Supplementary Materials for

Transcription

1 Supplementary Materials for Mapping the subcellular mechanical properties of live cells in tissues with fluorescence emission Brillouin imaging Kareem Elsayad,* Stephanie Werner, Marçal Gallemí, Jixiang Kong, Edmundo R. Sánchez Guajardo, Lijuan Zhang, Yvon Jaillais, Thomas Greb, Youssef Belkhadir* *Corresponding author. (K.E.); (Y.B.) This PDF file includes: Published 5 July 2016, Sci. Signal. 9, rs5 (2016) DOI: /scisignal.aaf6326 Note S1. Details of FBi. Note S2. Effect of anisotropy in n for calculated M. Note S3. Effect of uncertainty in n and for calculated M. Note S4. Experimental determination of the refractive index and density of Arabidopsis ECMs. Fig. S1. Schematic of the FBi setup. Fig. S2. Sketch of the used microscope and spectrometer setup. Fig. S3. FBi scans of epidermal onion cells. Fig. S4. FBi scans of Lti6-tdTomato root cells. Fig. S5. Width measurements of Arabidopsis hypocotyl longitudinal ECMs. Fig. S6. Effect of plasmolysis on ECM and nearby cytoplasm. Fig. S7. Determination of refractive index and density of Arabidopsis ECM by timeresolved fluorescence studies. Fig. S8. Cross-sectional Brillouin imaging scans of cells in deep layers of Arabidopsis roots. Fig. S9. Cross-sectional FBi scans of wild-type Arabidopsis root cells. Reference (69)

2 Supplementary Notes Note S1. Details of FBi Brillouin light scattering (BLS) spectroscopy involves measuring the spectral shift resulting from inelastic scattering of monochromatic light from density oscillations (acoustic phonons). The spectral shift is typically in the GHz frequency range, thus requires accurate spectroscopic techniques and narrow frequency probing excitation. The implementation of Virtually Imaged Phase Array (VIPA)-based spectrometers (US patent # US A) has enabled the application of Brillouin spectroscopy at laser intensities and acquisition times conducive to studying biological samples, and this approach has been implemented as an imaging technique for live cell studies (18). From a liquid-like sample, a typical Brillouin scattering spectra will consist of a symmetric pair of peaks the so-called Stokes and anti-stokes peaks - on each side of the elastic (Rayleigh) scattering peak, which correspond to the creation and annihilation of an acoustic phonon, respectively. Information on the hypersonic velocity may be extracted from the spectral shift of these peaks relative to the Rayleigh peak, called the Brillouin Frequency Shift (BFS) and denoted by ω B. In particular, for a scattering angle Ψ the BFS is proportional to the hypersonic velocity (v) and given by: ω B (Ψ) = 2ω 0 ( n ) v sin c (Ψ ) (Eq. 1) 2 where n is the refractive index of the sample and c is the vacuum speed of light. Note S2. Effect of anisotropy in n for calculated M In an optically anisotropic material, the angle(s) of probing and detection would affect the calculated values of M. Because the ECM likely has some anistropic optical properties, it is conceivable that, given the different orientations of the ECMs along the long and short sides of cells relative to the probing and detection angle(s), this can lead to a systematic offset in the calculated M. With our imaging geometry, the thickness of the cellulose microfibril layers probed in the ECMs along the long and short sides of cells will be comparable, with the only possible difference being in the orientation of microfibrils relative to the imaging axis for the two types of ECMs, which could cause a different effective refractive index for the calculation of M. The measured refractive index of microfibrils is along the ordinary axis (length of the fibril) and in the so-called extra-ordinary direction (perpendicular to the fibril) (70). Therefore, the orientation of the microfibrils should cause no more than a 5% deviation in the calculated M. Therefore, we consider it unlikely that optical anisotropy of the ECMs along the long and short sides of cells significantly contributes to the observed differences in the calculated elastic storage modulus M. Note S3. Effect of uncertainty in n and ρ for calculated M The uncertainty in the elastic storage modulus δm' will be determined largely by the uncertainty in the refractive index δn, density δρ, and measured BFS δω_b : δm'/m'=(2δω_b)/ω_b +2δn/n+δρ/ρ (Eq. 2) We found that the uncertainty in the fitted Brillouin peak averaged no more than a few percent (< 2%). Therefore, with the variation in the refractive index and density between the cytoplasm and the ECM (n = , and ρ = kg/m 3 ) (69), the maximum uncertainty would be <20% in the calculated M. The transitions in M from the ECM to the cytoplasm, even in the seemingly stiffer region of the cytoplasm in part of the cell closest the membrane and thus closest the ECM (Fig. 2 C and F), were larger than this and thus are unlikely to result from assigning incorrect values or unaccounted for variations in these parameters. Note S4. Experimental determination of the refractive index and density of Arabidopsis ECMs On the basis of their similar constituency, one would not expect the density or the refractive index to vary significantly between the ECMs along the long and short sides of elongating cells. Yet, variations between the two types of ECMs cannot be ruled out. Determining the local density variation is challenging, representing a main bottleneck in obtaining accurate elastic moduli from BFS. Because the calculated elastic moduli scale linearly with the refractive index, such differences in local density will not have a substantial effect. For example, even a change in the density from 1100 kg/m3 (as has been reported for onion ECMs) to that of water (1000 kg/m3) would only constitute a 10% change in the calculated elastic moduli, such that the observed difference in BFS cannot result from a difference in density. The refractive index has a larger impact in the calculation of the elastic modulus (Note S1, Eq. 1). The refractive index can be measured near an interface using either phase contrast or interference approaches. However, for distances further than a few wavelengths from an interface, these methods either are not applicable or no longer accurate. Thus, as a semi-quantitative check that the refractive index does not differ significantly between the ECMs of the long and short sides of cells, we exploited the fact that the fluorescence lifetime of many fluorophores depends on the local refractive index. We performed time-resolved confocal fluorescence lifetime measurements to confirm that refractive

3 index variations constituted no more than 5-10% deviations in the calculated values of the elastic modulus (fig. S7). Calculation of the hypersonic velocity (v) from the BFS (ωb) requires knowledge of the refractive index (n) in the probed region. Unaccounted for variations in the refractive index in the probed region will lead to inaccurate conclusions on the viscoelasticity parameters. In particular, an unaccounted for increase in the local refractive index will result in an underestimation of the hypersonic velocity and vice versa. To directly compare the stiffness of the ECMs of the long and short sides of cells we needed to determine to what extent the refractive index might differ between these two types of ECMs. We performed time-resolved fluorescence studies of fluorophores in the immediate vicinity of the ECM (at the plasma membrane). Because the decay rates are correlated to the local refractive index, these time-resolved fluorescence data indicated that there is negligible difference in the refractive index between the two types of ECMs in the studied Arabidopsis cells. The local photonic environment affects the spontaneous decay rate of dipole-like emitters (40). In particular, the radiative decay rate of a molecule will scale as the square of the local refractive index as predicted by the Strickler Berg equation (41-44). The coefficient depends on the transition dipole moment strength, which is also related to the absorption coefficient. Variations of <5% in the local refractive index can readily be measured in live cells with high brightness fluorophores (45). In many cases, the scaling of the decay rate with refractive index may differ from the theoretical quadratic dependence predicted for a pure electric-dipole-like transition due to additional effects, such as interactions with chemicals or solvents (45); however, the variations in the decay rate with refractive index would still be present. Furthermore, if one assumes similar chemical constituents in the probed regions (as is the case for the ECMs), the effects from refractive index variations should dominate. Fluorescence lifetime measurements, thus, offer a means of probing the local refractive index variations in and between ECMs. For studying the variation of refractive index of ECMs in Arabidopsis hypocotyl, we used plants with mcitrine labelled plasma membranes (Arabidopsis 35S::BRI1-mCitrine) (46,47). We chose mcitrine for several reasons. Its excitation and emission spectra have very little overlap with the autofluorescence in the studied cells. Its high quantum yield and extinction coefficient would result in notable variations in the lifetime for different refractive indexes. To perform our analysis, young seedlings were gently picked from the phytogel dish (on 7th day after germination), placed on a glass bottom dish (P35G C, MatTek), and covered with a phytogel pad. We imaged only the outer cells of the hypocotyl for direct comparison with our Brillouin scattering measurements. Time-correlated singlephoton counting (TCSPC) measurements were performed using an objective scanning confocal setup - MicroTime 200 (Picoquant, Berlin). Excitation was with a 510 nm pulsed laser diode (at 40 MHz repetition rate, pulse width ~100ps). For all measurements the confocal pinhole was fixed at 30 m, and the detection was through an appropriate interference bandpass filter (534/30 Semrock). For all imaging, we used a 60x 1.2NA water immersion objective, with lateral scans performed such that the resulting images had an effective pixel size of 0.4 μm. Data analysis was performed using SymPhoTime 64 v2 (Picoquant, Berlin). Prior to analysis, all data were re-convolved with the Instrument Response Function (IRF) measured at the probed excitation wavelength. Due to notable autofluorescent in the hypocotyl, we employed a double exponential fit. Chi-squared values were consistently <1.1 for all fits of the ECMs (fig. S7).

4 Figure S1. Schematic of the FBi setup. DIC = Dichroic Mirror, PMT = Photo-Multiplier Tube. Top Insets: transmitted light picture, viscoelasticity scans (red-black), FBi scans (blue-yellow).

5 Figure S2. Sketch of the used microscope and spectrometer setup. Three different types of spectrometer designs were used to obtain results in this study, because the spectrometer was being optimized and improved throughout the course of the study. Calibration studies (on distilled water, ethanol and glycerol) indicated that all spectrometers obtained the same results, except that designs B and C obtained a ~25% higher finesse (ratio of the free spectral range to the bandwidth) than A. Design C generally produced signals with less noise than B for transparent samples. Design B proved favorable in samples with more light scattering however yielded a lower spectral resolution. (A) Setup A, the basic setup, involves a single dispersion axis (out of page), an adjustable spatial mask (to remove unwanted and elastic scattering peaks), and magnifying optics (to guarantee adequate sampling on the EM-CCD chip). It offers poor background rejection, however achieves the highest throughout and is the easiest to align. It was used to obtain results from Fig. 1. (B) In setup B cross dispersion is provided by two large equilateral N-SF11 prisms. Note: Two back-to-back cylindrical lenses with one rotated 90-degrees around the optical axis relative to the others are used (instead of a single spherical lens) after the prisms to finely tune the focus of the intermediate image on Mask#2 in both perpendicular directions. It was used to obtain results in Fig. 2 and Fig. 4. (C) Setup C provides cross dispersion from a second VIPA and is based on the design described (32). It provides the cleanest and highest resolution spectra for fairly homogeneous samples, however was more challenging to align than A and not as effective as B in rejecting unwanted inelastic scattering in more highly scattering samples. It was used to obtain the data in Fig. 3 and 5. All lenses had a focal length of 200 m except for the magnifying lenses in front of the camera. The camera was mounted on a 3-axis manual translation stage to finely position the chip so it coincided with the region of interest. Analysis of 2D spectra imaged on the camera was performed as described (18). Routine calibration measurements on water and glycerol were performed to calibrate dispersion scales and axes.

6 Figure S3: FBi scans of epidermal onion cells. (A) Confocal fluorescence image of onion cells treated with FM4-64. (B) Left: Plot of BFS as a function of distance across ECM. Middle: Plots of fluorescence as a function of distance. Right: FBi correlative plots establishing the presence of pixels of high fluorescence intensity in epidermal onion cells treated with FM4-64. (C) same as in (B) but on samples not treated with FM4-64, showing no high fluorescence pixels.

7 Figure S4. FBi scans of Lti6-tdTomato root cells. (A) Fluorescence image of Lti6- tdtomato roots treated with either water (left panel) or 0.8 M mannitol for 10 minutes (right panel). (B) Map of BFS (top) in xz plane and plot of z- averaged change in x-direction (bottom) for Lti6- tdtomato hypocotyl ECM. (C) Corresponding change in fluorescence intensity. (D and E) same as (B and C) but for a wild-type sample, showing high BFS at ECM but no discernable fluorescent intensity above background. Data are representative of at least 3 independent experiments.

8 Figure S5. Width measurements of Arabidopsis hypocotyl longitudinal ECMs. (A) Wide-field transmitted light image showing laterally scanned region (dotted white line) across an ECM region. (B) BFS as a function of axial depth (z) at the center of the ECM region and in a region of the cytoplasm. (C) 2D BFS heatmap (yz-scan) of the ECM and surrounding cytoplasm. Heat map shows the BFS in units of GHz. (D) Same as in (C) but the GHz scale has been reduced to emphasize the region of higher BFS in the vicinity of the ECM. (E) Plot of the variation [in transverse direction (y)] of the BFS for different axial positions (z). Black, green, and red lines represent three different axial depths separated by 5 m, showing that the region of increased BFS near the ECM varies and extends over a distance of ~5 m on each side away from the cell borders.

9 Figure S6. Effect of plasmolysis on ECM and nearby cytoplasm. (A-B) BFS as a function of distance for a transverse scan (x) across a longitudinal ECM prior to and after plasmolysis. (C and D) Fluorescence intensity corresponding to (A and B) before and after plasmolysis. (E and F) Representative transmitted light pictures of hypocotyls subjected to water and mannitol, respectively.

10 Figure S7. Determination of refractive index and density of Arabidopsis ECM by time-resolved fluorescence studies. (A) Intensity averaged lifetime map of ECMs from both the short and long sides of a typical elongating hypocotyl cell. The blue dots correspond to the chloroplasts (autofluorescent organelles with a short autofluorescent lifetime). Mild local fluctuations in the lifetime on the submicron scale are apparent. Yet, the overall lifetime varies little along the short and long sides of the cells, indicating that the refractive indexes of the ECMs are very similar. (B) Increased magnifications on regions of the short and long sides of the cells. (C) Lifetime distributions for the entire image shown in (B). The short component centered at ~0.4 ns (purple line) can be attributed to autofluorescence, whereas the longer component (green line) centered at ~3.25 ns is from the mcitrine signal. The distribution of the long component is symmetric (in ), implying a single lifetime distribution and further confirming that on average the lifetime of the short and long sides ECMs are the same. (D and E, F and G, H and I) Lifetime distributions in the three defined plasma membrane regions highlighted in fluorescent green: short side (D and E), long side (F and G), and junction (H and I). In each case, the lifetime is normally distributed around the same value ( =3.25 ns), which suggests that there is no significant change in the refractive index between these three ECMs.

11 Figure S8: Cross-sectional Brillouin imaging scans of cells in deep layers of Arabidopsis roots. Cross-sectional yz scans at three different points (as indicated in transmitted light wide-field image) in the vicinity of a root hair showing (A) distinct high-bfs focal regions near cell intersections. (B) A large area cross sectional scan through the entire root revealing what appears to be the relatively stiff vascular system at the center. (C) Cross-sectional scan through the root hair, also showing distinct high-bfs focal patterns between cells.

12 Figure S9. Cross-sectional FBi scans of wild-type Arabidopsis root cells: BFS (blue, top), and fluorescence intensity (red, bottom) control measurements for Fig. 4. Representative data from 3 independent measurements.