Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red The fluorescent dye Nile red was used as a probe for the sensitive detection of large , denatured aggregates of the model protein β-galactosidase -LRB- E. coli -RRB- in solution . Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein 's unfolding temperature of 57.4 °C , and the presence of aggregates in heated solutions was confirmed by static light scattering . Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum -LRB- λmax -RRB- from 660 to 611 nm , and to an increase of fluorescence intensity . Time-resolved fluorescence and fluorescence correlation spectroscopy -LRB- FCS -RRB- measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm . By steady-state fluorescence measurements , it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution . The comparison with size exclusion chromatography -LRB- SEC -RRB- showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red . Large aggregates were not detected by SEC , because they were excluded from the column . The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC , and can detect the formation of large protein aggregates at early stages . Introduction Among the biological macromolecules , proteins have become an important source of active substances for medical purposes . Insulin and growth hormone are two prominent examples of the many proteins used in medicine nowadays -LSB- 1 -RSB- . Many protein formulations can cause immune reactions in patients , and the presence of protein aggregates , even in minute amounts , has been identified as an important factor leading to the breaking of immune tolerance -LSB- 2 , 3 -RSB- . Large protein aggregates are regarded as particularly immunogenic -LSB- 4 -RSB- , and for this reason , analytical procedures for their sensitive and selective detection are important . The most widely used technique for analyzing aggregates in protein formulations is size exclusion chromatography -LRB- SEC -RRB- -LSB- 2 -RSB- . However , large protein aggregates of more than about 5 million Da are out of the separation range of SEC and can even be excluded from separation columns . Moreover , protein-column interactions may cause aberrant elution leading to erroneous interpretations . Field flow fractionation and analytical ultracentrifugation have a greater capacity than SEC for detecting high molecular weight protein aggregates -LSB- 5 , 6 -RSB- , but until now , these techniques are not readily available in every laboratory . Analysis by light scattering techniques -LSB- 7 -RSB- and Fourier transform infrared spectroscopy -LRB- FTIR -RRB- -LSB- 8 -- 10 -RSB- is experimentally easy in the sense that these techniques do not rely on physical separation steps . However , the former is prone to artifacts when aggregates are present at low concentrations , and the latter is not very sensitive . Fluorescence spectroscopy with probes covalently labeled to proteins has been used for the direct detection of aggregated species -LSB- 11 -RSB- , but this approach suffers from the problem that the modified protein may exhibit an altered aggregation behavior . Methods without protein labeling are rarer , but environment sensitive fluorescent dyes can be useful for detecting aggregated structures . For example , Congo red is used for the detection and identification of amyloid fibrils -LSB- 12 , 13 -RSB- . Another potentially interesting dye is Nile red , which is established for the detection of hydrophobic areas on the surfaces of proteins -LSB- 14 -- 17 -RSB- . Recently , this dye has been used for the detection of aggregates of an IgG1 recombinant humanized monoclonal antibody , as well as human calcitonin fibrils by fluorescence microscopy -LSB- 18 -RSB- . The fluorescence of Nile red is strongly dependent on the polarity of its environment . Decreasing polarity leads to a substantial blue shift of the absorption and emission maxima , as well as a marked increase of quantum yield and fluorescence lifetime . This has been related to the large dipole moment of the molecules in the excited state and the existence of a non-emissive twisted intramolecular charge transfer -LRB- TICT -RRB- state , which is more likely to be formed in polar than apolar environments -LSB- 19 , 20 -RSB- . It has been proposed that the TICT state is non-emissive because of fast , non-radiative transition to the triplet state -LSB- 19 -RSB- . Nile red is a hydrophobic molecule with low solubility in water of ∼ 1 μM -LSB- 21 -RSB- . Therefore , it has the tendency to partition into hydrophobic phases -LSB- 22 -RSB- . β-Galactosidase from Escherichia coli is used as a model protein in the present study . This tetrameric enzyme -LRB- MW = 465 kDa -RRB- consists of four equal subunits . It contains four catalytic sites for the hydrolysis of lactose or other β-galactosides to monosaccharides . For maximal enzymatic activity , the presence of Mg2 + or , alternatively , Mn2 + is required -LSB- 23 , 24 -RSB- . It has been shown that Mg2 + increases the unfolding temperature of β-galactosidase in aqueous solution . In the presence of Mg2 + , heat induced unfolding is irreversible . It coincides with activity loss and leads to the formation of denatured monomers , dimers , and aggregates -LSB- 25 -RSB- . In this study , steady-state and time-resolved fluorescence spectroscopy experiments with Nile red are used for the sensitive detection of large β-galactosidase aggregates , present in solution at concentrations in the low nanomolar range . Denaturation and aggregation of native protein is induced by incubating aqueous solutions below and above the experimentally determined unfolding temperature of 57.4 °C . The presence of aggregates in heat-treated solutions is confirmed by light scattering . Time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy -LRB- FCS -RRB- are used for analyzing the size of the aggregates detected by Nile red . The results obtained with Nile red are compared with results from SEC analysis . Materials and methods Materials Freeze-dried β-Galactosidase from Escherichia coli -LRB- protein content ∼ 80 % -RRB- was purchased from Fluka -LRB- Buchs , Switzerland -RRB- and stored at − 20 °C . Phosphate buffer -LRB- pH 7.4 -RRB- containing 55 mM sodium-phosphate and 10 mM MgCl2 was prepared with reverse osmosis water , and rendered isotonic by the addition of NaCl . Upon preparation , buffer solution was filtered through a 0.2 μm cellulose acetate filter -LRB- Schleicher and Schuell , Dassel , Germany -RRB- , and stored at 4 °C for at most 4 weeks . 0.2 μm HPLC filters were obtained from Alltech -LRB- Deerfield , IL -RRB- . Nile red -LRB- 9-diethylamino-5H-benzo -LSB- α -RSB- phenoxazine-5-one -RRB- was obtained from Fluka . A stock solution containing 0.25 mM Nile red was prepared with water-free dimethylsulfoxide -LRB- DMSO -RRB- and stored at − 20 °C . Methods Preparation of β-galactosidase solution A fresh solution of β-galactosidase in phosphate buffer was prepared before each experiment . The protein was dissolved in the buffer and subsequently , the solution was filtered through a 0.2 μm HPLC filter . Protein concentration after filtration was determined by measuring absorption at 280 nm , assuming an absorption coefficient of 2.09 cm2/mg -LSB- 26 -RSB- . Reproducibly , a protein concentration close to 0.10 μM was found after filtration . For incubation at elevated temperatures , 1 ml aliquots of β-galactosidase solution containing 0.10 μM protein were filled into 1.5 ml polypropylene Eppendorf tubes . They were heated for a defined period of time without shaking using an Eppendorf Thermomixer -LRB- Eppendorf , Hamburg , Germany -RRB- , and then cooled at room temperature . Steady-state fluorescence measurements Instrument and measurement settings Steady-state fluorescence measurements were performed with a Fluorolog FL3-21 spectrofluorometer -LRB- Jobin Yvon -- Horiba , Edison , NJ -RRB- , equipped with a short-arc xenon lamp . The slit openings of the excitation and emission monochromators were set to a bandwidth of 3 nm . The integration time was 0.05 s , and the signals were corrected for lamp intensity fluctuations by a simultaneously recorded reference signal . Each sample was measured 3 -- 5 times , and the average spectrum was calculated . The sample temperature was controlled by a water bath with a temperature sensor connected to the sample holder . Samples were measured in quartz cuvettes -LRB- Hellma GmbH , Muellheim , Germany -RRB- . Intrinsic tryptophan fluorescence Measurements of intrinsic tryptophan fluorescence of β-galactosidase were performed by exciting 0.10 μM protein solutions at 298 nm and scanning emission between 310 and 450 nm . To establish a protein denaturation curve , the sample temperature was increased from 25 to 70 °C in one degree steps . At every temperature , the sample was equilibrated for 5 min and then measured . Nile red fluorescence Before every measurement , a fresh solution of 25 μM dye in phosphate buffer was prepared by dilution of Nile red stock solution . This solution was immediately added to heated 0.10 μM β-galactosidase solution , non-heated 0.10 μM β-galactosidase solution or phosphate buffer in a cuvette , and mixed well . The final concentration of Nile red was 18.75 nM , well below the solubility limit of Nile red in water of 1 μM -LSB- 21 -RSB- . Nile red fluorescence was measured by exciting at 550 nm and scanning emission between 565 and 750 nm . All measurements were performed at 25 °C . Fluorescence spectra were measured immediately after addition of Nile red . However , Nile red emission in water remained unchanged for at least 25 min upon sample preparation . Time-resolved fluorescence measurements Instrument and measurement settings Time-resolved fluorescence measurements were performed by time-correlated single photon counting , as described in detail elsewhere -LSB- 27 -RSB- . Excitation source was a mode-locked titanium : sapphire laser -LRB- model Verdi V10 , Coherent Inc. , Santa Clara , CA -RRB- , pumped by a CW diode-pumped , frequency doubled Nd : YVO4 laser -LRB- model Mira 900-D in fs mode , Coherent Inc. , Santa Clara , CA -RRB- , and tuned at 980 nm . An excitation wavelength of 490 nm was obtained by frequency doubling , using a second harmonic generation system -LRB- model 5-050 Ultrafast Harmonic Generation System , Inrad Inc. , Northvale , NJ -RRB- . The repetition rate of the excitation pulses was 3.8 MHz . The emission filters were a Schott 3 mm OG 530 nm cut-off filter -LRB- Schott AG , Mainz , Germany -RRB- , combined with a Schott IL 611.6 nm -LRB- Δλ = 11.0 nm -RRB- interference filter . Measurements were performed at 25 °C , and consisted of repeated 10 s sequences of measuring parallel and perpendicularly polarized fluorescence emission until a maximum peak content of at least 50,000 counts in the data files was reached . Samples of β-galactosidase solution without Nile red were measured for background correction . To minimize background luminescence , all solutions , including buffer , were prepared with fluorescence spectroscopy grade water -LRB- Fluka , Buchs , Switzerland -RRB- . For the performance of a deconvolution procedure in data analysis , the dynamic instrumental response of the experimental setup was recorded using the fast and single-exponential fluorescence decay of the reference compound erythrosine B in water . Data analysis was performed using the `` TRFA Data Processing Package '' of the Scientific Software Technologies Center -LRB- Belarusian State University , Minsk , Belarus -RRB- -LSB- 28 -RSB- . The decay of fluorescence intensity with time , I -LRB- t -RRB- , could be described by an exponential model with three independent fluorescence lifetimes -LRB- τi -RRB- -LSB- 29 , 30 -RSB- : where αi are the pre-exponential factors , which were normalized . I -LRB- t -RRB- itself was calculated from the measured parallel and perpendicular polarized fluorescence emission , III -LRB- t -RRB- and I ⊥ -LRB- t -RRB- : G in the above equation represents the g-factor , which had a value of 1 for the used experimental setup . The course of fluorescence anisotropy with time r -LRB- t -RRB- was calculated from the measured intensities III -LRB- t -RRB- and I ⊥ -LRB- t -RRB- : Fluorescence correlation spectroscopy measurements Instrument and measurement settings Fluorescence correlation spectroscopy measurements were performed with a system composed of a krypton-argon laser and an MRC1024 confocal laser-scanning microscope -LRB- Biorad , Hercules , CA -RRB- , a TE300D inverted microscope -LRB- Nikon , Tokyo , Japan -RRB- with a water immersion objective lens -LRB- Plan Apo 60 × , NA 1.2 , collar rim correction , Nikon -RRB- , and ALV-5000 / E avalanche photodiode detectors -LRB- ALV GmbH , Langen , Germany -RRB- . The 568 nm line of the krypton-argon laser was used for excitation of Nile red samples . Fluorescence emission above 585 nm was detected . Samples were prepared by mixing heated 0.10 μM β-galactosidase solution , non-heated 0.10 μM β-galactosidase solution or phosphate buffer with diluted Nile red stock solution in polypropylene Eppendorf tubes . Heated β-galactosidase solution was mixed with Nile red in the same tubes that were used for heating . The final concentration of Nile red in the samples was 18.75 nM . One hundred μl sample was transferred into a well of a 96 glass bottom well plate -LRB- Bio-one , Greiner , Frickenhausen , Germany -RRB- . For measurements , the focal volume was positioned 100 μm above the bottom of the well . Raw data , i.e. fluctuations of fluorescence intensity with time , were collected during 30 s . The laser power was minimized to prevent photobleaching during this period . Every sample was measured ten times , and ten experimental autocorrelation functions were subsequently obtained from the raw data . Analysis of the experimental autocorrelation functions was performed by non-linear regression , based on a theoretical expression that describes the decay of an autocorrelation function , G -LRB- τ -RRB- , in consequence of diffusion and fast , non-diffusive processes , such as excitation of fluorescent molecules to the triplet state -LSB- 31 -- 33 -RSB- : Here , Ei is the relative emission rate of species i , and 〈 Ni 〉 the average number of molecules of species i in the focal volume . Mi -LRB- τ -RRB- is the mobility term for free , 3-dimensional diffusion : where τD , i is the diffusion time of fluorescent species i . The coefficients and z0 are defined , respectively , as the distances from and along the optical axis at which the excitation light intensity has dropped by 1/e2 . These coefficients were determined from calibration of the focal volume with rhodamine green of known diffusion coefficient -LRB- 2.8 × 10 − 6cm2 s − 1 -RRB- . The coefficient F in Eq . -LRB- 4 -RRB- represents the fraction of molecules involved in a non-diffusive process , and τf is the decay time of this process . It is justified to assume the existence of a non-diffusive decay process , because the transition of excited state Nile red molecules to a non-emissive TICT state and to the triplet state has been proposed -LSB- 19 -RSB- . G -LRB- ∞ -RRB- is the value of the autocorrelation function at long time intervals -LRB- τ -RRB- . To reduce the number of fitting parameters , Eq . -LRB- 4 -RRB- was simplified . For one diffusing species , it can readily be written as : The assumption of two or more diffusing species gives : where Before data analysis , the experimental autocorrelation functions were truncated at very short time intervals below 0.001 ms and at very long time intervals above 600 -- 1000 ms. The autocorrelation functions were then normalized -LSB- 34 -RSB- : where G -LRB- 0 -RRB- is the start value of the autocorrelation function and G -LRB- ∞ -RRB- is its value at very long time intervals . G -LRB- 0 -RRB- and G -LRB- ∞ -RRB- were estimated individually for every experimental autocorrelation function by non-linear regression , using Eqs . -LRB- 6 -RRB- and -LRB- 7 -RRB- -LSB- 34 -RSB- . Normalization eliminated possible differences in experimental autocorrelation functions that were related to systematic errors , such as adsorption of dye molecules to the surfaces of the well plate during experiments . The above described normalization changes Eq . -LRB- 6 -RRB- to : Here , Gnorm -LRB- 0 -RRB- is the start value of the normalized autocorrelation function , which is expected to be one . The value at long time intervals , accounted for by Gnorm -LRB- ∞ -RRB- , is expected to be zero . Equation -LRB- 7 -RRB- changes to : where Equation -LRB- 12 -RRB- shows that fi , norm represents an intensity-weighted fraction of diffusing species i. Equations -LRB- 10 -RRB- and -LRB- 11 -RRB- were used for analyzing the normalized , experimental autocorrelation functions by non-linear regression using the Levenberg-Marquardt algorithm . The calculations were performed with IGOR Pro V. 4.04 -LRB- WaveMetrics , Inc. , Lake Oswego , OR -RRB- . When applicable , up to ten autocorrelation functions obtained from the same sample were concatenated into one data set and analyzed by a global analysis procedure contained in the software . The standard deviation -LRB- σ -RRB- of experimental autocorrelation functions , estimated from the measurement repeats , was used as the weighting factor during fitting -LSB- 34 -RSB- . Solutions with a heterogeneous distribution of particle sizes showed very complex decays of the experimental autocorrelation functions . These could not be adequately fit by the discrete multi-exponential model using a small number of diffusion times , and increasing the number of diffusion times above three in the model resulted in substantially increased error margins of the determined parameters . For such complex data , calculation of the distribution of diffusion times was performed by the maximum entropy method -LRB- MEM -RRB- using the MEMFCS software -LSB- 35 -RSB- . Translation of diffusion time -LRB- τD -RRB- to a diffusion coefficient -LRB- D -RRB- was done by the following relationship : Calculation of the hydrodynamic radius -LRB- Rh -RRB- was then possible using the Stokes-Einstein equation : Light scattering Detection of β-galactosidase aggregates in solution was done with static light scattering measurements using a fluorometer -LRB- Fluorolog FL3-21 , Jobin Yvon -- Horiba , Edison , NJ -RRB- . The excitation and emission wavelengths were set to 400 nm , and measurements were performed at 25 °C . Furthermore , the hydrodynamic radius of native β-galactosidase was determined by dynamic light scattering measurements of 0.10 μM protein solutions in phosphate buffer . Experiments were performed at 25 °C with a CGS-3 goniometer system and an LSE-5003 correlator -LRB- ALV , Langen , Germany -RRB- . The average hydrodynamic radius -LRB- Rh -RRB- of the β-galactosidase molecules was determined using the method of cumulants . Size exclusion chromatography -LRB- SEC -RRB- SEC measurements were performed using an Alliance 2695 system with a type 2487 dual wavelength detector and a type 2475 fluorescence detector -LRB- Waters , Milford , MA -RRB- . A Superose 6 column with a separation range of 5 × 103 to 5 × 106 Da , and an exclusion limit of 4 × 107 Da -LRB- GE Healthcare Europe GmBH , Roosendaal , The Netherlands -RRB- was used . Phosphate buffered saline -LRB- pH 7.4 -RRB- was used as an eluent , and the injection volume was 40 μl . Detection was by UV absorption at 210 nm or by fluorescence emission -LRB- λex 280 nm and λem 340 nm for intrinsic protein fluorescence ; λex 550 nm and λem 611 nm for Nile red fluorescence -RRB- . Before injection , samples were centrifuged at 10000 RPM for 5 min . A molecular weight calibration curve using chymotrypsin -LRB- 25 kDa -RRB- , human serum albumin -LRB- 66 kDa and 132 kDa dimer -RRB- , and thyroglobulin -LRB- 670 kDa -RRB- as molecular weight standards was established . Results and discussion Heat denaturation of β-galactosidase The temperature induced denaturation of β-galactosidase was followed by measuring intrinsic tryptophan fluorescence emission of this protein -LRB- not shown -RRB- . Denaturation started between 50 and 55 °C and was completed between 60 and 65 °C . An unfolding temperature of 57.4 °C was obtained by fitting the denaturation curve with a model for a two-state unfolding process -LSB- 36 -RSB- . This is in good agreement with DSC experiments and activity assays performed under comparable conditions -LSB- 25 -RSB- . Heating β-galactosidase solutions above the unfolding temperature leads to irreversible aggregation of protein molecules -LSB- 25 -RSB- . Accordingly , a 0.10 μM β-galactosidase solution incubated at 62 °C for 5 min was heavily aggregated and remained so upon cooling back to room temperature , as detected by light scattering -LRB- not shown -RRB- . The aggregates formed under these conditions were too large to be detected by SEC . This is shown in Fig. 1a , which depicts SEC chromatograms of a non-heated β-galactosidase solution and of a β-galactosidase solutions that was heated at 62 °C for 5 min . The former consists of peaks representing native , tetrameric β-galactosidase , oligomers of two and three β-galactosidase molecules , and even some aggregates of higher molecular weight . The latter consists only of a peak representing native , tetrameric β-galactosidase . However , the area under the curve of this peak is reduced to about 10 % of the area under the curve of the corresponding peak of non-heated β-galactosidase solution . The apparent loss of protein was caused by the formation of large aggregates that were excluded from the SEC column.Fig . 1 -LRB- a -RRB- SEC chromatograms of non-heated 0.10 μM β-galactosidase solution -LRB- solid line -RRB- and β-galactosidase solution incubated at 62 °C for 5 min -LRB- dotted line -RRB- . The peaks represent native -LRB- i.e. tetrameric -RRB- β-galactosidase -LRB- 1 -RRB- and oligomers of two -LRB- 2 -RRB- and three -LRB- 3 -RRB- β-galactosidase molecules . -LRB- b -RRB- Plot of the increase of scattered light intensity at 400 nm against incubation time of 0.10 μM β-galactosidase solution at 49 °C . Data points are averages of 5 -- 10 individual samples , and the error bars indicate ± 1 SD . -LRB- c -RRB- SEC chromatograms of non-heated 0.10 μM β-galactosidase solution and solutions heated at 49 °C for 15 and 60 min . The peaks represent native , tetrameric β-galactosidase -LRB- 1 -RRB- and oligomers of two -LRB- 2 -RRB- and three -LRB- 3 -RRB- β-galactosidase molecules . The insert shows that after 60 min incubation , high-order aggregates of β-galactosidase appeared , with a retention time of 29 min . There was no detectable difference between non-heated solutions and solutions heated at 49 °C for 15 min Static light scattering measurements using a fluorometer indicated that heating β-galactosidase solutions at 49 °C also led to the formation of aggregates . Figure 1b shows the intensity of scattered light of 0.10 μM β-galactosidase solutions heated for different times between 15 min and 6 h . The increase of scattered light intensity indicates the formation of protein aggregates . This is clearly an effect of heating , because scattered light intensity did not increase upon storage at 25 °C -LRB- not shown -RRB- . Figure 1b also shows that a maximum of scattered light intensity was reached after about 120 min of incubation . This is possibly related to the sedimentation of very large aggregates that had been formed after long incubation times , which would also explain the increasing error margins of the long time-points . Fig. 2 -LRB- a -RRB- Fluorescence emission spectra of Nile red added to phosphate buffer -LRB- broken line ; λmax = 662 nm -RRB- , non-heated 0.10 μM β-galactosidase solution -LRB- dotted line ; λmax = 659 nm -RRB- , and 0.1 μM β-galactosidase solution that was previously heated at 62 °C for 5 min -LRB- solid line ; λmax = 611 nm -RRB- . Excitation wavelength was 550 nm . -LRB- b -RRB- Fit of two Voigt-shaped peaks -LRB- broken lines -RRB- to an emission spectrum -LRB- circles -RRB- of Nile red added to β-galactosidase solution that was heated at 49 °C for 360 min . The solid black line is the fitted spectrum , and the residuals are indicated . The positions of the fitted peaks are 605.0 ± 2.2 nm and 658.3 ± 1.4 nm . -LRB- c -RRB- Plot of Nile red fluorescence intensity at 611 nm against incubation time at 49 °C of 0.10 μM β-galactosidase solution . Data points are averages of 5 individual samples , and the error bars indicate ± 1 SD The SEC chromatograms in Fig. 1c show the formation of large β-galactosidase aggregates by heating solutions at 49 °C . Non-heated solution mainly contained the native , tetrameric β-galactosidase , along with some dimer , trimer , and even higher order aggregates . Heating for 15 min at 49 °C did not lead to a change of the SEC chromatogram . However , after 60 min incubation an additional peak with a retention time of 29 min appeared , representing large β-galactosidase aggregates . The area of this peak was 1.5 % of the total area under the curve of the protein and increased with increasing incubation time at 49 °C -LRB- not shown -RRB- . The chromatograms shown in Fig. 1c were obtained by measuring absorbance at 210 nm . The use of an on-line fluorescence detector system -LRB- λex 280 nm and λem 340 nm -RRB- led to corresponding results , i.e. aggregation was only detected in β-galactosidase solutions that were heated at 49 °C for at least 60 min . Nile red steady-state fluorescence Figure 2a depicts the emission spectra of Nile red in buffer , in non-heated 0.10 μM β-galactosidase solution , and in 0.10 μM β-galactosidase solution that was previously heated at 62 °C for 5 min . The emission of Nile red in the presence of non-heated protein was almost identical to the dye 's emission in buffer . This indicates that the surface area of native β-galactosidase is highly polar , and that possible apolar sites are not accessible to Nile red . Storage of unheated β-galactosidase solutions containing Nile red for up to 25 min at room temperature did not lead to a change of the dyes fluorescence , showing that the presence of Nile red does not lead to a change of β-galactosidase surface hydrophobicity over time . Interaction of Nile red with heat-denatured β-galactosidase resulted in a shift of emission maximum from 660 to 611 nm and in a substantial increase in fluorescence intensity -LRB- Fig. 2a -RRB- . This is typically observed when Nile red molecules bind to hydrophobic , apolar sites on the surface of a protein -LSB- 14 -RSB- , and shows that irreversible denaturation and aggregation by heating above the unfolding temperature has increased the surface hydrophobicity of β-galactosidase . Upon 15 min incubation of 0.10 μM β-galactosidase solution at 49 °C , the emission spectrum of Nile red reproducibly showed an increased intensity below 640 nm . Upon longer incubation between 1 and 6 h , a shoulder between 605 and 615 nm emerged . Figure 2b shows a two-peak fit of an emission spectrum obtained with Nile red added to β-galactosidase solution that was previously heated at 49 °C for 6 h . In Fig. 2b , the λmax of the main peak is 658 nm , whereas the peak responsible for the shoulder is positioned at 605 nm . Emission at 658 nm originates from free Nile red molecules , and emission at 605 nm from Nile red interacting with denatured β-galactosidase . The latter is comparable with the λmax of Nile red in β-galactosidase solution that had been heated above the unfolding temperature -LRB- Fig. 2a -RRB- . This indicates that the surface polarity of protein molecules denatured at different temperatures is similar . The fluorescence of Nile red did not depend on the time that protein solutions were kept at room temperature upon heating at 49 °C -LRB- not shown -RRB- , which indicates that irreversibly denatured β-galactosidase species were detected . The effect of increasing incubation time at 49 °C on Nile red fluorescence intensity is shown in Fig. 2c . An observation wavelength of 611 nm was chosen , which corresponds to the average emission wavelength of Nile red interacting with irreversibly denatured β-galactosidase . The profile of Fig. 2c is similar to that of Fig. 1b , suggesting that Nile red interacted with aggregated β-galactosidase . A clear decrease of Nile red emission at 611 nm upon centrifugation at 10000 RPM -LRB- not shown -RRB- supported this notion . However , it was not possible to confirm this by SEC , neither by adding Nile red to the samples before injection and performing on-line fluorescence detection -LRB- λex 550 nm and λem 611 nm -RRB- , nor by staining fractions from SEC runs with Nile red and performing off-line fluorescence detection . For this reason , time-resolved fluorescence spectroscopy and FCS were used to further investigate the interaction between Nile red and aggregated β-galactosidase . Fig. 3Fluorescence intensity decay , I -LRB- t -RRB- , of Nile red in 0.10 μM β-galactosidase solution that was heated at 49 °C for 60 min . The fitted decay curve and the weighted residuals are shown . Fitting gave three lifetimes -LRB- τ1 = 0.11 , τ2 = 1.11 , and τ3 = 4.36 ns -RRB- and three -LRB- normalized -RRB- pre-exponential factors -LRB- α1 , norm = 0.40 , α2 , norm = 0.24 , and α3 , norm = 0.36 -RRB- . The value of χ2 was 1.056 Table 1Lifetime analysisSampleχ2 totala 〈 τ 〉 bbuffer1.0390.20non-heated1.0110 .4215 -- 49 °C 1.0621.3060 -- 49 °C 1.0561.89120 -- 49 °C 1.0642.48240 -- 49 °C 1.0002.105 -- 62 °C 1.0321.50 aχ2 obtained with Eq . -LRB- 1 -RRB- using three fluorescence lifetimes.b Time-resolved fluorescence spectroscopy with Nile red Figure 3 shows the analysis of the fluorescence intensity decay , I -LRB- t -RRB- , of Nile red in 0.10 μM β-galactosidase solution that was heated at 49 °C for 60 min . The data points could be adequately described using Eq . -LRB- 1 -RRB- with three fluorescent lifetimes , as can be seen from the regular distribution of the residuals around zero and the proximity of χ2 to unity . The use of three lifetimes was necessary for the analysis of all intensity decays , and it was not possible to relate particular lifetimes to the free and the protein-bound state of Nile red . In Table 1 , the results are summarized by listing 〈 τ 〉 values , which are proportional to steady state intensities . In agreement with the increase of Nile red steady state intensity at 611 nm , heating of β-galactosidase solution at 49 °C for 15 min led to a clear increase of 〈 τ 〉 as compared to Nile red in buffer or non-heated protein solution . The tendency of 〈 τ 〉 to further increase with increasing heating time at 49 °C corresponds nicely to the increase of fluorescence intensity upon prolonged heating shown in Fig. 2c . Also with β-galactosidase solution that was heated at 62 °C for 5 min , 〈 τ 〉 was substantially increased as compared to non-heated protein solution . However , it was lower than expected from the steady-state fluorescence intensity at 611 nm -LRB- Fig. 2a -RRB- . Possible reasons for this difference are the variation between individual experiments with solutions containing highly aggregated protein and , since there is a lot of light scattering in such samples , different sensitivities of the steady-state and time-resolved instrumentation for scattered light . The course of fluorescence anisotropy with time , r -LRB- t -RRB- , of Nile red in 0.10 μM β-galactosidase solution that was heated at 49 °C for 15 , 60 , 120 , and 240 min was measured -LRB- not shown -RRB- . With all samples , anisotropy decreased at short times , then increased again , and finally remained at an almost constant value . The practically constant value of anisotropy at long times indicated rotational diffusion that was too slow to be detected by Nile red fluorescence . Therefore , although indicating that Nile red was in fact bound to large particles such as aggregates of β-galactosidase , it was not possible to estimate the size of these particles by time-resolved fluorescence anisotropy . Fluorescence correlation spectroscopy -LRB- FCS -RRB- measurements with Nile red Figure 4a depicts an overlay of ten normalized , experimental autocorrelation functions that were obtained with Nile red in buffer . These data were analyzed simultaneously using the one-component model -LRB- Eq . -LRB- 10 -RRB- -RRB- . The fitted curve included in Fig. 4a , the regular distribution of the weighted residuals around zero , and the proximity of χ2 to unity show that Eq . -LRB- 10 -RRB- was appropriate for describing the experimental data . The determined decay time for non-diffusive process -LRB- τf -RRB- was 0.022 ms , the fraction of dye molecules involved in this process -LRB- F -RRB- was 0.45 , and the determined diffusion time -LRB- τD ,1 -RRB- was 0.309 ms -LRB- Table 2 -RRB- . A diffusion time of Nile red around 0.3 ms is in agreement with reported values -LSB- 37 -RSB- . It corresponds to a diffusion coefficient of 2.9 × 10 − 6 cm2 s − 1 , which is reasonable for a molecule with a molecular weight of 318 Da . Therefore , τD ,1 can be associated with the diffusion of free dye molecules . Gnorm -LRB- 0 -RRB- deviated slightly from the expected value of unity -LRB- Table 2 -RRB- . Forcing it to unity increased χ2 slightly , but had no effect on the determined parameters τD ,1 , τf , and F. Also Gnorm -LRB- ∞ -RRB- deviated somewhat from the expected value of zero -LRB- Table 2 -RRB- . This is related to the background subtraction described in the materials and methods section . Leaving out background subtraction did not lead to different values of the coefficients τD ,1 , τf , and F. Measurement of Nile red in non-heated 0.10 μM β-galactosidase solution gave the same parameters as Nile red in buffer -LRB- Table 2 -RRB- , confirming that there was no substantial interaction between the dye and non-heated protein.Table 2Coefficients from autocorrelation function analysisSample withNile redModelτD ,1 a -LRB- ms -RRB- f1 , normaτD ,2 a -LRB- ms -RRB- f2 , normaτfa -LRB- ms -RRB- FaGnorm -LRB- 0 -RRB- aGnorm -LRB- ∞ -RRB- aPhosphate bufferOne-Component0.309 ± 0.008 -- -- -- 0.022 ± 0.0020.451 ± 0.0100.920 ± 0.016 − 0.0049 ± 0.00031.03β-Galactosidase non-heatedOne-Component0.291 ± 0.009 -- -- -- 0.018 ± 0.0020.434 ± 0.0150.915 ± 0.021 − 0.0047 ± 0.00041.08β-Galactosidase 62 °C / 5 minTwo-Component0.287 ± 0.0170.335 ± 0.00545.7 ± 1.020.690 ± 0.0070.019 ± 0.0010.246 ± 0.008 -- − 0.0368 ± 0.00211.44 aEstimates ± 1 SD.Fig . 4Analysis of FCS data . -LRB- a -RRB- Simultaneous fit of 10 normalized autocorrelation functions obtained with Nile red in buffer . Analysis was performed by non-linear regression using Eq . -LRB- 10 -RRB- . The fitted curve -LRB- black -RRB- and the weighted residuals for one of the normalized autocorrelation functions are shown . Determined parameters -LRB- value ± 1 SD -RRB- were F = 0.451 ± 0.010 , τf = 0.022 ± 0.002 ms , τD ,1 = 0.309 ± 0.008 ms , Gnorm -LRB- 0 -RRB- = 0.920 ± 0.016 , and Gnorm -LRB- ∞ -RRB- = − 0.0049 ± 0.0003 . The value of was 1.03 . -LRB- b -RRB- Simultaneous fit of 9 normalized autocorrelation functions obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62 °C for 5 min . Analysis was performed by non-linear regression using Eq . -LRB- 11 -RRB- for two diffusing species . The fitted curve -LRB- black -RRB- and the weighted residuals for one of the normalized autocorrelation functions are shown . Determined parameters -LRB- value ± 1 SD -RRB- were F = 0.246 ± 0.008 , τf = 0.019 ± 0.001 ms , f1 , norm = 0.335 ± 0.005 , τD ,1 = 0.287 ± 0.017 ms , f2 , norm = 0.690 ± 0.007 , τD ,2 = 45.70 ± 1.02 ms , and Gnorm -LRB- ∞ -RRB- = − 0.037 ± 0.0021 . The value of was 1.44 Fig. 5Analysis of FCS data . -LRB- a -RRB- MEM analysis of a single autocorrelation function obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62 °C for 5 min . The non-weighted residuals are shown . -LRB- b -RRB- Distribution of diffusion times -LRB- τD -RRB- calculated by MEM analysisFig . 6Raw data of two FCS measurements obtained with 0.10 μM β-galactosidase solution that was heated at 62 °C for 5 min -LRB- upper curve -RRB- and at 49 °C for 15 min An overlay of nine normalized , experimental autocorrelation functions , obtained with Nile red in 0.10 μM β-galactosidase solution that was previously heated at 62 °C for 5 min , is shown in Fig. 4b . There is an additional decay of the autocorrelation functions between 20 and 200 ms , which was not observed with Nile red in buffer . Data analysis was therefore performed using a two-component model , i.e. Eq . -LRB- 11 -RRB- assuming two diffusing species . The fitted line and the weighted residuals in Fig. 4b show that up to time intervals between 1 and 10 ms , the data could be well described by the two component model . Within this range , the decay of the autocorrelation functions is governed by τf and τD ,1 . The determined values were 0.019 and 0.287 ms , respectively -LRB- Table 2 -RRB- , which is in good agreement with the values found for Nile red in buffer . This shows that also in the heated β-galactosidase solution , not all Nile red was bound to denatured protein . The fraction of dye molecules -LRB- F -RRB- involved in non-diffusive process was 0.25 , which is substantially lower than for Nile red in buffer -LRB- Table 2 -RRB- . A possible explanation for this is that in solutions with large amounts of denatured protein , less Nile red molecules undergo transition to the TICT and triplet state , because they can interact with hydrophobic surfaces -LSB- 19 -RSB- . The second diffusion time -LRB- τD ,2 -RRB- of 46 ms -LRB- Table 2 -RRB- governed the decay of autocorrelation functions between 20 and 200 ms. It corresponds to a diffusion coefficient -LRB- D -RRB- of 1.9 × 10 − 8 cm2 s − 1 and a hydrodynamic radius -LRB- Rh -RRB- of 130 nm , which deviates substantially from the values of native β-galactosidase -LRB- D = 3.1 × 10 − 7 cm2 s − 1 ; Rh = 8 nm -RRB- . This shows that incubation at 62 °C for 5 min had led to very large , denatured aggregates of β-galactosidase , which could be detected by Nile red . However , the two-component model -LRB- Eq . -LRB- 11 -RRB- -RRB- was not able to fully describe the very complex autocorrelation function decays shown in Fig. 4b , as reflected by the residuals and the relatively high χ2 value of 1.44 -LRB- Table 2 -RRB- . For this reason , the distribution of diffusion times was determined by the maximum entropy method -LRB- MEM -RRB- -LSB- 35 -RSB- . Such an analysis is shown in Fig. 5a for an individual experimental autocorrelation function . The distribution of diffusion times -LRB- Fig. 5b -RRB- shows 3 peaks , centered at 0.02 , 0.36 , and 39 ms. The 0.02 and 0.36 ms peaks correspond to τf and τD ,1 determined with Eq . -LRB- 11 -RRB- . The peak around 39 ms is very broad and ranges from 5 to 150 ms , which corresponds to hydrodynamic radii between 14 and 420 nm . The peak maximum of 39 ms corresponds to an Rh of 109 nm , in good agreement with τD ,2 obtained with Eq . -LRB- 11 -RRB- . This is well above the hydrodynamic radius of small products of β-galactosidase denaturation , i.e. denatured monomers , dimers , and tetramers -LSB- 25 -RSB- , and confirms that interaction with large β-galactosidase aggregates was the reason for the change of Nile red fluorescence . Nile red also detected large aggregates in 0.10 μM β-galactosidase solutions that had been incubated at 49 °C . As for protein samples heated at 62 °C , these aggregates were detectable by an additional decrease of FCS autocorrelation functions at long times . However , aggregate concentration in samples heated at 49 °C was much lower than in samples heated at 62 °C . This is shown in Fig. 6 , which compares the raw FCS data obtained with Nile red in β-galactosidase solutions heated for 5 min at 62 °C and for 15 min at 49 °C . The former is full of high-intensity peaks that were caused by large aggregates with bound Nile red crossing the focal volume . The latter contains only one high-intensity peak , representing a single large aggregate crossing the focal volume . As a consequence of the low aggregate concentration in samples heated at 49 °C , only 3 out of 100 measurements contained high-intensity peaks . In spite of their rare occurrence , these peaks were not artifacts . No high-intensity peaks were detected in a total of 150 control experiments with Nile red in buffer and in non-heated β-galactosidase solution . The combined results of steady-state fluorescence spectroscopy , time-resolved fluorescence spectroscopy , and FCS show that Nile red fluorescence gave complementary information to SEC about the aggregation of β-galactosidase . Native protein molecules and small oligomers could be detected by SEC -LRB- Fig. 1a and c -RRB- , but did not substantially affect Nile red fluorescence . It is possible that both the surface hydrophobicity and the surface area of these protein species were not sufficient for an effective interaction with the dye . However , Nile red was able to interact with large , denatured aggregates of β-galactosidase and could therefore be used as a probe for their specific detection . Although some higher order aggregates could be detected by SEC upon heating at 49 °C -LRB- Fig. 1c -RRB- , Nile red fluorescence was sensitive to the presence of even larger aggregates , which were missed by SEC -LRB- Fig. 1a -RRB- . The interaction of Nile red with large aggregates of β-galactosidase makes it very difficult to use the SEC results for estimating the sensitivity of the Nile red method . Figure 1c shows that some large aggregates were first detected by SEC in protein samples that had been heated at 49 °C for 60 min . However , the inability of Nile red to stain these aggregates fractionated from SEC runs -LRB- see results above -RRB- and the possible exclusion from the SEC column of even larger protein aggregates , which are detectable by Nile red , render the use of SEC as a reference method questionable . Furthermore , Nile red fluorescence was clearly more sensitive than SEC , as it already detected irreversible changes in samples that were heated at 49 °C for 15 min . Therefore , to estimate the sensitivity of Nile red , non-heated β-galactosidase solutions were doped with defined amounts of β-galactosidase solution that had been irreversibly denatured and aggregated by heating at 62 °C for 5 min . The lower limit of detection by Nile red steady-state fluorescence was 1 % -LRB- v/v -RRB- of heated protein solution , corresponding to 1 nM denatured and aggregated β-galactosidase . The fluorescence intensity of this solution at 611 nm corresponded to that of β-galactosidase solution that had been heated at 49 °C for 15 min -LRB- Fig. 2c -RRB- . This comparison is justified , because nearly all β-galactosidase molecules had formed large aggregates upon heating at 62 °C for 5 min -LRB- Fig. 1a -RRB- , and because the fluorescence intensity of the detected aggregates did not depend on the incubation temperature -LRB- Fig. 6 -RRB- , which means that Nile red was equally sensitive to aggregates formed by heating at 49 and 62 °C . Conclusions Using the model protein β-galactosidase , it was shown that the presence small amounts of large , denatured protein aggregates in solution can be detected by Nile red fluorescence . Aggregates detected by Nile red had hydrodynamic radii around 130 nm with a broad size distribution . Native protein and small aggregates thereof had no substantial effect on Nile red fluorescence intensity . By steady-state fluorescence measurements , it was possible to detect 1 nM denatured and highly aggregated β-galactosidase in solution . The spectroscopic detection of protein aggregates by Nile red is potentially useful for formulation screening or quality control of protein pharmaceutics . The presence of even minute fractions of aggregates in protein formulations needs to be avoided , because this can cause immune reactions in patients . Since large aggregates are particularly potent for breaking immune tolerance , their analytical detection is very important . SEC , which is the standard method for studying protein aggregation , is not reliable for detecting large aggregates , because they may be excluded from the separation column . In this work we showed that an analytical detection method with Nile red is a possible approach to overcome this shortcoming of SEC . After establishing the method with time-resolved fluorescence spectroscopy and FCS , as was done for β-galactosidase , steady-state fluorescence measurements , which can be performed in most labs , may enable an experimentally simple and sensitive detection of large protein aggregates .

O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

protein aggregation
fluorescence spectroscopy
fluorescence correlation spectroscopy
nile red
size exclusion chromatography