A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

bioRxiv preprint doi: https://doi.org/10.1101/275230; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Biophysical Journal-Biophysical Letters

A fluorescence correlation spectrometer for measurements in cuvettes

B Sahoo†, TB Sil†, B Karmakar‡ and K Garai†

ABSTRACT We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single molecule measurements on samples inside regular cuvettes. We built this by using an Extra Long Working Distance (ELWD), 0.7 NA, air objective with working distance > 1.8 mm. We have achieved counts per molecule > 44 kHz, diffusion time < 64 s for rhodamine B in aqueous buffer and a confocal volume < 2 fl. The cuvette-FCS can be used for measurements over a wide range of temperature that is beyond the range permitted in the microscope-based FCS. Finally, we demonstrate that cuvette- FCS can be coupled to automatic titrators to study urea dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette-FCS in the experiments that are regularly performed in fluorimeters but are generally avoided in microscope-based FCS.

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Fluorescence correlation spectroscopy (FCS) is a high S/N can enable applications of FCS in the above- powerful single molecule technique with wide spread mentioned biophysical experiments conveniently. applications in biophysics and biology. The high spatio- A major obstacle for performing FCS in a cuvette is that temporal resolution of FCS allows measurements of fast the thickness of the optical windows of commercially processes in dilute solutions. Some of the common available cuvettes are about 1.25 mm. Conventional FCS applications of FCS are measurements of molecular size, setups employ high numerical aperature (NA ≥ 1.2) chemical kinetics, conformational dynamics of objective lenses, which are carefully corrected for both biomolecules, protein-ligand interactions and protein spherical and chromatic aberrations. These objectives have aggregation in vitro (1-4). Due to its single molecule working distances less than 0.31 mm, and hence can’t be sensitivity, FCS techniques have been developed used for measurements inside cuvettes. While high NA extensively over the past two decades for measurements in objectives are required for high S/N, FCS measurements live cells and tissue samples. For example, several flavors have been demonstrated using optics with much lower NA. of imaging FCS techniques have been developed for For example, Garai et al performed FCS measurements measurements of membrane dynamics, cellular and nuclear using a single mode optical fiber (NA = 0.13) for detection localization, and transport of biomolecules in live cells (5- of protein aggregates in samples placed remotely (14). 8). Furthermore, development of 2-photon FCS has Furthermore, Banachowicz et al. have shown that FCS extended applications of FCS in tissues and other thick measurements can be performed using objectives with NA samples (9). equal to 0.4 (15). Conventionally, FCS setups are attached to confocal Here we report building a highly senstitive FCS setup microscopes for the high spatial resolution and the signal to capable of performing measurements inside regular noise (S/N) achieved in confocal microscopy. cuvettes. This setup is built using an extra long working Consequently, applications of FCS have several limitations distance (ELWD) objective can yield molecular brightness in many of the biophysical experiments. For example, > 44 kHz and a diffusion time (τD) < 64 μs for Rhodamine folding-unfolding of proteins using chemical or thermal B in phosphate buffered saline (PBS) at pH 7.4. The denaturation are rarely studied using FCS (10-12). Kinetic confocal volume obtained in this setup is < 2 fl. experiments which require stirring of the samples can’t be Figure 1 shows the schematic of the cuvette-FCS setup. performed using microscope based FCS setups. This setup is similar to the conventional FCS with two Furthermore, experiments that require non-aqueous or major differences. First, the objective is mounted corrosive solvents are seldom performed by using FCS horizontally. Such geometry is required for FCS (13). However, the above mentioned experiments are measurements inside a cuvette. Second, we have used an performed regularly in most biophysics and biochemistry ELWD plan achromat objective with NA equal to 0.7. laboratories using fluorimeters. Therefore, an FCS setup These objectives are corrected for both spherical and capable of performing measurements inside a cuvette with chromatic aberrations but the corrections are less extensive than the conventionally used plan apochromat objectives.

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data. It is clear that the Hellma cuvette is the most suitable for cuvette-FCS. Hence, all the FCS measurements reported in the rest of this article are performed using a Hellma cuvette. We then examined the effects of the relative angle between the face of the objective and the optical window of

Figure 1: Schematic of the cuvette-FCS setup. Laser (543 nm, CW), L1 and L2: lenses, DCM: Dichroic mirror, AD: Figure 2: Effects of quality and angular misalignment (θ) of Achromatic doublet lens, EF: emission filter, BS: 50/50 beam the cuvette on the autocorrelation, G(τ) data. A) G(τ) splitter, FC: fiber coupler, TS: micrometer translation stage, obtained from aqueous solution of rhodamine B using objective: ELWD, air objective, 0.7 NA, WD = 2.6-1.8 mm. cuvettes from three different manufacturers. G(τ) using Hellma cuvette is the best. B) G(τ) at different θ. G(τ) 0 However, the advantage of using the ELWD objective is worsens drastically with even a small (by ±0.40 ) increase of that it’s working distance is > 1.8 mm and the correction θ. Hence, the cuvette must be placed parallel to the objective. The symbols represent data and the solid lines are collar can be adjusted for cover slips of thickness up to 1.3 fits using single diffusing component (suppl. Eq. S1). mm. Therefore, these objectives may be suitable for use with common cuvettes. the cuvette. Figure 2B shows that G(τ) changes drastically We have used a solution of rhodamine B in PBS to with the change of the angle. It may be seen that G(0) characterize the sensitivity and the spatial resolution of the decreases by 4 fold for an angular misalignment of 0.40 cuvette-FCS setup. Supplementary Eq. S1 shows that FCS degree. Supplementary Figure S3A and B show that autocorrelation (G(τ)) data can be analyzed to determine corresponding increase in τD and decrease in CPM are the average number of the molecules () and the about 2 and 4-folds respectively. Hence, the best sensitivity diffusion time (τD) of the fluorophore in the FCS in the cuvette-FCS can be achieved when the cuvette observation volume. The confocal volume (Vconfocal) and the surface is parallel to the face of the objective. axial resolution (σxy) can be determined from and τD We then calibrate the cuvette-FCS setup by measuring respectively (Supplementary Eq. S2 and S3). Furthermore, the CPM, and τD using solutions of rhodamine B in S/N of the G(τ) is dependent on the number of photons PBS buffer at pH 7.4. Figure 3A shows that CPM of collected per molecule per unit time, viz, the counts per rhodamine B increases monotonically with the incident molecule (CPM) (16). CPM is calculated from the ratio of laser power. The increase of CPM deviates from linearity total photon count rate and . above 50 µW of incident power but it doesn’t reach First, we investigate the factors that affect the complete saturatation even at the highest intensity (114 performance of the cuvette-FCS most ctirically. We find μW) used here. It may be seen that the maximum CPM that τD, and CPM obtained are critically dependent on obtained here from rhodamine B is 44 kHz. Supplementary two factors, viz, quality of the cuvettes, and alignment of Figure S4B shows that average τD obtained from fitting all the cuvette with respect to the objective. Figure 2A the G(τ) data is 64±2 μs. compares the autocorrelation data obtained from an Figure 3B shows the plot of as a function of aqueous solution of rhodamine B placed in quartz cuvettes concentration (C) of rhodamine B. As expected, procured from three different manufacturers referred here varies linearly with C over the entire range of concentration as Hellma, MF1 and MF2. Dimensions of all the three of 3-265 nM. Linear fit of the data yields a slope = 1.1 cuvettes are the same, e.g., thickness of the optical window molecule/nM. Hence, the estimated value of Vconfocal in = 1.25 mm and the optical pathlength = 10 mm. cuvette-FCS is 1.8 fl (see Supplementary Eq. S2). Amplitudes of the autocorrelation traces (i.e., G(0)) Supplementary Figure S5B shows that mean τD of obtained from the different cuvettes differ by about 1.4- rhodamine B obtained from fitting of all the G(τ) data is fold. Furthermore, supplementary Figure S2A and S2B 63±2 μs. Using the known diffusion coefficient (D = -6 2 show that τD and CPM differ by 1.2 and 2.0-fold 4.510 cm /s) of rhodamine B in water, the axial respectively. Therefore quality of the optical windows of resolution in cuvette-FCS may be estimated as 340 nm (see the cuvettes is extremely important for the S/N of the FCS supplementary Eq. S3). The axial resolution expected for a

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Figure 3: Characterization of sensitivity and resolution of the cuvette-FCS Setup. A) CPM as a function of incident laser power obtained using aqueous solution of rhodamine B. Symbols represent data and the solid line is fit using a parabola. CPM increases linearly till ≈ 50 µW of incident power. B) Average number of molecules () in the confocal observation volume (Vconfocal) as a function of concentration of rhodamine B. Symbols represent data. Slope of the fitted line is 1.1 molecules/nM indicating Vconfocal = 1.8 fl. C) log-log plot of τD versus molecular weight (MW) of free dye and several TMR labeled-proteins. Symbols represent data and the solid line is linear fit. The slope obtained is 0.36±0.01. Expected slope for globular proteins is 0.33 (17). diffraction limited confocal setup is between 285 to 422 nm larger range of temperature can be used for measurements for λex= 543 nm and NA = 0.7 (see supplementary Eq. S4). using cuvette-FCS. Hence, axial resolution of the cuvette-FCS setup is within We then examine if the cuvette-FCS can be used for the expected range. measurements in urea. Urea is widely used as a chemical We then examine if cuvette-FCS is suitable for denaturant in experiments involving folding-unfolding of measurements using proteins. The proteins used here are proteins. While FCS can yield single molecule level amylin, α-Synuclein, T4-Lysozyme, an N-terminal information, there are major difficulties in measurements of fragment of apolipoprotein E4 (Nt-ApoE4) and bovine molecular size in urea in conventional FCS setups. The serum albumin (BSA). All the proteins are fluorescently difficulties arise due to mismatch of refractive index of the labeled with tetramethylrhodamine (TMR). Figure 3C solution, requiring adjustment of the distance between the shows that log(τD) varies linearly with log(MW). The slope objective and the glass coverslip for samples containing of the line is equal to 0.36 ± 0.01. The expected scaling urea. These complications have been discussed in detail exponent is 0.33 for globular proteins (17). Hence our data elsewhere (10). Cuvette-FCS has certain advantages in are consistent with the scaling law for globular proteins. these experiments. For example, the distance between the The τD of α-synuclein is found to be somewhat higher, objective and the cuvette is fixed during the entire duration which is consistent with the extended conformation of α- of the experiment. Concentration of urea can be changed by synuclein in native conditions (18). manual pipetting or by using autotitrators as has been done We then examine suitability of the cuvette-FCS setup for here. Furthemore, mixing of the samples can be performed measurements in conditions that are generally avoided in by stirring using magnetic stirrers. Therefore, titration microscope based FCS. First, we perform FCS experiments in the cuvette-FCS setup can be automated measurements using rhodamine B in a range of temperature fully. Figure 4B shows the effect of concentration of urea 0 0 starting from 15 to 60 C. In confocal microscopes on the τD of rhodamine B. These experiments are temperature range is generally restricted to 250-370C due to performed using an automatic titrator coupled to the incompatibility of the objectives at the higher or the lower cuvette, which is placed in a temperature controlled cell temperatures. Since we are using an air objective the holder. The entire experiment is performed in an automatic temperature of the sample doesn’t affect the resolution. As manner. It may be seen that the τD of rhodamine B viscosity of water changes with temperature we have increases linearly with increase of viscosity of the urea plotted τD of rhodamine B as a function of viscosity of the solution as expected from SE relationship shown earlier by solution in Figure 4A. It may be seen that τD varies linearly others (11). We note here that in this experiment FCS with the viscosity of water as may be expected from measurements have been performed on a total of 77 Stokes-Einstein (SE) relationship (19). Hence, Cuvette- intermediate concentrations of urea between 0-7.1 M. FCS is suitable for measurements over a wide range of Measurements using such large number of concentrations temperature. While here we have used a limited range of of urea are possible in this setup due to compatibility of temperature, with appropriately designed cell holder even cuvette-FCS with automatic titrators.

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Figure 4: Applications of cuvette-FCS in a range of temperature (A) and in urea (B). A) τD of rhodamine B versus viscosity. Here visocity changes due to change of temperature of the solution. (B) τD versus viscosity of urea. Symbols represent data and the solid line is linear fit of the data. Both A and B show that τD increases linearly with viscosity, consistent with the Stokes-Einstein relationship (11). (C) Urea dependent unfolding of TMR-Nt-ApoE4. The circles represent hydrodynamic radius (Rh) measured by FCS and the squares represent fraction folded measured by CD at 222 nm. The solid lines are fits using a two-state model (21). Clearly, increase of Rh is consistent with loss of secondary structure due to unfolding of the protein.

Finally, we examine if cuvette-FCS can be successfully measurements over a large range of temperature. employed to study unfolding of proteins induced by urea. Furthermore, cuvette-FCS can be integrated with automatic Here we have used a folded protein, viz, TMR-labeled-Nt- titrators for fully automated titration experiments. Use of apoE4). The Nt-apoE4 is a four helix bundle (PDB ID, automatic titrators and temperature controlled cell holders 1gs9). The hydrodynamic radius (Rh) of Nt-apoE4 are particularly advantageous in acquiring large number of calculated using Hydropro10 is 2.67 nm (20). Figure 4C highly accurate data points as may be seen in Figure 4B shows that Rh of Nt-apoE4 increases monotonically from and C. Sherman and Haran have used single molecule 2.6 nm to 4.8 nm with increasing concentrations of urea FRET and FCS to measure GdnCl induced expansion of from 0 to 7.1 M. The Rh of Nt-apoE4 shows a sigmoidal protein L to determine the coil-globule (CG) transition transition consistent with cooperative unfolding of the point and calculated the per residue average solvation protein in urea. Furthermore, urea dependent changes of Rh energy (19). Furhermore, Schuler and coworkers have agree well with the changes in secondary structure content demonstrated that single molecule studies of protein measured by Circular Dichroism (CD). The midpoint of unfolding can be used to estimate net intrachain interaction transition measured by FCS and CD respectively are 4.0 energy of the unfolded form of a protein in a particular and 3.9 M of urea when fitted with a two-state model (21). solvent (12). However, there are only a few studies of However, FCS data show that Rh continues to increase even protein unfolding using FCS reported in the literature (10, after complete unfolding of the protein, indicating 11, 19). We have demonstrated that Cuvette-FCS can be expansion of the unfolded chain at higher concentrations of used to study chain collapse or expansion respectively due the denaturant (11, 12, 22). to folding or unfolding of proteins with high sensitivity and Here we demonstrate that FCS measurements can be accuracy (see figure 4C). Protein unfolding data reported performed inside regular cuvettes with high sensitivity. In here show that changes in secondary structure and the our current setup we have obtained CPM greater than 44 hydrodynamic size of the Nt-apoE4 are almost kHz from aqueous solution of rhodamine B and a confocal concomitant. These results are consistent with those volume less than 2 fl. Expectedly, the sensitivity and the reported earlier for denaturation of IFABP (10). However, spatial resolution are lower than that can be achieved in our Sherman et al have found that the midpoints of transition homebuilt microscope-based FCS setup (data not shown). measured by FCS and by CD are somewhat different in However, the maximum CPM obtained using commcercial case of adenylate kinase (AK) protein (11). We speculate microscope based FCS are reported to be about 30 – 50 here that cuvette-FCS can be useful in addressing several kHz (from alexa488 in aqueous buffer) and the confocal other problems. For example, FCS has been used to volume obtained is about 1.0 fl (11, 23) . Therefore, the monitor formation of oligomers and protofibrils in the early sensitivity and the spatial resolution of cuvette-FCS is stages of protein aggregation (24). However, nucleation comparable to some of the commercially available dependent aggregation kinetics are generally slow, microscope-based FCS. occuring over several hours, days or weeks. Therefore, in Cuvette-FCS offers several advantages over microscope vitro these processes are accelarated by agitations such as based FCS instruments. Two major advantages have been stirring and/or by raising the temperature of incubation demonstrated here. For example cuvette FCS allows (25). Cuvette-FCS would be suitable to monitor such processes. Cuvette-FCS can also be used for measurements

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in non-aqueous solvents including corrosive solvents such 11. Sherman, E., A. Itkin, Y. Y. Kuttner, E. Rhoades, D. Amir, E. as those experiments involving solvent dependent dynamics Haas, and G. Haran. 2008. Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured of colloids, polymers or biopolymers such as intrinsically Proteins. Biophysical Journal 94:4819-4827. disordered proteins. Furthermore, cuvette-FCS can offer 12. Hofmann, H., A. Soranno, A. Borgia, K. Gast, D. Nettels, and B. significant advantage in measurements at very low Schuler. 2012. Polymer scaling laws of unfolded and intrinsically concentrations of biomolecules due to the low surface to disordered proteins quantified with single-molecule spectroscopy. Proceedings of the National Academy of Sciences of the United volume ratio of cuevttes and due to compatibility of its States of America 109:16155-16160. surfaces to passivation by covalent linkage of PEG to 13. Zettl, H., W. Häfner, A. 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