Fluorescent plasma membrane probes are indispensable tools for biological studies, enabling the visualization of the fine structure and dynamics of plasma membranes, and, by extension, the overall morphology of living cells. However, their use has been mostly limited to imaging cultured cells or fixed tissue slices. Indeed, few probes have been optimized for visualizing cellular morphologies in intact tissues or organs. Here, we report a new bright squaraine-based membrane probe, dSQ12AQ, which incorporates two anionic anchor groups (sulfonate and long alkyl chain) to ensure high water dispersibility without precipitation-even at concentrations exceeding 10 mg mL-1. This highly concentrated probe solution was intravenously administered to living mice without the need for dimethyl solfoxide or other solubilizing agents. Combined with two-photon microscopy, dSQ12AQ enabled clear visualization of whole-cell morphology in vivo, allowing dynamic imaging of flowing, rolling, and/or remaining stationary blood cells in the bone marrow vasculature. Moreover, dSQ12AQ extravasated from blood vessels, enabling further staining and visualization of cells in the perivascular bone marrow region. This extravasation was also observed in the hind paw skin, enabling clear visualization of keratinocytes in the epidermis, as well as fibroblasts and eccrine sweat duct cells in the dermis. These results highlight the potential of dSQ12AQ as a valuable tool for in vivo studies of various cellular processes and for investigation of refractory or poorly understood diseases and their treatments.

Correction for 'Synthesis and photophysical properties of a new push-pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging' by Kazuki Inoue et al., J. Mater. Chem. B, 2022, 10, 1641-1649, https://doi.org/10.1039/D1TB02728J.

Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4-5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget's disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER® and HistoBright®) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.

Sweat is an essential protection system for the body, but its failure can result in pathologic conditions, including several skin diseases, such as palmoplantar pustulosis (PPP). As reduced intraepidermal E-cadherin expression in skin lesions was confirmed in PPP skin lesions, a role for interleukin (IL)-1-rich sweat in PPP has been proposed, and IL-1 has been implicated in the altered E-cadherin expression observed in both cultured keratinocytes and mice epidermis. For further investigation, live imaging of sweat perspiration on a mouse toe-pad under two-photon excitation microscopy was performed using a novel fluorescent dye cocktail (which we named JSAC). Finally, intraepidermal vesicle formation which is the main cause of PPP pathogenesis was successfully induced using our "LASER-snipe" technique with JSAC. "LASER-snipe" is a type of laser ablation technique that uses two-photon absorption of fluorescent material to destroy a few acrosyringium cells at a pinpoint location in three-dimensional space of living tissue to cause eccrine sweat leakage. These observatory techniques and this mouse model may be useful not only in live imaging for physiological phenomena in vivo such as PPP pathomechanism investigation, but also for the field of functional physiological morphology.

Pyrene derivatives bearing substituents at positions 1, 3, 6, and 8 find numerous applications, as exemplified by their use in lasers, sensors, and bioimaging probes. However, these derivatives typically have point-symmetric or short-axially symmetric structures, whereas long-axially symmetric derivatives remain underexplored because of the difficulty in obtaining their precursor, 1,3-dibromopyrene. To address this problem, we herein synthesized 1,3-dibromopyrene from 1-methoxypyrene in an overall yield (71 % over four steps) considerably exceeding those of existing methods. 1,3-Dibromopyrene was converted into 13OPA, a long-axially symmetric pyrene dye with electron-donor (alkoxy) groups at positions 1 and 3 and electron-acceptor (formyl) groups at positions 6 and 8. 13OPA exhibited photophysical properties distinct from those of its point-symmetric and short-axially symmetric isomers, featuring a broad and strongly redshifted absorption, strong fluorescence with reduced sensitivity to protic solvents, and small dipole moment change upon photoexcitation. The derivatization of 13OPA into a Schiff base and its functionalization via Lewis acid-base pairing were also demonstrated. Thus, our work expands the design scope of pyrene-based molecules, particularly those used as emitters.

Bright polymethine dyes, typified by carbocyanines, are employed in various fluorescence techniques such as the 3D visualization of living cell morphology and the tracking of extracellular vesicles in the blood vessels of a zebrafish. However, they often exhibit low photostability, particularly for dyes with red-shifted absorption/fluorescence wavelengths due to extended polymethine length, and limited photofunctionality. This limitation restricts their utility in specific applications requiring high-power excitation and/or a wash-free approaches. This study introduces novel merocyanine dyes, MCPY3 and MCPY5, comprising a newly developed pyrene-fused dioxaborine and polymethine chain. Despite their minimal polymethine lengths, their absorption/fluorescence wavelengths reside in the red to near infra-red regions due to the substantial pi-conjugation system of pyrene. Moreover, they exhibit a considerably superior photostability to carbocyanine dyes and fluorogenic behavior between low (ON) and high (OFF) polar solvents, while maintaining brightness comparable to carbocyanine. Leveraging these advantages, the hydrophilic analogs of MCPY3, MCPY3S, were applied to two-photon microscopy imaging of the skin tissues on the finger of a living mouse. The dye clearly visualized the individual cell morphology in the epidermis and the elastin within the dermis, highlighting the potential of the new dye as a valuable tool for fundamental dermatological and histological studies. We have developed novel merocyanines, MCPY3 and MCPY5, based on pyrene-fused dioxaborine and polymethine chain. These dyes exhibit red to near-infrared emission depending on polymethine length, considerably superior photostability to carbocyanines, and fluorogenic behavior, along with high brightness. The hydrophilic and red-emissive derivative, MCPY3S, was successfully applied to fluorescence imaging of skin tissues on the finger of a living mouse.

Bacterial infections pose significant global health concerns, necessitating precise and sensitive detection methods. This study introduces a multifunctional probe for bacterial detection. Bifunctional magnetoplasmonic nanoparticles (NPs) serve a dual purpose as carriers for magnetic separation (MS) and enhancers for light scattering of the target bacteria. We achieved exceptional selectivity at the bacterial species level by bioconjugating them with bacteriophages (phages). Optimal phage coverage was carefully determined to enhance the capture efficiency of the target bacterial cells. Additionally, we identified the ideal mixing ratios of the phage probe to bacteria, ensuring efficient MS and high light-scattering intensity. The use of the phage probe enabled the successful separation of captured target bacteria from other bacterial strains in the sample mixture. Bacterial detection was accomplished through dark-field light-scattering imaging, eliminating the need for additional labeling with other probes. This method showed a remarkable sensitivity, achieving a detection limit of similar to 102 colony-forming units ml-1. This value is 2 orders of magnitude lower than that achieved using our previously reported technique using monofunctional plasmonic NPs. These phage probes hold promise for healthcare, biotech, and environmental monitoring owing to their adaptability to diverse bacterial species. Graphical Abstract This study introduces a novel multifunctional probe for selective bacterial detection using bioconjugated magnetoplasmonic nanoparticles (MagPlas NPs). Employing phage-modified NPs enhances bacterial species specificity through efficient magnetic separation and light scattering. The method achieves high sensitivity (limit of detection: 102 colony-forming units ml-1), promising versatile applications in healthcare, biotech, and environmental monitoring.

Current biomedical applications of nanocarriers are focused on drug delivery, where encapsulated cargo is released in the target tissues under the control of external stimuli. Here, we propose a very different approach, where the active toxic molecules are removed from biological tissues by the nanocarrier. It is based on the drug-sponge concept, where specific molecules are captured by the lipid nanoemulsion (NE) droplets due to dynamic covalent chemistry inside their oil core. To this end, we designed a highly lipophilic amine (LipoAmine) capable of reacting with a free cargo-aldehyde (fluorescent dye and 4-hydroxynonenal toxin) directly inside lipid NEs, yielding a lipophilic imine conjugate well encapsulated in the oil core. The formation of imine bonds was first validated using a push-pull pyrene aldehyde dye, which changes its emission color during the reaction. The conjugate formation was independently confirmed by mass spectrometry. As a result, LipoAmine-loaded NEs spontaneously loaded cargo-aldehydes, yielding formulations stable against leakage at pH 7.4, which can further release the cargo in a low pH range (4-6) in solutions and living cells. Using fluorescence microscopy, we showed that LipoAmine NEs can extract pyrene aldehyde dye from cells as well as from an epithelial tissue (chicken skin). Moreover, successful extraction from cells was also achieved for a highly toxic aliphatic aldehyde 4-hydroxynonenal, which allowed obtaining the proof of concept for detoxification of living cells. Taken together, these results show that the dynamic imine chemistry inside NEs can be used to develop detoxification platforms.

In extramammary Paget's disease (EMPD), Paget cells are sometimes detected outside the clinical border (subclinical extension) with a skipping pattern. Therefore, “mapping biopsy” is performed to evaluate the clinical border before complete resection. To increase the proportion of Paget cells in biopsy specimens, it is necessary to increase the number of biopsies. However, this is associated with increased pain and discomfort to the patient. The tumor border decision is also a critical factor for prognosis. If we could detect the EMPD tumor cells from the skin surface directly without cutting the patient skin, we could check numerous points where the mapping biopsy is necessary for detecting the tumor border, and it may bring a good prognosis with a skin surgery. Recently, we have established a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions.1, 2 The new technique is very good at describing the skin tissue structure in the epidermis and dermis, for example, acrosyringium, individual keratinocytes, and nerve fibre.1 Hence, to resolve the abovementioned issue, we evaluated whether a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions is applicable for mapping biopsy in EMPD. EMPD samples resected during surgery were used in this study. Briefly, skin tissue samples were collected via 2-mm disposal biopsy punches from six areas around the main EMPD lesion. After fixation with 4% paraformaldehyde in PBS for 30 min, the samples were processed for propidium-iodide staining, and optically cleared using the transparency-enhancing ilLUmination of Cleared organs to IDentify target molecules method (LUCID),3 and newly established push-pull pyrene probe bearing a ketone acceptor group (PK), which is a solvatochromic dye.4 The samples were evaluated by two-photon excitation fluorescent microscopy (TPM), as described previously.1 All procedures were performed with prior approval from the Ethics Committees of Ehime University Graduate School of Medicine. This study was conducted in accordance with the principles of the Declaration of Helsinki. As shown in Figure 1A, typical histopathological findings of EMPD tumor cells include clear cytoplasm and a “shotgun” pattern on haematoxylin and eosin staining (HE) in the epidermis. In the same sagittal slice view, TPM confirmed similar LUCID/PK staining of the EMPD tumor cells (Figure 1B). The tumor cells had a dark, clear cytoplasm and nucleus, and there were tumor nests in the epidermis (Figure 1B, Video S1). On horizontal slices, EMPD tumor cells could be detected in the epidermis (Figure 1C) and dermis (Figure 1D, Video S2), suggesting that this tumor was already an invasive carcinoma as opposed to an in situ lesion. The major advantage of this new method is the ability of deep imaging to show the whole area from the surface to the deep lesion. This new technique has several advantages over the routine pathological procedure with HE staining. First, although we could obtain cell and tissue information with slices only 5 μm thick per slide via HE staining, our method can determine histological information over a thickness of 100–500 μm, which allowed us to obtain a three-dimensional image of each tissue slice. In addition, the tissue slices do not require processing; the thin tissue can be directly observed after LUCID/PK staining. Second, preparing the HE pathological specimen usually takes at least 1 week because a number of procedures are required, including making a paraffin block, cutting slices, staining with HE, dehydration, and mounting. However, the new technique only requires that the sample be placed in the LUCID/PK staining cocktail for several hours (overnight if needed), with no requirement for histopathological preparation. Two-photon microscopy is a very useful application of multiphoton microscopy in dermatological studies.5 Conventional skin tissue slices have autoimmunofluorescence and the basic structure of the epidermis, which can reveal the dermis and skin appendages. However, the resolution of this procedure is inferior to conventional HE staining. Therefore, a new technique using TPM with immunohistochemistry was developed that can reveal the detailed structure of skin appendages.6 Two-photon microscopy with immunohistochemical analysis has already been applied to EMPD,7 and the results revealed the EMPD tumor border histologically. Compared to their method, our new technique requires only a LUCID/PK cocktail and TPM; there is no requirement for immunohistochemical staining or sample processing, such as making paraffin blocks or slices. Our procedure also has s higher resolution and is very easy to perform in any laboratory where TPM is available. Furthermore, confocal laser microscopy can detect the signal in tissue (<100 μm thick). Further experiments are underway to establish the non-invasive “mapping biopsy” technique. MM, RK, and YN involved in conceptualization; MM, RK, and TT involved in data curation; MM and RK involved in methodology and formal analysis, writing—original draft preparation, writing—review and editing, and funding acquisition; MM, RK, and TT involved in validation, investigation, and visualization; RK, TI involved in technical supervision and advisory. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/RycH7j. The authors have no conflict of interest to declare. This study was supported by JSPS KAKENHI Grant number 20 K08691, Grant-in-Aid for Scientific Research on Innovative Areas_Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support,” and AMED under Grant Number JP22ym0126810. Research data are not shared. Video S1. EMPD lesion in the epidermis with three-dimensional observation. Video S2. EMPD lesion scanned from the surface to the dermis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.

Infectious disease mortality has decreased due to effective drugs and healthcare. However, global health remains threatened by infectious diseases. New methods of rapid and accurate bacterial detection have attracted considerable attention. Fluorescence detection of whole bacterial cells offers high sensitivity, quantitative analysis, and simple operation. A highly fluorescent bioconjugated probe improves sensitivity and selec-tivity. This study presents a novel, bright fluorescent probe comprising a bacteriophage and a fluorescent nanoemulsion (fNE) as biorecognition and signal transduction elements, respectively. We demonstrate that fluorescence microscopy imaging using the S. aureus-specific phage, S13 & curren;-fNE (phage-fNE), detects S. aureus in the presence of E. coli or S. pseu-dintermedius, another closely related Staphylococci, in a highly selective manner. Furthermore, fNEs with high dye loadings exhibit considerably greater brightness compared to the fluores-cent dye alone, making them suitable for sensitive fluorescence imaging. Phage-fNEs can quantitatively detect S. aureus at 104-108 colony-forming units per milliliter (CFU mL11), with a limit of detection of 8 (c) 104 CFU mL11. This result is com-parable to the lowest value achieved by microscopic bacterial detection, with no preconcentration or enzymatic signal en-hancement methods used. Bioconjugated fNEs open new avenues for highly selective and sensitive fluorescent detection of bacteria.

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

Herein, we discuss a new pyrene-based push-pull dye (PC) and our investigation of its photophysical properties and applicability to biological studies. The newly synthesized dye exhibits highly polarity-sensitive fluorescence over a significantly wide range (i.e., the green to far-red region), accompanied by high fluorescence quantum yields (ΦFL > 0.70 in most organic solvents) and superior photostability to that of the commonly used Nile Red (NR) dye, which also fluoresces in the green to red region. When human prostate cancer cells stained with PC were imaged using a confocal laser scanning fluorescence microscope, PC was found to selectively stain the lipid droplets. Under the cell conditions where the formation of droplets was inhibited, PC could be distributed to both the remaining droplets and the intercellular membranes, which could be distinguished based on the fluorescence solvatochromic function of PC. Furthermore, PC efficiently stained normal human skin tissue blocks treated with a transparency-enhancing agent and enabled clear visualization of individual cells in each tissue architecture by means of two-photon fluorescence microscopy (2PM). Interestingly, PC provides bright 2PM images under tissue-penetrative 960 nm excitation, realizing much clearer and deeper tissue imaging than conventional pyrene dyes and NR. These results suggest that PC could replace several commonly used dyes in various biological applications, particularly the rapid and accurate diagnosis of tissue diseases, typified by biopsy.

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.

The therapeutic and diagnostic applications of nanoemulsions (NEs) are restricted by a lack of suitable methods to modify their surfaces, specifically the surfactant-stabilized oilwater interface. Herein, we developed a modification technique using a lipophilic nitrile N-oxide compound (LipoCNO). LipoCNO-loaded NEs underwent efficient 1,3-dipolar cycloaddition with functional molecules bearing a terminal C=C or COC bond under mild, catalyst-free conditions at the oilwater interface, thus producing surface-functionalized NEs that could be used without purification. To exemplify the applicability of this technique, NEs encapsulating fluorescent molecules (Cy3.5LP) and LipoCNO were surface functionalized with pheophorbide a (PpA), a singlet-oxygen-generating photosensitizer. These NEs acted as light-harvesting nanoantennas, with the excitation energy gathered by Cy3.5LP in the NEs migrating to PpA on the NE surfaces via Forster resonance energy transfer. This system showed 7-18 times more efficient singlet oxygen generation than direct PpA excitation. Furthermore, PpA-functionalized dye-loaded NEs induced cancer cell death under photoillumination. These results highlight the utility of LipoCNO for functionalizing NE surfaces toward the realization of new biological applications.

We developed glycolipid-type amphiphiles with a small anilinochloromaleimide-based luminogen, H-AAC-C6-Ph-MS (monosaccharide, MS=alpha or beta-D-glucose (Glc), alpha or beta-D-galactose (Gal), or alpha-D-mannose (Man)) exhibiting aggregation-induced emission (AIE) features. Interestingly, the AIE properties of the amphiphiles depend on the epimerism of MS moiety. For instance, H-AAC-C6-Ph-alpha Man did not show fluorescence in the aggregate state, whereas the other amphiphiles exhibited fluorescence in the aggregate state. The findings strongly suggest that the design of molecular packing is important to control the AIE features.

Chemosensors are molecules capable of monitoring changes in the concentration, structure, or location of chemical species based on a detectable physical signal and can therefore be used in quantitative analysis or for the monitoring and/or visualization of targeted analytes (ions, biomolecules, organelles, etc.). Besides the analyte itself, chemosensors can be used indirectly to observe chemical reactions, biological events, or specific phases in materials where the analyte appears (e.g., the concentration of reactive oxygen species in mitochondria is related to apoptosis). As functional dyes show inherent optical properties such as photon absorption/emission at distinct wavelengths, they are potential optical signal indicators in chemosensors. In fact, in the more than 150 years since F. Goppelsrönder invented a morin-based chemosensor to detect aluminum anion using fluorescence signal (Wu et al. in Chem Soc Rev 46:7105–7123, 2017), a number of such “fluorescent chemosensors” have been developed. Some of them are widely used in practical scientific fields such as biology, physiology, pharmacology, food chemistry, and environmental chemistry, as well as in industrial and military/defense fields. This chapter describes the representative principles and molecular designs of fluorescent chemosensors, and several historically important progresses are introduced. The reason to limit the discussion to fluorescence is that fluorescence-based techniques generally exhibit superior sensitivity to absorption-based ones, and therefore many dyes have been reported for the development of fluorescent chemosensors.

The push-pull solvatochromic pyrene derivatives PA and PK have been applied to the study of model membrane vesicles, cells and purified human serum lipoproteins, using both confocal fluorescence microscopy and fluorescence spectroscopy. These polarity-sensitive probes provide information similar to that obtained by Laurdan or Prodan, i.e. mainly lipid order in biomembranes, but they have the essential advantage of being excitable by a standard 405 nm laser light, bypassing the use of multiphoton excitation. In addition, they are brighter and much more photostable than those dimethylamino naphthalene derivatives. Our results with model membrane spectroscopy (multilamellar vesicles) and with microscopy (giant unilamellar vesicles) showed the capacity of PA and PK to report differently on liquid-disordered, liquid-ordered and gel phase bilayers. Moreover, a ratiometric parameter, the Red/Blue Intensity Ratio (RBIR) could be used for inter-domain, inter-vesicle and even inter-technique comparison, and the appropriate microscopy-spectroscopy conversion coefficients could be estimated. In studies at the cellular level, PA probe stained almost exclusively the plasma membrane of red blood cells, revealing its high degree of lipid order. Using Chinese Hamster Ovary cells PA was shown to be an excellent probe for the detection of cytoplasmic lipid droplets, superior to Nile Red in that PA provides simultaneously a detailed information of membrane order in the whole cell, in which the lipid droplets appear with a very good contrast. Moreover, spectrofluorometric data of PA-stained serum lipoproteins indicated an essentially identical value of RBIR for lipid droplets and for high-density lipoproteins.