 470-560 nm / 550-700 nm (See below)
  • Lipid composition of lipid droplets can be evaluated using fluorescence lifetime imaging microscopy (FLIM)

Fluorescent characteristics

The absorption maximum is between 470–520 nm, but excitation is also possible with light sources in the 520–560 nm range. For details, please refer to the excitation and emission spectra as well as data on applicable excitation wavelengths. Multiplex staining with blue- or green-fluorescent dyes is also possible, but careful wavelength selection is required. In particular, when performing multiplex staining with green dyes (e.g., FITC, GFP) and using a 488 nm laser to excite the green dye, LipiDye™ RED will also be excited. Therefore, to selectively detect green dyes in multiplex experiments, use a bandpass filter that blocks fluorescence above 520 nm.
Conversely, to selectively detect LipiDye™ RED, excite with a 514 nm or 532 nm laser and use a filter that blocks fluorescence below 560 nm.


Examples of light sources

  • Lasers: 458 nm, 473 nm, 488 nm, 514 nm, 532 nm, 561 nm
    561 nm laser can excite LipiDye™ RED but shows weak fluorescence compared with other excitations. When using a 561 nm laser, empirically optimize imaging conditions such as dye concentration, etc., for your experiments.
  • Light source (Xenon lamp or LED) + filters: Commercial Alexa555 or RFP filters are available.
  • STED super resolution microscopy: Recommended excitation light: 488 nm laser, STED light: 775 nm laser.

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Reference Data

Excitation / Fluorescence spectrum

吸収スペクトル

Absorption spectrum of LipiDye™ RED
The absorption spectrum of LipiDye™ RED is largely unaffected by the solvent and shows absorption in the 430–550 nm range.

蛍光スペクトル

Fluorescence spectrum of LipiDye™ RED
LipiDye™ RED is a solvatochromic dye, meaning its fluorescence properties change depending on the polarity of the surrounding environment. In hydrophobic environments such as cyclohexane or toluene, it exhibits strong orange-to-red fluorescence. In contrast, in highly polar environments such as acetonitrile or DMSO, the fluorescence maximum shifts to longer wavelengths and the fluorescence intensity is significantly reduced. This property enables the selective observation of red fluorescence derived from the hydrophobic environment of lipid droplets.

脂肪滴の蛍光スペクトル

Fluorescence spectrum of Lipid Droplets stained with LipiDye™ RED
When cells are stained with LipiDye™ RED and the lipid droplet regions are analyzed by spectral scanning microscopy, a fluorescence spectrum with a maximum around 600 nm is observed.


Applicability of excitation wavelength

励起波長適用性

LipiDye™ RED was used to stain LDs in HepG2 cells, and red fluorescence (λem = 580-750 nm) intensity was measured with several different excitation lasers was detected using a confocal laser scanning microscope.


Photostability

光安定性

After staining HeLa cells with LipiDye™ RED, Nile Red, and BODIPY493/503, images were repeatedly acquired using a high-power laser (500 nm) using a confocal laser scanning microscope, and changes in fluorescence intensity were observed. In contrast to Nile Red and BODIPY493/503, whose fluorescence attenuated significantly after multiple image acquisitions, LipiDye™ RED showed almost no change in fluorescence intensity even after a total of 200 image acquisitions.


Changes in fluorescence lifetime depending on lipid composition

蛍光寿命イメージングによる脂肪滴組成の解析

Artificial LDs with varying composition ratios of triolein (TO), a typical TAG, and diolein (DO), a typical DAG, were stained with LipiDye™ RED, and their fluorescence lifetime imaging was observed. The results showed that the higher the DO ratio, the shorter the fluorescence lifetime, and the higher the TO ratio, the longer the fluorescence lifetime (τ = 4.1 ns to 7.5 ns). This indicates the DAG/TAG ratio of LDs can be evaluated using the fluorescence lifetime of LipiDye™ RED. Fluorescence lifetime is expressed in pseudo-color.


Cytotoxicity

細胞毒性

HeLa cells were treated with various concentrations of LipiDye™ RED for 24 hours. After incubation, cell viability was evaluated by MTT assay. The concentration range (0.2-5 μM) showed little cytotoxicity in cells.


Live/Fixed cells staining

生細胞・固定細胞染色

Huh-7 cells treated with oleic acid to form LDs were stained with LipiDye™ RED and fixed with 4% paraformaldehyde. Almost no change in the fluorescence signal was observed before and after the fixation treatment. It has been shown that LipiDye™ RED can be used for co-staining with immunostaining after fixing cells.



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Application Data

LDs imaging after medium exchange

血清培地交換後の観察

Adipocytes differentiated from 3T3-L1 cells (3T3-Adi) were stained with LipiDye™ RED, LipiDye™ II, Nile Red, and BODIPY493/503, and a fluorescence image of LDs was observed using a confocal laser scanning microscope. The fluorescence intensity of Nile Red and BODIPY493/503 decreased after cell washing, and almost no fluorescence signal was observed after culturing for 24 hours in an FBS-containing medium (DMEM+). LipiDye™ II maintained its fluorescent signal after washing, but the fluorescent signal significantly weakened after culturing in DMEM+ for 24 hours*. On the other hand, the fluorescence intensity of LipiDye™ RED did not decrease after washing, and remained even after culturing in DMEM+. This indicates that LipiDye™ RED exhibits greater intracellular retention compared to conventional reagents.

*Long-term fluorescence observation with LipiDye™ II is also possible by continuous culturing in a medium containing LipiDye™ II. In details, please see here.

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Live-cell STED super-resolution microscopy imaging

STED超高解像度顕微鏡による微小脂肪滴の可視化

HeLa cells were treated with 0.5 μM LipiDye™ RED, washed, and cultured in medium. The cells were imaged by confocal laser microscopy (Ex 488 nm/ Em 560-750 nm) and STED microscopy (Ex 488 nm/ Em 560-750 nm, depletion laser 775 nm). STED imaging detected -92 nm (FWHM) small LD, which was not clearly detected by confocal microscopy.


Fluorescence intensity and lifetime imaging of non-adipocyte cells

非脂肪細胞の蛍光強度および蛍光寿命イメージング

Huh-7 cells treated with oleic acid to form LDs were treated with 0.5 μM LipiDye™ RED, and fluorescence imaging and fluorescence lifetime imaging (Ex 488 nm/Em 560-750 nm) were performed using a confocal laser microscope. FLIM images are pseudo-colored with fluorescence lifetime (blue: t = 5.5 ns, red: t = 7.5 ns)



Fluorescence intensity and lifetime imaging of non-adipocyte cells

Adipocytes differentiated from 3T3-L1 cells (3T3-Adi), undifferentiated 3T3-L1 cells, HepG2 cells, Huh-7 cells, COS-7 cells, and HeLa cells were stained with LipiDye™ RED (0.5 μM), and fluorescence lifetime imaging was performed (excitation 488 nm/fluorescence 560-750 nm). Cells other than 3T3-Adi were treated with oleic acid for 1 day to form LDs and then observed. The fluorescence lifetimes of LipiDye™ RED-stained LDs in hepatoma-derived HepG2 cells and Huh-7 cells were not uniform, suggesting that the DAG/TAG composition of each LD differs largely. On the other hand, the fluorescence lifetimes of LDs in other cells were relatively uniform and longer than those of hepatoma cells, indicating that the overall lipid composition had a high proportion of TAG.

さまざまな細胞種での蛍光寿命イメージング


Evaluation of the influence of lipase activity on lipolysis

LipiDye™ RED fluorescence lifetime imaging was performed on Huh-7 cells in which adipose triglyceride lipase (ATGL) was knocked down using siRNA. The fluorescence lifetime of LDs in WT Huh-7 cells was heterogeneous, whereas the fluorescence lifetime in ATGL knockdown cells was uniform and long. The same phenomenon was also observed when HepG2 cells were treated with the ATGL inhibitor NG-497. These suggest the heterogeneity of the lipid composition of LDs in hepatoma cells is due to an increase in the proportion of DAG generated by LDs degradation (lipolysis) catalyzed by ATGL.

脂肪滴分解における加水分解酵素の影響を評価


Time-lapse analysis of the change of lipid composition during lipolysis induction

Forskolin, an adenylate cyclase activator, was added to adipocytes differentiated from 3T3-L1 cells (3T3-Adi) to induce lipolysis, and time-lapse analysis of fluorescence lifetime imaging of LipiDye™ RED -stained LDs was performed for 100 minutes. The fluorescence lifetime of LDs became heterogeneous and shorter over time after drug treatment, suggesting that the proportion of DAG increased due to LDs degradation.


脂肪滴分解における加水分解酵素の影響を評価

Time-lapse



Evaluation of the change of lipid composition during lipolysis induction

脂肪滴分解における加水分解酵素の影響を評価

Huh-7 cells, derived from liver cancer, were treated with Forskolin and the phosphodiesterase inhibitor IBMX (100 nM) to induce lipid droplet degradation (lipolysis), and fluorescence lifetime imaging was performed using LipiDye™ RED. As a result, the number of lipid droplets with shorter fluorescence lifetimes increased following drug treatment, suggesting that the proportion of DAG rises during lipid droplet degradation.



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Reference

  1. Wang, J., et al., "Single-Cell Fluorescence Analysis of Lipid Droplet Compositional Dynamics during Triacylglycerol Catabolism", J. Am. Chem. Soc.、 147, 41514-41523(2025). [PMID:41065230]

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Product Information

[Date : March 22 2026 00:07]

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[Date : March 22 2026 00:07]


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