Spectrofluorometric studies of the lipid probe, nile red Phillip Greenspan and Stanley D. Fowler Department of Pathology, School of Medicine, University of South Carolina, Columbia, SC 29208 Abstract We found that the dye nile red, 9-diethylamino-5H- benzo[cu]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry 1985. u. Cell. Biol. ing, we examined the fluorescence properties of nile red in the 100 965-973). To understand the selectivity of the stain- presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of shifted nile red to However, the fluorescence of nile red was quenched in aqueous shorter wavelengths with decreasing solvent polarity. medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemul- sions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluores- cence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin.MThese results demonstrate that nile red fluorescence accompanied by a red in spectral blue shift reflects the presence of nile a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. serves as excellent fluorescent lipid probe. Nile red thus an and S. -Greenspan, P., D. Fowler. Spectrofluorometric studies of the lipid probe, nile red. J. Lipid Res. 1985. 26: 781-7. Supplementary key words 9-diethylamino-5H-benzo[cr]phenaxazine-5- one neutral lipids fluorescent hydrophobic probe lipid histo- chemistry lipoproteins membranes phospholipids Nile red is a red phenoxazone dye that is present in trace amounts in commercial preparations of the lipid stain nile blue (1). Nile red is responsible for the meta- chromatic coloring of tissue lipids by staining procedures that utilize the nile blue dye (2). We have recently synthe- sized nile red and found that it can be used on living cells as a fluorescent stain for the detection of intracellular lipid droplets by fluorescence microscopy (3). An example of the application of nile red as a lipid stain is illustrated in Fig. 1. Stained cultured macrophages previously incu- bated with acetylated low density lipoprotein to induce intracellular lipid droplets, as described by Goldstein, Brown and colleagues (4, 5), are shown in Fig. la; stained control macrophages are shown in Fig. lb for comparison. Numerous large fluorescent bodies are evident in the cells incubated with acetylated low density lipoprotein, thus demonstrating that the nile red staining reveals cytoplas- mic lipid droplets. We have also successfully employed nile red staining of these same cells to quantitate by flow cytofluorometry the degree of cellular lipid overloading (3). We found that the best selectivity for cytoplasmic lipid droplet staining is obtained when nile red-treated cells are examined for yellow-gold fluorescence (528 nm) rather than for red fluorescence (>610 nm) (3). With the latter conditions, a diffuse staining of the entire cell is observed; while with the former, a more selective staining of lipid droplets is obtained. To understand these observations, we conducted spectrofluorometric studies on the interaction of nile red with various lipid models including phospho- lipid vesicles, serum lipoproteins, hepatic microsomes, and isolated adipocytes. Our investigations, which we present here, demonstrate that nile red acts as a hydro- phobic probe in which the fluorescence maxima exhibit a blue-shift proportional to the hydrophobicity of the en- vironment. This, and the preferential solubility of the dye in lipid, account for the selective staining of the intracellu- lar lipid droplets by nile red. Our studies also suggest that the unique fluorochromic properties of nile red could be of great use to lipid chemists as a tool to investigate the hydrophobic domains of lipoproteins and cell membranes. A preliminary report of this work has been presented previously (6). MATERIALS AND METHODS Chemicals Nile blue chloride (CI 51180, dye content approximately 95%) was purchased from Sigma Chemical Co. Phospha- tidylcholine (egg lecithin) was obtained from Lipid Prod- ucts, Surrey, England; the other lipids were purchased from Sigma Chemical Co. Metrizamide was purchased from Nyegaard and Co., Oslo, Norway. Organic solvents were reagent grade and were obtained either from Fisher Scientific or Sigma Chemical Co. Bovine serum albumin Journal of Lipid Research Volume 26, 1985 781 Downloaded from www.jlr.org by guest, on July 21, 2013Fig. 1. Nile red fluorescence of lipid-loaded mouse peritoneal macrophages. (a) Cultured macrophages previously incubated 40 hr with acetylated low density lipoprotein (50 pg protein/ml) in 10% fetal calf serum to induce production of cytoplasmic lipid droplets (4, 5). (b) Control cultured macrophages incubated a similar period in the prucnce of 10% fetal calf serum only. Prior to staining, the cells were fixed with 1.5% glutaraldehyde in phosphate-buffered saline and washed with buffered saline. Nile red concentration: 100 nglml. Excitation wavelength, 450-500 nm; emission wavelength, >528 nm. Magnification, ~1040. (essentially fatty acid-free) was purchased from Sigma were centrifuged for 15 min at 20,000 rpm in a Beckman Chemical Co. and further purified from fatty acids by the Ti-75 rotor (Reckman Instruments, Inc.) to remove any method of Chen (7). titanium particles produced by the sonication. Microemulsions of phosphatidylcholine and trioleoyl- Preparation of nile red glycerol (molar ratio 1:l) were prepared by a 1-hr sonica- Nile red' was prepared from nile blue by dissolving tion at 45OC as described by Burrier and Brecher (12). 0.92 g of the latter in 500 ml of 0.5% H2SOI and boiling The resultant mixture was centrifuged for 1 hr at 40,000 it under reflux for 2 hr (Fig. 2) (8). After cooling, nile red Tm in a Be'kman Ti-75 rotor* The top 1-2 m1 Of solution was separated by repeated extraction into equal volumes was removed and the remaining infranatant was recentri- of xylene until only a faint pink color appeared in the fuged 'or l6 hr at 40,000 Vm. The upper ' m1 Of the organic phase. The organic solvent was removed by flash resu1ting suPernatant was used as Our microemulsion evaporation, leaving a dark purple residue (0.18 g). The Preparation. The dimYristoYIPhosPhatidY1choline and latter was identifie,, as nile red on the basis of ultraviolet cholesteryl linoleate (molar ratio 1:l) microemulsion was and infrared spectra (9, 10). The compound exhibited a Prepared as described bY Ginsburg, Smal1, and Atkinson sharp melting point (192-193OC). The preparation ap- (13) with minor modifications, and was generously Pro- peared to be >98% pure based on high pressure liquid vided to us by Dr. John Parks, Bowman Gray School of chromatography in three solvents: acetonitrile-water Medicine, Winston-Salem, NC. 9O:lO (v/v); acetonitrile-tetrahydrofuran 80:20 (v/v); and a linear gradient of acetonitrile-0.1% acetic acid in water Isolation of lipoproteins (25:75 (4.) initial condition to 75:25 (v/v) final condition) Lipoproteins were isolated from human plasma by on a pBondapak C18 column (Waters Associates, Inc.). sequential UltracentrifWPtion in KBr (14). The IiPOPro- teins were exhaustively dialyzed against saline containing Preparation of lipid vesicles and microemuleions 0.01% EDTA, pH 7.4. The purity of the preparation was Unilamellar vesicles of phosphatidylcholine (egg leci- confirmed by cellulose acetate gel electrophoresis. thin or dimyristoylphosphatidylcholine), or phosphatidyl- choline (egg lecithin) and trioleoylglycerol (molar ratio Isolation of adipocytes 66:l) were prepared by sonication at 45OC for 12 min as Adipocytes were kindly provided by Dr. Donald o. described by Brecher et al. (11). Phosphatidylcholine (egg lecithin)/cholesterol (molar ratio 1:0.8) vesicles were pre- pared by sonication at 45OC for 1 hr. These preparations lIn view of its heterocyclic StructUrr, M handled nile red as a careinom gen, though no evidence exists that such is the case. 782 Journal of Lipid bearch Volume 26, 1985 Downloaded from www.jlr.org by guest, on July 21, 2013CI- NILE BLUE NILE RED V Fig. 2. Oxidation of nile blue to form nile red. After boiling nile blue in dilute HzSO,, the nile red produced is extracted into xylene. Nile blue, 5-amino-9-diethylamino-benzo[a]phenoxazie, is a basic dye, the chloride and sulfate salts of which are blue while the fm base is red. Nile red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is a neutral dye, red in color, and intensely fluorescent in the visible range. Allen, Department of Pharmacology, School of Medicine, wavelength settings of 517 and 584 nm, respectively. A University of South Carolina. The cells were obtained 10-nM solution of nile red in iso-amylacetate was em- from epididymal fat pads of adult rats by collagenase ployed as a standard. The fluorescence was found to be enzymic digestion as described by Lech and Calvert (15). linearly proportional to nile red content up to a concen- tration of 15 pM. Nile red concentrations in organic sol- Preparation of microsomal membranes vents were similarly measured following evaporation of A microsomal (P) fraction was prepared from a rat liver the solvent and dissolving the dye in 3 ml of iso-amyl- homogenate as described by de Duve et al(16). The prepa- acetate. ration was diluted to 1 mg of protein/ml in 100 mM Na carbonate, pH 11.5, incubated at O°C for 1 hr, and then Other assays centrifuged 1 hr at 50,000 rpm in a Beckman Ti-75 rotor. Protein was determined by the method of Lowry et al. The pellet was resuspended with a Dounce homogenizer (18) using bovine serum albumin as the standard. Phos- in 0.85% NaCl (pH 7.4). The carbonate treatment effec- pholipids were quantitatively extracted by the method of tively strips ribosomes and peripheral proteins from the Bligh and Dyer (19); they were ashed and the total phos- membranes (17). phate was measured according to Ames and Dubin (20). Fluorescence spectroscopy RESULTS Excitation and emission fluorescence spectra were de- termined with a Perkin-Elmer 650-40 fluorescence spec- Physical properties of nile red trophotometer (Perkin-Elmer Corp) equipped with a 150 Nile red is a benzophenoxazone dye (Fig. 2); it is some- watt xenon lamp, concave grating monochromators, and times referred to in older literature as nile blue A-oxazone. a red sensitive Hamamatsu R928 photomultiplier tube. Table 1 lists some properties of nile red helpful for its Except as indicated, spectra were recorded at room tem- identification and use. The dye dissolves in a wide range perature with both slits set at 5 nm. The spectra presented of organic solvents, but negligibly in water. It is also sub- are partially corrected by use of a ratio quantum counter stantially less soluble in heptane and higher alkanes than system (ratio mode). They are shown plotted as wave- in other solvents such as xylene, chloroform, or acetone. length versus normalized fluorescence intensity in which The partition coefficients of nile red in several organic the y-axis scale units have been adjusted to place maxi- solvents relative to water were found to be approximately mum peak heights at 70% of full chart scale. The relative 200 at 4OC. An exception was n-heptane for which the fluorescence intensity of nile red in the presence of the partition coefficient was found to be 58. Similar ratios various samples was obtained after subtraction of both the were obtained when the solvent partitions were performed autofluorescence of the samples and the fluorescence in- at room temperature. tensity of nile red alone in buffer. Nile red is a dye that exhibits solvatochromism (9), its absorption band varies in spectral position, shape and Assay for nile red intensity with the nature of the solvent (24). The dye is The content of nile red in aqueous solutions could be also highly fluorescent in organic solutions.* The fluores- determined by extraction of the dye into 3 ml of iso- cence intensity of nile red approaches that of rhodamine amylacetate with vigorous vortexing followed by brief B (Table 1). centrifugation. The iso-amylacetate solvent is particularly useful if the aqueous sample contains protein. The fluores- cence intensity of nile red in iso-amylacetate was mea- *A variety of phenoxazone dyes have been examined for their Auores- sured using 10-nm slit widths at excitation and emission cence, and, of those studied, nile red was found to be the most intensely fluorescent (25). Greenspan and Fowler Nile red: spectrofluorometric studies 783 Downloaded from www.jlr.org by guest, on July 21, 2013TABLE 1. Phvsical DroDerties of nile red Molecular formula\" CZ~HL8N~OZ, m.w. = 318 Melting point 192-193OC Solubility 1 mg/ml acetone 62 pg/ml n-heptane <1 pg/ml water Partition coefficient (4OC) xylene/water 210 chloroform/water 196 iso-amylacetate/water 198 n-heptane/water 58 Absorption maximab (HCCI:, solvent) ultraviolet, 2 nm; visible, 538 nm Infrared maxima (HCC13 solvent)' 1000, 1105, 1265, 1305, 1550, 1580, 1612 (C=O), 1710 cm-' Fluorescence intensity relative to rhodamine B (HCCI, solvent)d 0.36 \"For reviews of the chemistry of phenoxazone dyes see refs. 21 and 22. For separation of nile red from nile blue by thin-layer chromatography, see ref. 23. bSolvent dependent, visible absorption maxima range from 487 nm (isooctane) to 5 nm (formamide) (9, 23). 'Similar to data of Stuika et al. (10). dSolutions (0.5 pM) measured at the excitation/emission maxima of nile red and rhodamine B, which were 543/595 nm and 554/579 nm, respectively. Solvent effects on the fluorescence spectra of nile red The emission spectra of many fluorescent compounds, especially those containing polar substituents on the aromatic rings, are known to be sensitive to the chemical and physical properties of solvents (26). As demonstrated in Fig. 3, nile red is a fluorophore of this kind. Table 2 summarizes the fluorescence properties of nile red in eight different solvents. Both the excitation and emission maxi- ma shift over a span of 100 nm, the colors ranging from golden yellow to deep red. The red shift of the maxima parallels increases in the dielectric constant of the solvent as expected for general solvent effects on fluorescence spectra (26). The relative fluorescence intensity of the dye is equal in all the nonpolar solvents examined. The fluores- cence intensity of nile red in water, however, is 40-fold less. Examination of the fluorescence spectra of nile red in organic solvents (Fig. 3) shows alterations in the shape of the spectral curves. This suggests the presence of specific solvent effects, such as hydrogen bonding or some other interaction. Confirming this notion, addition of small amounts of ethanol (0.1-1.0%) to a heptane solution of nile red resulted in red shifts in the fluorescence emission maxima of up to 50 nm (data not shown). Nile red fluorescence in the presence of lipid In view of likely complex interactions of nile red with organic solvents, we chose a more empirical approach to understand selective staining of lipids by nile red. We 784 Journal of Lipid Research Volume 26, 1985 began by examining the fluorescence of the dye in the presence of aqueous suspensions of lipids. Similar to the fluorescence of nile red in various solvents, the excitation and emission spectra were different for various lipid preparations (Fig. 4, Table 3). For example, the fluores- cence spectra of nile red in the presence of phosphatidyl- choline (egg lecithin) vesicles are sharp and symmetrical, with excitation and emission maxima of 549 nm and 628 nm, respectively (Fig. 4a). In contrast, the fluorescence spectra of nile red in the presence of a phosphatidylcho- line-trioleoylglycerol 1:l microemulsion are shifted toward the blue region of the spectrum; the emission maximum of the microemulsion is at 576 nm, although the emission spectrum exhibits a broad shoulder that extends almost to the nile red maximum associated with phosphatidylcholine vesicles alone. Fig. 4b shows the interaction of nile red with a micro- emulsion composed of cholesteryl linoleate and dimyris- toylphosphatidylcholine. The fluorescence spectra of nile red in the presence of dimyristoylphosphatidylcholine vesicles alone is nearly identical to that recorded for egg lecithin vesicles (Fig. 4a). At 25OC, a small but significant blue shift is seen in the nile red fluorescence maximum in the presence of the dimyristoylphosphatidylcholine-choles- teryl linoleate 1:l microemulsion, indicative of an inter- action between the dye and the liquid crystalline choles- teryl linoleate. This interaction can be enhanced by heating the sample to 46OC. At this temperature, the cholesteryl linoleate core is in an isotropic liquid, dis- ordered state (28). The fluorescence spectrum of nile red is then markedly shifted toward the blue region in a 460 500 000 560 000 700 WAVELENGTH (nm) Fig. 3. Excitation and emission fluorescence spectra of nile red in iso- octane and ethanol. A 1 @ml solution of nile red in each solvent was used. The excitation and emission spectra of nile red were recorded at their corresponding emission or excitation maxima. Downloaded from www.jlr.org by guest, on July 21, 2013TABLE 2. Fluorescence properties of nile red in various solvents Fluorescence of nile red-stained adipocytes and Relative microsomal membranes Didectric Excitation Emission Fluorescence Solvent Constant' Maximum Maximum Intensity As a final test of the fluorochromic differences attain- nm nm able with nile red coloring, we compared nile red staining Water 78.5 591 657 of two distinctly different biological systems: adipose Ethanol 24.3 629 tissue cells laden with neutral lipids, especially triacylglyc- Acetone 20.7 559 608 355 18 erols, and microsomal membranes in which phospholipid Chloroform 595 687 iso- Amylacetate 4.8 536 543 517 584 748 690 is the principal lipid class present. The fluorescence spec- Xylene 2.4 - 565 tra of the nile red-stained preparations are shown in Fig. n-Dodecane 2.0 523 531 685 n-Heptane - 492 484 529 739 585 5. Nile red excitation and emission fluorescence maxima Nile red was analyzed at a concentration of 1 pg/ml in all solvents. Excitation and emission maxima were determined and the relative I EXCITATION SPECTRA 1 1 fluorescence intensity in each solvent was measured at the corresponding - EMISSION SPECTRA PCTO excitation and emission maxima. PC 'Derived from (27). manner nearly identical to the spectral shift observed for the nile red fluorescence of the phosphatidylcholine-tri- oleoylglycerol 1:l microemulsion. The temperature-depen- dent blue shift in the fluorescence spectra of nile red was not observed with dimyristoylphosphatidylcholine vesicles alone nor with the lipid preparations listed in Table 3. As indicated in Table 3, the fluorescence properties of nile red in the presence of lipid suspensions are dependent on the concentration of nile red employed: u) the emission maxima vary depending on the amount of nile red used, Z-4 0and b) the relative fluorescence intensities observed in the LL EXCITATION SPECTRA EMISSION SPECTRA presence of all the lipid preparations are dependent on the DUPC CL (1 1) ! DYPC CL(1 1) nile red concentration. We found that, at the concentra- tions of lipid used in this study, the fluorescence obtained increased linearly with dye concentration up to 0.5 pg of nile red/ml. Nile red fluorescence in the presence of proteins Nile red also fluoresces when combined with serum lipoproteins or proteins bearing hydrophobic domains. As shown in Table 4, the fluorescence of nile red bound to the neutral lipid-rich lipoproteins is strong. The excita- tion and emission maxima of nile red fluorescence in the presence of both very low density and low density lipo- proteins are shifted to shorter wavelengths compared to 400 600 eo0 600 a00 nile red fluorescence in the presence of high density lipo- WAVELENGTH (nm) protein. Defatted albumin also induces nile red fluores- Fig. 4. Excitation and emission fluorescence spectra of nile red in the cence, but the intensity is one-tenth that attained with presence of phospholipid vesicles or phospholipid-neutral lipid micro- very low density lipoprotein. In contrast, immunoglobulin emulsions. (A) Nile red fluorescence spectra in the presence of phospha- G and gelatin, two hydrophilic proteins, did not induce tidylcholine (egg lecithin) (TG) (PC) vesicles or phosphatidylcholine-trioleoyl- glycerol 1:l (molar ratio) microemulsion. (B) Nile red fluorescence nile red fluorescence. At the highest concentrations of nile spectra in the presence of dimyristoylphosphatidylcholine (DMPC) red used (1.66 pg/ml), distinct differences in the fluores- vesicles or dimyristoylphosphatidylcholine cholesteryl linoleate (CL) 1:l cence intensity of nile red with the different proteins are (molar ratio) microemulsion. The lipid suspensions were diluted to a concentration of 250 nmol of phosphorus/ml in phosphate-buffered observed. The very low density lipoproteins, carrying the saline. Five pl of an acetone solution of nile red (100 pg/ml) was mixed greatest mass of lipid, produce the most intense nile red with 3 ml of the lipid suspension and the fluorescence spectra were fluorescence. recorded. The excitation and emission spectra were measured at their corresponding emission or excitation maxima. Grempan and Fowler Nile red: spectrofluorometric studies 785 Downloaded from www.jlr.org by guest, on July 21, 2013TABLE 3. Fluorescence of nile red in the presence of aqueous suspensions of lipid 0.166 pg Nile Redlml 1.66 gg Nile Red/ml Excitation, Relative Excitation, Relative Emission Maxima Fluorescence Emission Intensity Maxima Fluorescence Intensity nm nm Phosphatidylcholine vesicles 549,628 19 548,633 225 Phosphatidylcholine-trioleoylglycerol 66: 1 vesicles 549,629 23 548,633 207 Phosphatidylcholine-trioleoy lgly cero1 1 : 1 microemulsion 529,576 23 534,623 210 Phosphatidylcholine-cholesterol 1:0.8 vesicles 549,605 22 550,621 206 Aqueous suspensions of lipid were prepared as described in Methods. The phosphatidylcholine was egg lecithin. Each preparation was diluted with phosphate-buffered saline to give a final concentration of 250 mol of phosphomdml. Five pl of an acetone solution of nile red (0.1 mg/ml or 1 mg/ml) were mixed with 3 ml of a lipid suspension and the fluorescence spectra were recorded. The relative fluorescence intensity of nile red in the presence of each model lipid was measured at the corresponding excitation and emission maximum. were recorded at 521 nm and 582 nm, respectively, for amino] naphthalene) (30): a) quenched fluorescence in stained adipocytes (Fig. 5a). In contrast, the maxima aqueous media, and 6) blue shift of the fluorescence spec- occur at 540 and 624 nm, respectively, for nile red-stained trum with decreasing polarity of the solvent environment. microsomes (Fig. 5b). The spectra of the latter are similar Both TNS and PRODAN have been widely used as hy- to those observed for phosphatidylcholine vesicles colored drophobic probes. Our studies show that nile red can be with nile red whereas the former are similar to phospha- similarly used as a hydrophobic probe for the detection of tidylcholine-trioleoylglycerol 1:l microemulsions treated neutral lipids or mixtures of lipids and proteins that with nile red (Fig. 4a). create a hydrophobic environment. We found that nile red will fluoresce intensely in the presence of phospholipid vesicles and that a blue shift in the fluorescence spectra DISCUSSION will occur if neutral lipids such as cholesteryl esters or triacylglycerols are incorporated with the phospholipid to Nile red exhibits spectral properties similar to the form microemulsions. Serum lipoproteins also induce nile fluorescent probes TNS (2-p-toluidinylnaphthalene-6-sul- red fluorescence, especially very low density lipoprotein fonate) (29) and PRODAN (6-propionyl-2-[dimethyl- and low density lipoprotein which also cause a spectral TABLE 4. Fluorescence of nile red in the presence of proteins 0.166 pg Nile Redml 1.66 pg Nile Redd Excitation, Relative Excitation, Relative Emission Emission Protein Maxima Fluorescence Intensitv Maxima Fluorescence Intcnsitv nm nm Very low density lipoprotein\" 530,576 33 526,586 424 Low density lipoprotein 53 1,584 33 532,603 286 High density lipoprotein 543,610 27 543,619 165 Albumin 553,620 3 568,0 40 Gelatin - 593,657 Immunoglobulin G - - - 1 Hepatic microsomal membranes 540,624 537,632 162 With the exception of hepatic microsomal membranes, proteins were diluted to a concentration of 100 pg/ml with phosphate-buffered saline. Hepatic microsomal membranes were diluted to the same concentration with phosphate- buffered saline containing 12% metrizamide. Five pl of an acetone solution of nile red (0.1 mg/d or 1 mg/ml) were mixed with 3 ml of the protein solution and the fluorescence spectra were recorded. The relative fluorescence intensity of nile red in the presence of each protein was measured at the corresponding excitation and emission maxima. \"Isolated as lipoproteins of density lipoprotein. < 1.019 g/ml, this preparation may also contain some intermediate density 786 Journal of Lipid Research Volume 26, 1985 Downloaded from www.jlr.org by guest, on July 21, 2013> aA ADIPOCYTES 8 B HEPATIC MICROSOMAL MEMBRANES 400 500 600 700 400 500 800 700 WAVELENGTH (nm) Fig. 5. Excitation and emission fluorescence spectra of nile red-stained rat adipocytes and hepatic microsomal membranes. (A) Aliquots of adipocytes, to which nile red was added to obtain a final concentration of 10 pg/ml, were placed in a 96-well black cell culture plate (Dynatech Corp.). The cells were allowed to float to the solution surface. The excitation (left) and emission (right) spectra were then recorded by measuring surface fluorescence using IO-nm slit width settings with a Perkin-Elmer TLC scanning attachment. The dashed lines represent the nearly superimposable fluorescence spectra of adipocytes in the absence of nile red and of an aqueous solution of nile red alone. (B) Hepatic microsomal membranes were diluted to a concentration of 100 pg protein/ml in phosphate-buffered saline containing 12% metrizamide (to slow sedimentation of the vesicles). Five pl of an acetone solution of nile red (100 pg/ml) was mixed with 3 ml of microsomal membranes and the excitation (left) and emission (right) fluorescence spectra were recorded using 5-nm slit width settings. The dashed lines represent the fluorescence of microsomal membranes in the presence of metrizamide and in the absence of nile red. All excitation and emission spectra were determined at their corresponding emission or excitation maxima. blue shift. The selectivity of the spectral shifts, even in microemulsions, this effect was most pronounced. The complex systems, is illustrated by the difference in fluores- physical structure of the particles with which the nile red cence spectra of nile red-stained adipocyte (triacylglycerol- interacts may explain in part the shifts seen. At low con- rich) compared to microsomal membranes (phospholipid- centrations of nile red, both the core hydrophobic and rich) (Fig. 5). coat amphipathic components could be interacting with It is important to recognize that nile red fluorescence is nile red, giving an emission maximum of approximately an indication of lipid hydrophobicity and physical state 570 nm. With increasing concentrations of nile red, the and not just chemical structure. Thus, if the temperature hydrophobic core may become saturated and the excess is raised to 46OC, microemulsions containing cholesteryl nile red could then only interact with the coat phospho- ester produce blue shifts in the nile red spectra compara- lipids or hydrophobic proteins. The emission maximum ble to those obtained with microemulsions containing should then shift toward that observed with phosphatidyl- triacylglycerol. Nile red will even fluoresce in the presence choline vesicIes and microsomal membranes (620-630 of cholesterol crystals (unpublished observations) and nm), just as is observed (Tables 3 and 4). Other observa- cholesterol incorporated into phosphatidylcholine vesicles tions support this notion. u) The solubility of nile red in induces a blue shift of the nile red fluorescence. Nile red n-heptane was found to be approximately 1/16 of that in fluorescence is also induced with oleic acid in aqueous acetone (Table 1). Thus, nile red could more easily attain medium, the fluorescence spectra at acid pH appearing at saturation in an extremely nonpolar environment. b) shorter wavelength (approximately 30 nm) than the fluores- Patton et al. (31) have described limited solubilities of cence spectra of the mixture at alkaline pH (unpublished hydrophobic molecules in liquid fat. c) Comparisons of observation). the nile red fluorescence intensities of high density lipo- The emission and excitation maxima of nile red in lipid proteins under spectral conditions that preferentially suspensions and in various proteins appear to be depen- detect strongly hydrophobic domains suggest that nile red dent upon the concentration of the dye. The emission saturation of core lipids does occur (3). From the above maxima at the lower concentration of the dye tend to be discussion it is clear that the dye concentration depen- of a shorter wavelength than that observed in the presence dence of nile red fluorescence must be carefully considered of higher concentrations. In the presence of low density when interpreting nile red staining of lipoproteins, mem- lipoproteins or phosphatidylcholine-trioleoylglycerol 1:l branes, or whole cells. Greenspan and Fowler Nile red: spectrofluorometric studies 787 Downloaded from www.jlr.org by guest, on July 21, 2013In conclusion, the results of the studies described here provide a rational basis to explain the selective fluorescent staining of cellular lipids by nile red. When the fluores- cence of nile red-stained cells is examined at wavelengths of 580 nm or less, the fluorescence of nile red interacting with extremely hydrophobic environments (i.e., neutral lipid droplets) is maximized while the fluorescence of nile red interacting with cellular membranes is minimized (Fig. 1). This is not the case when the fluorescence is de- tected at emission wavelengths greater than 590 nm. Under the latter conditions, the interaction of nile red with phospholipid-rich intracellular membranes becomes a more important determinant of cellular fluorescence. By proper selection of spectral conditions, nile red should be a powerful fluorescent reagent to examine intracellular lipid loading in various pathologic conditions such as atherosclerosis, genetic lipid storage diseases, and drug- induced phospholipidoses. I The authors express their thanks to Ms. Indhira Handy, Susan Maness, and Janice Warfel for excellent technical assistance and to Mrs. Sarah Gable of the University of South Carolina School of Medicine Library for helpful literature searches. The authors are also very grateful to Dr. Donald 0. Allen, School of Medi- cine, University of South Carolina, for providing the adipocytes and to Dr. John Parks, Bowman Gray School of Medicine, for providing the preparation of cholesteryl linoleate-phospholipid microemulsions. These studies were supported by Public Health Service Grant HL-29940 from the National Heart, Lung, and Blood Institute, and by grants from the Juvenile Diabetes Foundation and the American Health Assistance Foundation. This research support is gratefully acknowledged. Manuscript received 12 December 1984. REFERENCES 1. Conn, H. J. 1953. Biological Stains. Sixth Edition. Williams and Wilkins Co., Baltimore. 2. Adams, C. W. M., and In 0. B. Bayliss. 1975. Lipid histo- chemistry. Techniques of Biochemical and Biophysical Morphology. Vol. 2. D. Glick and R. M. Rosenbaum, editors. Wiley Interscience, New York. 99-156. 3. Greenspan, P., E. P. Mayer, and S. D. Fowler. 1985. Nile red: a selective fluorescent stain for intracellular lipid drop- lets. J Cell Biol. 100: 965-973. 4. Goldstein, J. L., Y. K. Ho, S. K. Basu, and M. S. Brown. 1979. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc. 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