Live-cell 3D super-resolution imaging in thick biological samples

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1 Nature Methods Live-cell 3D super-resolution imaging in thick biological samples Francesca Cella Zanacchi, Zeno Lavagnino, Michela Perrone Donnorso, Alessio Del Bue, Lauria Furia, Mario Faretta & Alberto Diaspro Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Results 1 Supplementary Results 2 Supplementary Results 3 Supplementary Results 4 Set-up used for IML-SPIM Selective plane photoactivation Background comparison between widefield and SPIM imaging schemes Localization accuracy for IML SPIM images within cellular spheroids expressing Connexin43-PAmCherry 3D super resolution imaging in depth by means of elliptical PSF distortion 3D super resolution imaging in depth of nuclear PAmCherry protein. Scattering effects on deep single molecule detection capability Light sample interaction along the illumination path Point spread function characterization in thick scattering samples Background comparison between widefield SPIM Localization precision and resolution degradation in complex biological samples 3D super resolution by astigmatism-based technique Scattering effects on the single molecule detection and localization Note: Supplementary Video 1 is available on the Nature Methods website.

2 Supplementary Figure 1 Schematic representation of the IML-SPIM set-up.

3 Supplementary Figure 2 Selective plane photoactivation Activation process initiated by violet laser (405 nm) in single plane illumination system: photoactivation experiments have been performed on cells expressing H2B-PAmCherry fusion protein (A). For photoactivation an intensity of 0.2 kw/cm 2 is employed and a readout laser (561 nm) of 0.5 kw/cm 2 is used to excite the sample. The exposure time is 500 ms. Both the activation and the readout lasers have been maintained continuously running after the initiation of photoactivation (t = 13 s). Images related to different total irradiation times are shown in (A). The mean fluorescence intensity (emitted by PAmCherry molecules within the cell nucleus) is reported over time, showing the competition of the photoactivation and the photobleaching processes (B). A 40x detection lens of NA = 0.8 is used and the light sheet thickness is approx. 4 µm.

4 Supplementary Figure 3 Background comparison between widefield and SPIM. Signal to noise ratio improvement provided by SPIM. Imaging of FITC labeled nanocapsules performed using single plane illumination microscopy (A). Comparison between single plane illumination microscopy (B) and widefield imaging (C) of nanocapsules. Images are acquired in SPIM and widefield using the same excitation intensity at 488 nm (0.9 kw/cm 2 ) and same exposure time (100 ms). (B) and (C) clearly show the background reduction, provided by the single plane illumination scheme compared to widefield, due to out of focus contributions. The thickness of the light sheet is approximately 4 µm.

5 Supplementary Figure 4 Localization accuracy for IML SPIM images within cellular spheroids expressing Connexin43- PAmCherry Direct estimation of the effective localization accuracy within the cellular spheroid by repeated measurements on targeted sub-resolved molecular aggregates or on individual molecules. The localization accuracy was determined from repeated localization of point like objects in the spheroid which provide SNR conditions typical of IML SPIM experiments. The typical number of collected photons/molecule in IML SPIM imaging ranges from 100 to 500 photons and the measured background photons within spheroids (which is typically in the range of b = 3 4 photons/pixel) remain constant with depth (up to 100µm). Images are performed under these conditions (histogram of the collected photon number is shown in (B) and the image of a single localized molecule is shown as an example (A)). The spatial distribution of localizations in the radial (C) and axial (D) dimensions are shown. As expected, the localization accuracy in the axial direction was approximately twice the localization accuracy measured in the radial one. The histogram of localizations was generated by aligning different localized clusters by the mean value (each cluster contains more than ten localization events). Fitting with a gaussian function yielded to a standard deviation of 26 nm in the radial direction and 60 nm in the axial one. The corresponding FWHM provided an estimation of the localization accuracy within complex samples such as cellular spheroids (63 nm and 141 nm in the radial and in the axial direction, respectively). Images are performed using the experimental conditions typically used for single molecule detection in IML-SPIM experiments (frame rate of 50 ms and at readout laser power of 5 kw/cm 2 ). The thickness of the light sheet used to perform this experiment is approximately 1.8µm and the detection lens used is a 100x water dipping objective lens (NA = 1.1).

6 Suppplementary Figure 5 3D Super resolution imaging in depth by means of elliptical PSF distortion (A) The intensity point spread function obtained imaging sub-resolved fluorescent beads (Supplementary Results 3) at different depths showed the asymmetry due to astigmatism (induced inserting a 500 mm cylindrical lens in the detection path). IML SPIM is used to image human mammary MCF10A cell spheroids expressing Connexin43 PAmCherry 36 in 2D (B) or in 3Dsuperresolution configuration (C). Total amount of events: 30,000. The average localization precision obtained is 28 nm and the axial resolution obtained using astigmatic PSF is approximately 65 nm (Figure 1g). Photoactivation is achieved by 405 nm employing an activation intensity I = 0.25 kw/cm 2. Images have been acquired using 561 nm excitation at 5 kw/cm 2 and exposure time t = 40 ms. Total acquisition time is approximately 2 min. To increase the optical sectioning capabilities of the system, the thickness of the light sheet used to perform this experiment is approximately 1.8 µm and the detection lens used is a 100x water dipping objective lens (NA 1.1). Scale bar = 1 µm.

7 Supplementary Figure 6 Scattering effects on deep single molecule detection capability (A) Top view schematic diagram of the imaging geometry based on the calculations of the total optical path length within the sample (Supplementary Results 4). The red dot indicates a representative position of the sample within the gel cylinder. SPIM images of MCF10 acini at different depths within the samples (B). Sparse photoactivation has been induced by 405 nm radiation and the signal collected upon continuous 561 nm illumination. Single frame images showing the IML SPIM capability to detect single molecules (C) at different imaging depths (10 μm, 40 μm, 70 μm and 100 μm). Frequency histograms of number of photons collected for each molecule (D) show that single molecules can be detected without any appreciable loss in term of collected photons. For single molecule imaging of PAmCherry nuclear protein the activation intensity used is 0.05 kw/cm 2 and the readout one is 11 kw/cm 2. Scale bar 10 μm.

8 Supplementary Figure 7 Light sample interaction along the illumination path Images of the light sheet thickness for different optical pathways through a phantom sample with scattering coefficient 50 mm -1 (see Online Methods for sample preparation details). Images of the light sheet thickness has been collected rotating (90 ) the cylindrical lens in the conventional SPIM architecture. The position of the light sheet has been centered at 50 µm (A), 100 µm (B), 200 µm (C) deep within the sample. On the right, the intensity profiles along the direction of the light propagation (Y) show that no significant decay occurs over the entire field of view (Intensity fluctuations are within 5%). The excitation wavelength used was λ = 488 nm and laser intensity 0.3 kw/cm 2. Scale bar 10 µm.

9 Supplementary Figure 8 Point Spread Function characterization in thick scattering samples Lateral and axial experimental point spread function in non scattering samples (A) and in a phantom -1 sample (B) with scattering coefficient 50 mm (as described in Online Methods). PSF are measured imaging sub- diffraction sized beads (40 nm). Images are acquired 100 µm deep into the sample. The FWHM of the lateral PSF in non scattering samples (485 ± 4 nm) and in samples with scattering properties (489 ± 7 nm) remained constant. The axial extent of the PSF is (2.69 ± 0.25 µm) in case of non scattering sample and is (2.71 ± 0.33 µm) in the scattering one. Excitation wavelength used was λ = 488 nm and the laser intensity 0.15 kw/cm 2. Scale bar = 1 µm.

10 Supplementary Figure 9 3D Super resolution imaging in depth of nuclear PAmCherry protein. IML SPIM is used to create 3D super resolution images of a nucleus within a human mammary MCF10A cell spheroids expressing H2B-PAmCherry. IML SPIM imaging (A) of cell nuclei deep within a spheroid. The final image has been reconstructed after the collection and localization of 31,000 molecules. As a comparison the SPIM image obtained by adding the total signal over the frames is shown in (B). A z color coded map of the cell represented in the right region of (A) and (B) is shown in (C) to visualize z localizations corresponding to µm. XY section of the very same cell nucleus (D) and the 3D volume corresponding to the ROI in (D) is represented in (E) showing the XZ intensity projection. Supplementary Video 1 steps through XY slices within the sample. Imaging depth is 50 µm. Images are acquired with a frame rate of 50 ms and at a laser power of 5 kw/cm 2. Activation laser intensity I = 0.04 kw/cm 2. The activation and excitation wavelength used are λ = 405 nm and λ = 561 nm respectively. The thickness of the light sheet used to perform this experiment is approximately 1.8 µm and the detection lens used is a 100x water dipping objective lens (NA = 1.1). Total acquisition time for (A) is 3 minutes.

11 Supplementary Results 1 BACKGROUND COMPARISON BETWEEN WIDEFIELD SPIM Supplementary Figure 3A shows the sample as imaged by conventional SPIM. Supplementary Figure 3B, obtained using SPIM configuration, demonstrates the improvement in terms of background signal when compared to the image produced by conventional widefield illumination (Supplementary Figure 3C). The average number of photons/pixel in the background, calculated in regions inside the nanocapsules (Supplementary Figure 3B, C), is reduced in case of single plane illumination configuration (3.3 ± 0.4 photons/pixel) compared to the widefield illumination scheme (6.4 ± 0.6 photons/pixel). The measurement has been obtained by repeating the measurements statistically over 20 samples. Measurements are obtained by calculating the background in the center of the nanocapsules, and the obtained results are in agreement with measurements previously performed in the single molecule regime and presented in the literature 27. Supplementary Results 2 LOCALIZATION PRECISION AND RESOLUTION DEGRADATION IN COMPLEX BIOLOGICAL SAMPLES In order to provide a demonstration of the IML SPIM imaging capabilities we reported in the manuscript the value of the localization precision loc which was found to be less than 35 nm. This value represents the error due to the localization process and was calculated according to the analytical model earlier presented in literature 28, 29 : 2 loc 2 2 a s sb 2 N 9 a N where the term due to the background noise can be neglected thanks to the background suppression provided by single plane illumination. a represents the pixel size, N is the number of collected photons, s is the radial point spread function of the system and b is the background noise. For practical imaging of large scattering biological samples several limiting factors, mainly related to scattering and aberration effects, can contribute to a decreased effective localization precision. To consider additional errors induced in the localization process, the precision can be redefined by considering also the standard deviation inst of the instabilities of the system loc inst where the factor of the EMCCD. 2 takes into account for the excess noise introduced by electron multiplying process Concerning the axial accuracy of IML SPIM, tested on model samples by repeated localizations of subresolved fluorescent beads (Supplementary Results 3), the measured value was found to be approximately 65 nm. However, localization of a bead, composed of bright and randomly oriented fluorophores, does not provide the same localization performance of single molecules. Again, in case of

12 localization of single emitters within large scattering samples the effective localization precision will be limited by the low photon regime. In fact, even if the axial accuracy provided by the system was found to be 65 nm in model samples, the real localization accuracy could be affected by background signal and low light conditions when imaging biological complex samples. In order to experimentally evaluate the effective accuracy of the axial coordinate determination and to estimate the overall localization precision, point-like fluorescent objects have been repeatedly localized directly inside the cellular spheroids (Supplementary Figure 4). The measurement has been performed in the same experimental conditions used for IML SPIM imaging and led to an effective accuracy of about 63 nm in the radial direction and 141 nm in the axial one. This measurement provides the closest estimate of the effective resolution 31 obtained by IML-SPIM in large samples, since it takes into account for aberrations due to light-sample interactions and for additional errors in the localization precision due to instabilities of the system. Therefore, the experimental measure of the localization accuracy deep within cellular spheroids proves the super-resolution imaging capabilities of IML SPIM within large scattering samples. In this case, even if the localization precision itself strongly differed from the localization accuracy, a radial resolution of 63nm and axial resolution of 140 nm has been demonstrated (Supplementary Figure 4). Supplementary Results 3 3D SUPER RESOLUTION BY ASTIGMATISM BASED TECHNIQUE 3D super-resolution techniques, based on multiplane detection 32 and on astigmatic distortions, have been presented recently in literature 33, 34. In order to extend the imaging capabilities of the system to 3D super-resolution we implemented the method based on astigmatic distortions 33,35. The introduction of weak astigmatism, by inserting a cylindrical lens (f = 500 mm), into the detection path provided an elliptically stretched point spread function along a lateral axis depending on the axial position (Supplementary Figure 5A). The experimental 3D point spread function can be obtained by imaging subresolved fluorescent beads by scanning the sample through the focus of the detection lens. Calibration has been performed by acquiring a z stack of 40nm fluorescent beads (Invitrogen) with z step of 50 nm. Model images of 40 nm fluorescent markers at sampled z position (the 0 coordinate is referred to the light sheet center) are shown in Supplementary Figure 5A. Images are acquired in a scattering phantom sample (50 mm -1 scattering coefficient) at an imaging depth of 60 µm. Although the PSF was found not to be depth dependent in case of homogeneously scattering samples (Supplementary Figure 8), sample-dependent variations in the astigmatic PSF, due to irregular shape of the sample and scattering variations, can occur while imaging biological samples. This can induce small distortions in the elliptical shape of the PSF which cannot be easily taken into account by a theoretical PSF model. In order to remove errors induced in the assignment of the axial position of single molecules, the calibration was always performed by acquiring beads in the surroundings of the imaged region. To this end fiducial fluorescent markers are embedded together with the sample in the agarose gel. The 3D intensity distribution obtained is fitted to an elliptical Gaussian function and used to extract calibration curves. In such a way the relation between the x and y dimensions of the radial PSF and the z position of the particle is made explicit. Once the calibration curves are available it is possible to parameterize the x and y dimensions in terms of z. Non linear regression is then used to fit the intensity distributions in order to extract the z coordinate. The spatial resolution along the z direction provided by the system can be calculated by localizing sub-resolved fluorescent beads several times.

13 The histogram of the distribution of z coordinates (Figure 1g) is obtained localizing at least six beads 300 times each 33. The z coordinate is calculated and each distribution is aligned by the mean value (the obtained distribution is shown in Figure 1g). The standard deviation of the obtained gaussian distribution is 27 nm, which corresponds to an effective resolution of about 65 nm. IML-SPIM is used to create 3D super-resolution images in depth of overexpressed Connexin43-PAmCherry (Supplementary Figure 5C) and H2B- PAmCherry in infected human mammary MCF10A cell spheroids. Supplementary Figure 9A, 9B show the comparison between SPIM and 3D superresolution images. In addition, 3D images are shown using both a z-color coded map (Supplementary Figure 9C) and orthogonal projections (Supplementary Figure 9D, 9E). Supplementary Video 1 shows XY slices through the sample with 116 nm z step. Supplementary Results 4 SCATTERING EFFECTS ON THE SINGLE MOLECULE DETECTION AND LOCALIZATION The single molecule imaging capability can be affected by scattering phenomena and light-sample interactions while imaging large biological samples. Since image quality degradation with the optical path length through the sample is expected, we investigated the scattering effects both in the illumination and the detection pathways. We considered that the total optical path length is composed by the contribution due to the illumination and to the distance covered by the emitted photons to the imaging lens. To schematically show the geometry of the SPIM imaging architecture in relation to the length of the optical pathway (Supplementary Figure 6A) we calculated the total optical path length within an object having cylindrical symmetry (i.e. the agar cylinder experimentally used). The optical path length (OPL) is related to the image quality and is defined as: C Optical Path Length n s ds (1) where n(s) represents the local refractive index of the sample and C is the pathway through the sample. The optical path length has been calculated (Wolfram Mathematica 7), according to eq.1, for a homogeneous sample. The imaging geometry is sketched in Supplementary Figure 6A. A density map schematically visualizes the relationship between sample position and path length. Dark regions correspond to a short OPL and a better image quality while bright regions represent a long OPL and correspond to a degraded imaging capability. Since the imaging performance degrades with the increased total travel of the light within the sample, the contributions due to scattering processes along the detection path and the illumination path needed to be investigated separately. To this end, the number of photons collected for each single molecule has been verified at different depths within a large biological sample. Supplementary Figure 6B showed a series of selected planes of an early-stage mammary acinus formed by MCF10A infected cells, 30 μm spaced through its entire extension and positioned 50 μm apart from the agar edge. In Supplementary Figure 6B SPIM optical sections show the growing cells with the formation of a cavity leading to generation of the internal duct. The ability to perform individual molecule detection (Supplementary Figure 6C) is shown at different depths (10 µm, 40 µm,70 µm and 100 µm) within the whole 3D spatial organization of the sample. In order to evaluate the effects due to scattering through the detection path, the effective number of photons for each single event is calculated at different depths within the

14 entire spheroid. Despite a small fraction of emitted photons lost due to scattering effects, histograms in Supplementary Figure 6D showed that no appreciable loss occurs in the depth range µm within the spheroid. Nevertheless, is worth noting that scattering effects could play a relevant role in single molecule detection at greater imaging depths (> 100 µm), affecting the number of collected photons/molecule and leading to a depth-dependent localization precision. Here we showed that no appreciable loss of collected photons/molecule through the entire volume of the spheroid occurs (Supplementary Figure 6D). This implies a negligible effect of the imaging depth on the localization precision which strictly depends on the number of collected photons, according to analytical models presented in the literature 28,29. To investigate the effects due to the scattering processes through the illumination path, the light sheet intensity distribution at different positions is acquired (Supplementary Figure 7). Distortions in the shape of the illumination intensity distribution due to light-matter interaction may occur while imaging scattering and non transparent samples. To investigate this phenomenon, direct imaging of the emitted fluorescent intensity is performed using fluorescent phantom samples. The cylindrical lens is rotated by 90 in order to image the thickness of the light sheet directly onto the CCD camera (Supplementary Figure 7). Images of the light sheet intensity profile are acquired at different light sheet positions (50 µm, 100 µm and 200 µm). The intensity distribution along the direction of the light propagation (y) at different illumination path lengths clearly shows that no significant decay occurs over the entire field of view (intensity fluctuations are within 5%). The obtained result demonstrated that, under these experimental conditions, the illumination intensity distribution can be assumed as uniformly distributed along the entire field of view (80 µm x 80 µm). Furthermore it also confirms a negligible distortion in the light sheet shape. Since the localization precision depends on the number of collected photons/event as well as on the characteristics of the imaging system used 28, the experimental measurement of the PSF in scattering phantom sample is also provided (Supplementary Figure 8). Experimental point spread functions are measured by imaging sub-resolved 40 nm fluorescent beads (Invitrogen). Images are acquired at 100 µm imaging depth. Lateral and axial experimental point spread functions have been experimentally measured in non scattering samples (Supplementary Figure 8A) and in a phantom scattering sample (Supplementary Figure 8B) having a scattering coefficient of 50 mm -1 (prepared as described in Online Methods). The x z section of the intensity point spread functions showed weak aberration effects in both the cases. The PSF FWHM, which influences the localization precision value 28, is calculated by gaussian fitting. The lateral PSF in non-scattering samples (485 ± 4 nm) and in samples with scattering properties (489 ± 7 nm) remained constant. The invariance of the radial PSF, coupled with the neglectable loss of collected photons/molecule along the detection path, assured an invariant localization precision (within an imaging depth range of µm). The experiments described in this section have been performed using a light sheet thickness of approximately 4 µm and a 40x imaging lens (NA = 0.8). REFERENCES 27. Ritter et al. Opt. Express 16, (2008). 28. Thompson, R.E., Larson, D.R. & Webb, W.W. Biophys. J. 82, (2002) 29. Mortensen, K.I. et al. Nat Methods 7, (2010)

15 30. Aquino, D. et al. Nat Methods 8, (2011) 31. Hess, S.T. et al. Methods Mol Biol 544, (2009) 32. Juette, M.F. et al. Nat Methods 5, (2008) 33. Huang, B. et al. Science 319, (2008) 34. York, A.G. et al. Nat Methods 8, (2011) 35. Kao, H.P. & Verkman, A.S. Biophys. J. 67, (1994) 36. McLachlan, E. et al. Cancer Res 66, (2006)