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Three Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging Anna M Chizhik, Daja Ruhlandt, Janine Pfaff, Narain Karedla, Alexey I. Chizhik, Ingo Gregor, Ralph Heinz Kehlenbach, and Joerg Enderlein ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04671 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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Three Dimensional Reconstruction of Nuclear Envelope Architecture Using DualColor Metal-Induced Energy Transfer Imaging Anna M. Chizhik1,#, Daja Ruhlandt1,#, Janine Pfaff2,#, Narain Karedla1, Alexey I. Chizhik1, Ingo Gregor1, Ralph H. Kehlenbach2,*, Jörg Enderlein1,*

1

University of Göttingen, Third Institute of Physics, Göttingen, Germany

2

Universitätsmedizin Göttingen, University of Göttingen, Department of Molecular Biology, GZMB,

Göttingen, Germany

#

Equal contribution

*Corresponding authors

ABSTRACT

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color Metal-Induced Energy Transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

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The eukaryotic cell nucleus is surrounded by a membrane system comprising the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), the latter being continuous with the endoplasmic reticulum (ER). The space between INM and ONM, which is topologically equivalent to the lumen of the ER, has a typical width of 30 nm - 50 nm.1-3 Both membranes fuse at the nuclear pore complexes (NPCs), which gate the transport of macromolecules between the nucleus and the cytoplasm (for review see refs.4, 5). NPCs are composed of a core that is embedded between the INM and the ONM and nuclear extensions forming a basket-like structure. Under certain conditions, filament-like extensions may also emanate into the cytoplasm.4 The NPC has a diameter of ~120 nm and a height of the core structure (perpendicular to the plane of the nuclear membrane) of ~80 nm, or of ~150 nm 200 nm when its nuclear and cytoplasmic extensions are taken into account. Most of the ~30 protein components of the NPC, the nucleoporins, are arranged in a symmetric manner with respect to the plane of the nuclear envelope.4 Some of them, however, are found exclusively on either the nuclear (e.g. Nup153 and Tpr) or the cytoplasmic side of the NPC. Nup358, for example, with 358 kDa the largest of all nucleoporins, localizes specifically on the cytoplasmic side,6 where it seems to function as an assembly or disassembly platform for transport complexes in transit.7-9 According to recent data obtained by cryoelectron tomography, Nup358 localizes to the cytoplasmic ring of the NPC.10 The protein composition of the ONM is very similar to that of the ER, although a number of ONM-specific proteins have been described as well. Proteins of the INM, in contrast, are clearly distinct and several hundred proteins that are enriched at this location have been identified, mostly by proteomic screens.11, 12 Many of these INMproteins interact with the nuclear lamina, a network of intermediate filament-type proteins called lamins and associated proteins that is found underneath the INM of metazoan cells (for review see ref.13). One of the best-described proteins of the INM is LAP2β (lamina-associated polypeptide 2 β; also referred to as thymopoietin β), a 452-amino-acid protein containing a single transmembrane domain close to the Cterminal end.14, 15 Several methods to demonstrate localization of a protein to the INM have been described. One method involves differential permeabilization of cellular membranes, the idea being that proteins of the INM are only accessible to antibodies upon permeabilization of the nuclear envelope. Hence, treatment of cells with digitonin, which permeabilizes the plasma membrane but leaves the nuclear membrane intact,16 exposes epitopes on the ONM but not on the INM. Treatment of cells with Triton X-100, on the other hand, permeabilizes essentially all cellular membranes, allowing detection of proteins both at the ONM and INM. Thus, a protein that can be detected upon Triton X-100 treatment but not upon digitonin treatment is likely to be associated with the INM. In addition to these studies in fixed cells, functional


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assays can be performed in living cells where a nuclear reporter protein can be recruited to the periphery of the nucleus upon rapamycin-induced dimerization with a protein that localizes to the INM.17, 18 Immuno-electron microscopy can be considered as the gold standard for protein localization studies, because of its high resolution which is basically only limited by the size of antibodies and metal particles used for labeling. It allows one to distinguish proteins at the INM and the ONM or, for example, at the nuclear and the cytoplasmic side of the NPC.6, 19 This method requires, however, specific antibodies of very high quality and rather harsh and complex fixation procedures. In light microscopy, the lateral and axial diffraction limits are in the range of 250 nm and 800 nm, respectively. Hence, conventional light microscopy cannot faithfully localize a protein of interest at the INM or ONM, because the distance between them is well below these limits. However, optical superresolution microscopy techniques achieve a resolution of the order of tens of nanometers in routine measurements,20-22 making them applicable for studies of the cell nucleus. In particular, dSTORM23 and STED microscopy24 have been used to analyze the structure of the NPC at a lateral resolution of about 20 nm. In a recent paper, Yang and co-workers used single-point Fluorescence Recovery After Photobleaching (FRAP) in conjunction with single-molecule localization (SML) for measuring the lateral INM-ONM distance in an equatorial section through the nucleus to be 36 nm - 40 nm,25 exploiting the high lateral resolution of SML. However, almost all of these super-resolution techniques have an axial resolution which is by a factor 3-5 worse than their lateral resolution, which typically does not allow them to distinguish between the INM and ONM along the optical axis. The only methods which indeed achieve nanometer resolution in fluorescence microscopy along the optical axis are interferometric PALM (iPALM)26 and 4Pi-STORM,27 but for the cost of requiring an exceptionally complex and difficult to operate interferometric setup. We have recently developed a simpler method to precisely measure distances of fluorescent molecules from a surface, which is termed Metal-Induced Energy Transfer (MIET).28 In a MIET measurement, the sample of interest is placed on a substrate that has been coated with a thin, semitransparent metal film. The electromagnetic near-field of fluorescent emitters close to the metal couples to surface plasmons of the metal film, thus transferring energy from excited molecules to the metal. This leads to a decrease in an emitter’s fluorescence lifetime τ, which can be measured using a fluorescence-lifetime imaging microscope (FLIM). Up to a distance of ∼150 nm above the metal, there exists a monotonic relationship between the distance h of an emitter to the metal surface and its ACS Paragon Plus Environment

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fluorescence lifetime τ, yielding an unambiguous measure for an emitter’s distance from the surface. The well-known relationship between lifetime and distance can be used to directly convert the FLIM image into a height profile. So far, this technique has been used to measure height maps of the basal membrane of different cell types,28, 29 and to determine the axial position of single molecules with an accuracy of 2.5 nm.30 In this study, we use Lap2β and Nup358 as proteins with defined localizations at the INM and the cytoplasmic side of the NPC, respectively, and determine the axial distance between the two proteins by dual-color MIET.28-30 The nanometer axial resolution of MIET allows us to measure the distance between the INM and ONM throughout the whole basal area of the nuclear membrane (i.e. the part that is close to the surface of the microscopic slide) and to reconstruct its 3D architecture.

RESULTS AND DISCUSSION Lap2β as a marker for the INM15 and Nup358 as a marker for the cytoplasmic side of the NPC (i.e. in approximation also for the ONM10) were labeled with antibodies coupled to two different organic dyes that allowed us to spectrally separate their signals. To assess the quality of our protein detection procedure, we first analyzed the localization of our proteins of interest by conventional confocal microscopy. HeLa cells were fixed and subjected to standard indirect immunofluorescence, detecting Nup358 and LAP2β within a horizontal plane through the center of the nucleus. Accordingly, a clear rim staining was observed for both proteins, characteristic for nucleoporins or proteins associated with the nuclear envelope (Figures 1c,d and 2c,d). Hence, the two proteins and our specific antibodies seem very appropriate for a MIET-approach. Measuring the fluorescence lifetimes of the two fluorophores should allow the determination of the distance of both proteins from the metal surface (i.e. the “height”). The height difference immediately yields the distance between the INM and the ONM. One of the key prerequisites of MIET imaging is a precise characterization of the fluorophores in the absence of a metal film (free space parameters). In particular, the emission spectra, fluorescence lifetimes, and quantum yields are required. Therefore, we determined the above parameters of AlexaFluor 488 (A488) and AlexaFluor 633 (A633) dyes under conditions close to those in our samples (see Methods for further details), allowing the calculation of exact calibration curves for our samples (Figure 6).


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For MIET imaging, cells were seeded on metal-coated substrates. To use the same sample preparation procedures as for glass surfaces as used for standard immunofluorescence, we coated the gold layer with a 10 nm thick silica (SiO2) layer. For better adhesion of the gold layer to the glass and the SiO2 layer, 2 nm thick intermediate titanium layers were added. Focusing the excitation light with a 1.49 NA objective lens onto the metal film allowed us to excite only molecules that are located within ca. 200 nm distance above the sample surface, that is within the working range of MIET. As a result, only fluorescence from the basal nuclear membrane was detected. Out of focus fluorescence was filtered out by a confocal pinhole. Two types of samples were examined: sample type I consists of Lap2β labeled with A633 and Nup358 labeled with A488 (Figure 1a), whereas in sample type II the fluorophores were switched (Figure 2a). Although Lap2β is expected to localize predominantly to the INM, a small proportion could be found at the ONM as well, e.g. prior to translocation of the protein via the NPC to the INM. This could lead to Förster Resonance Energy Transfer (FRET) between A488 and A633 at the locations on the ONM where the distance between Lap2β and Nup358 would be within FRET range, that is near or closer than 5 nm for the two dyes. Because FRET opens an additional de-excitation pathway for the donor (A488), this may lead to a decrease of its fluorescence lifetime (Figure 7). However, for MIET measurements, a reduction of fluorescence lifetime due to FRET would lead to wrong height values. To exclude any impact of FRET, fluorescence lifetime measurements of A488 were done after photobleaching of A633. Once the acceptor molecules are bleached, they cannot receive energy from the donor anymore, which results in a complete suppression of FRET. Typical fluorescence confocal images of basal nuclear membranes are shown in Figures 1e,f and 2e,f for sample type I and sample type II, respectively. Using FLIM imaging, for every pixel of an image we obtained both the fluorescence intensity and fluorescence lifetime. The latter was determined as the average arrival time of photons after an exciting laser pulse (see Methods for further details). Using the fluorescence lifetime images and the MIET calibration curves (Figure 6), we converted fluorescence lifetime values for every pixel into height values. Figures 1g,h and 2g,h show the height images obtained for the two types of samples. All the height images show that the profiles of the INM and ONM follow each other, resulting in an approximately constant distance between the two membranes of nearly 30 nm. However, some variations result in a broadening of the distance distributions (see Figures 1b and 2b). Similar variations were obtained using electron microscopy, where the distance between the two membranes was shown


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to vary from 30 nm to 50 nm.1, 2 Figure 3 shows the 3D reconstruction of the INM and ONM for samples of both types, yielding a view of the architecture of the nuclear membrane system. For each sample type, measurements were repeated for six cells, and the resulting histograms are shown in Figure 4. The red circles show the mean distance between the INM and the ONM calculated for each of the distributions. Using the obtained mean values and standard deviations for each of the distance distributions, we calculated the weighted arithmetic mean (dashed red line in Figure 4) of the distance for each sample type and its standard deviation (red rectangle in Figure 4). The results for the distance between Lap2β and Nup358 are (35 ± 19) nm for sample type I, and (31 ± 16) nm for sample type II. The values are in excellent agreement with the values obtained by electron microscopy1-3 and with recent single-point FRAP measurements.25 It should be noted that the higher standard deviation in comparison with those obtained in ref.25 is a consequence of the fact that we measure the height difference between the ONM and INM over the whole area of the basal nuclear membrane, thus including regions where the ONM does not follow the profile of the INM perfectly.

CONCLUSIONS In summary, using dual-color MIET, we determined the axial distance between the proteins Lap2β and Nup358 as components of the nuclear envelope and the NPC, with defined localizations at the INM and the cytoplasmic side of the protein complex, respectively. The obtained thickness of the nuclear envelope of 30 – 35 nm is in excellent agreement with the values obtained using electron microscopy. We have shown that optical microscopy allows one not only to measure the distance between the outer and inner nuclear membrane, but to reconstruct its 3D profile over the whole basal area. This result demonstrates that optical imaging can come close to nanometer resolution of electron microscopy in cell imaging. Technical simplicity of MIET and its nanometer axial resolution make it applicable for a broad range of biological applications. MATERIALS AND METHODS Cell culture and immunofluorescence HeLa P4 cells31 were grown in DMEM (1X; Gibco) supplemented with 10% calf serum (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (Gibco) under 5% CO2 at 37 °C. For indirect immunofluorescence followed by MIET-analysis, cells were seeded onto metal-coated glass coverslips


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(see below) and incubated until they reached 70% confluency. Alternatively, cells were seeded onto standard glass coverslips. Cells were washed with PBS, fixed with 3.7% (vol/vol) formaldehyde in PBS for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing, the cells were blocked with 2% BSA in PBS for 30 min and incubated with rabbit anti-Lap2β (Millipore, 06-1002, 1:100) and goat anti-Nup3587, 32, 1:500) in PBS with 2% BSA for 2 h. The samples were rinsed three times with PBS and incubated with highly cross-absorbed secondary antibodies (Molecular Probes, diluted 1:1000 in PBS with 2% BSA) for one hour. We used either a combination of goat anti-rabbit A633 and donkey anti-goat A488 (sample type I), or goat anti-rabbit Alexa 488 and donkey anti-goat Alexa 633 (sample type II). After three final washing steps with PBS and one with H2O, samples were mounted in Mowiol 4-88 (Calbiochem).

Experimental setup

- Confocal microscope Images were acquired with an LSM 510-Meta confocal microscope (Zeiss) based on the Axiovert 200M fluorescence microscope using an LCI Plan-Neofluar 63x/1.3 Imm Corr DIC M27 water-corrected objective and appropriate filter settings. For excitation, Argon488 and HeNe633 lasers were used.

- MIET Photoluminescence (PL) measurements were performed with a homebuilt confocal microscope equipped with an objective lens of high numerical aperture (Apo N, 60x oil, 1.49 NA, Olympus Europe, Hamburg, Germany). A pulsed, linearly polarized white light laser (SC400-4-20, Fianium Ltd., UK, pulse width ~50 ps, repetition rate 20 MHz) equipped with a tunable filter (AOTFnC - 400.650-TN, Pegasus Optik GmbH, Wallenhorst, Germany) served as excitation source. In our experiments, we used the 488 nm and 635 nm wavelengths for fluorescence excitation. The light was reflected by a non-polarizing beam-splitter towards the objective, and the back-scattered excitation light was blocked with long pass filters (EdgeBasic BLP01-488R, Semrock, inc., New York, USA, for the green channel, and BLP01-635R, Semrock, for the red channel). An additional band-pass filter 550/88 (BrightLine FF01-550/88, Semrock) was used for the A488 measurements. Emission light was focused onto the active area of an avalanche photo diode (PDM Series, MicroPhoton Devices), and data recording was performed with a multichannel picosecond event timer (HydraHarp 400, PicoQuant GmbH, Berlin, Germany).


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Samples were scanned with a focused laser spot using a piezo nano-positioning stage (P-562.3CD, Physik Instrumente GmbH, Karlsruhe, Germany). PL spectra of A488 and A633 molecules inside the cells were recorded using a spectrograph (SR 303i, Andor Technology ltd., Belfast, UK) equipped with a CCD camera (iXon DU897 BV, Andor). For MIET measurements, glass cover slides were coated with the following multilayer structure: 2 nm Ti, 15 nm Au, 2 nm Ti, 10 nm SiO2. The metal and silica films were prepared by vapor deposition onto a cleaned glass cover slide (thickness 170 µm) using an electron beam source (Univex 350, Leybold) under high-vacuum conditions (~ 10-6 mbar). During vapor deposition, film thickness was monitored using an oscillating quartz unit, and afterwards verified by atomic force microscopy.

- FRET For FRET measurements, ‘donor-acceptor’ images were first acquired by exciting the A488 donor molecules with the white-light laser at 488 nm as described in the previous subsection. Subsequently, the acceptor molecules A633 were bleached using a diode laser at 640 nm (MRL-FN-639, CNI Laser, CNI Optoelectronics Tech. Co., Ltd., Changchun, P.R. China) with a power of ~1 mW after the objective lens. The bleaching time was usually around 10 min per area. Thereafter, ‘donor only’ images were acquired on the same area using the 488 nm excitation.

Quantum yield measurements Quantum yield measurements were done with our recently developed absolute metal nanocavity method33. The metal nanocavity consists of two silver mirrors with sub wavelength spacing. The bottom silver mirror was prepared by vapor deposition of a 30 nm silver film (see above for details) onto a commercially available cleaned microscope glass coverslip (thickness 170 µm). The top silver mirror was prepared by vapor deposition of a 60 nm thick silver film onto the surface of a plano-convex lens (focal length of 150 mm) under the same conditions. The spherical shape of the upper mirror allowed for reversibly tuning the cavity length by moving the laser focus laterally away from or towards the contact point between the lens and the cover slide. It should be noted that across the size of the diffraction-limited laser focus, the cavity can be considered to be a plane-parallel resonator. For a detailed presentation of the theoretical background, refer to ref. 33.


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Fluorescence lifetime data evaluation Fluorescence lifetimes were determined as described previously28. Briefly, fluorescence photons were recorded in time-tagged, time-resolved mode, which allows gathering all photons from a single pixel and sorting them into a histogram according to their arrival time after the last laser pulse. These timecorrelated single-photon counting (TCSPC) histograms were corrected for detector and electronics deadtime effects34, and finally an average decay rate was extracted from each histogram. The lifetimes used in the calculations are always the inverse values of these average decay rates. In our previous work28, we found an empirical relationship between the number of photons N, the lifetime and the uncertainty of

the lifetime , namely ≈ 4.8 ⁄√ . Using the MIET calibration curves described in the following

section, this value can be translated to a height uncertainty for each pixel. In this article, however, we always show distributions of height values from many different pixels, which is why we do not take the height uncertainty of each single pixel into account.

MIET calibration curves and axial localization For the calculation of MIET calibration curves, a detailed knowledge of the optical parameters of the sample is required. In all MIET measurements, the sample consisted of glass cover slides (refractive index n = 1.52), coated with 2 nm titanium, 15 nm gold, 2 nm titanium, 10 nm silicon dioxide (n = 1.5) and finally the cell in its mounting medium (Mowiol). The wavelength-dependent refractive indices of the metal layers were taken from35, while the mean refractive index of a HeLa cell was assumed to be 1.3736, 37

. At first glance, it is unclear whether the complex structure of a cell with different refractive indices in

various cell compartments, the cytosol and the plasma membrane has to be taken into account for an accurate MIET measurement. The refractive index of the plasma membrane has been reported as n = 1.4638 or n = 1.4839, while the refractive index of the nuclei of four different cell lines, including HeLa cells, has been determined as n = 1.3637 and values for the cytosol range between 1.36 and 1.3938, 40, 41. In order to estimate the magnitude of the effect these different refractive indices have on the MIET result, MIET calibration curves were calculated for three different situations, as shown in Figure 5: a) a homogeneous refractive index of n = 1.37 everywhere above the SiO2 spacer, b) a 10 nm thick layer of Mowiol (the refractive index seems to vary from batch to batch. We found reports of values between 1.41 and 1.49 and used the “worst case” of 1.49) between the SiO2 spacer and the cell with n = 1.37, and c) a homogeneous refractive index of n = 1.37 almost everywhere, except for a 6 nm thick lipid bilayer


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(n = 1.46) situated 2 nm below the fluorescent label of the inner nuclear membrane or 2 nm above the fluorescent label of the outer nuclear membrane. The results for the average height distance between INM and ONM, calculated as described in the main text, are: sample

a)

b)

c)

INM-A488, ONM-A633 (type I)

31(16) nm

29(15) nm

31(16) nm

INM-A633, ONM-A488 (type II)

35(19) nm

35(18) nm

34(19) nm

These results show no significant difference between the three sample geometries. Therefore, we decided to use the simplest situation a) for all further calculations.

As described in28, both the quantum yield and the lifetime of the dye in the same surroundings as in the MIET measurement, but without presence of the metal layer (called “free space quantum yield” and “free space lifetime”) are needed for calculating the MIET calibration curves. Both quantities can be determined with the help of a metal nanocavity as explained in the previous section. Previous measurements found the quantum yield of A488 in water (refractive index n0 = 1.33) to be Φ0 = 0.94, with a lifetime of τ0 = 4.4 ns33. Since the manufacturer did not publish the quantum yield of A633, we measured it as well and obtained Φ0 = 0.59, τ0 = 3.2 ns in water. However, when a dye is placed in a different medium, the nonradiative decay rate of the excited state can change unpredictably, while we assume that the radiative decay rate changes with the refractive index n of the surrounding medium as described by the empty-cavity model42. By measuring the lifetime of the dye in the cell (in the absence of any metal layers), the quantum yield Φ in this new environment can be calculated via: = ∙

2 + 1 ∙ ∙ 2 + 1

We measured samples without metal coating and averaged over all pixels within one cell to get an estimate of the lifetimes of the dyes conjugated to the different antibodies and in the cell environment. With n = 1.37, we arrive at the following table: measured lifetime [ns]

calculated QY Φ [-]

gA488 (ONM, sample type I)

2.6

0.59

rbA633 (INM, sample type I)

2.4

0.47

rbA488 (INM, sample type II)

2.4

0.54

gA633 (ONM, sample type II)

1.9

0.37

Sample


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Using these values for Φ and and assuming the layered structure a) described above, MIET calibration curves for the four different cases were calculated as described previously28. They are shown in Figure 6. Finally, these curves were used to convert measured lifetime values from the metal-coated samples into height values (where h = 0 corresponds to the interface between silicon dioxide and the cell medium). All MIET calculations presented in this work were performed using custom-written MATLAB (The MathWorks, Inc.) routines, which are available for free download at https://projects.gwdg.de/projects/miet in the form of a graphical user interface.

FRET distance analysis For FRET measurements, lifetime images of the donor molecules (A488) in the presence of the acceptor molecules (A633) were recorded, as described in the sections above. Figure 7a shows one such fluorescence lifetime image. Thereafter, the acceptor was photo-bleached by performing multiple scans with an excitation wavelength of 640 nm with high excitation power. After ensuring that the acceptor was completely bleached, lifetime images of the donor (A488) were recorded again in the same area (Figure 7b). These FLIM images were corrected for any dead-time related artifacts using the correction algorithm mentioned above. Repeated scanning using a piezo stage is likely to produce a drift which needs to be taken into account before estimating the FRET efficiency at each pixel. The z-position of the objective was adjusted by focusing the excitation light onto the surface of the substrate, while a part of the reflected excitation light was focused onto a sensitive camera. The back-reflection image was used to estimate and correct the z-position of the objective. For correcting any lateral drift between the two lifetime images, we maximized the correlation between these images by shifting one with respect to the other in the x- and y-directions. In this way, the relative lateral position of scan images was corrected with one-pixel accuracy. Next, we identified the region of interest which contains the nucleus by applying an intensity threshold. This region of interest was divided into clusters of roughly 4x4 pixels for noise reduction. The TCSPC curves from the donor-acceptor and donor-only data for each cluster were tailfitted with a bootstrapping procedure taking 104 photons at a time. In this way, mean values τda and τd of donor-acceptor lifetime and donor-only lifetime, respectively, from each cluster are obtained. Only if the difference of these mean values (Δτ) was larger than the errors of the fitted lifetime from the bootstrapping procedure, the average FRET distances (r) in the cluster was calculated, which is given by


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Δ = where R0 is the Förster radius for the dye pair. Neglecting any interface based effects43, R0 is related to the refractive index of the medium n and the quantum yield of the donor ϕd as: ∝

! ∙ " #

Using the quantum yield of the donor ϕd determined from the previous section and assuming an isotropic distribution of the orientations of the donor and acceptor molecules (κ2 = 2/3), we obtained a Förster radius of around 50 Å. In this way, the FRET distance image is computed, as shown in Figure 7e. The distribution of mean values τda and τd and the FRET distance are shown in Figures 7d and c, respectively. All the data analysis was performed using custom-written MATLAB routines.

Author Contributions A.M.C, J.P., A.I.C, R.K., and J.E. conceived and designed the experiments. J.P. performed cell culture and antibody-labeling experiments. A.M.C. performed the MIET-experiments. A.M.C., D.R., and N.K. analyzed the data. A.I.C, I.G., R.K., and J.E. contributed materials/analysis tools. A.M.C, D.R, A.I.C, R.K., and J.E. wrote the paper.

Acknowledgments This work was supported in part by the DFG through the Cluster of Excellence “Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB)”. A.I.C. and D.R. are grateful to the DFG for financial support via SFB 937 (project A14). N.K. is grateful to the DFG for financial support of his position via SFB 860 (project A06). R.H.K. and J.P. acknowledge funding by the DFG through SFB1190 (project P07).


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FIGURES

Figure 1. Sample type I. Detection of proteins at the INM and the cytoplasmic side of the NPC. HeLa cells were fixed and subjected to indirect immunofluorescence using goat anti-Nup358 (c, e, g) and rabbit anti-Lap2β (d, f, h) as primary antibodies. Goat anti-rabbit A633 (to detect Lap2β) and donkey anti-goat A488 (to detect Nup358) were used as secondary antibodies. a – schematic of the positions of Lap2β and Nup358 in the INM and the NPC, respectively. b – diagram depicting the height differences of individual pixels in g and h. c, d – confocal images of nuclei. Note that the laser focus was placed in the middle of the nuclei. e, f – fluorescence intensity images of an individual nucleus with the laser focus on the gold surface. g, h – height image of the same nucleus as in e, f, as analyzed by MIET. The measured height is the distance from the silica spacer.


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Figure 2. Sample type II, with switched secondary antibodies compared to sample type I. Detection of proteins at the INM and the cytoplasmic side of the NPC. HeLa cells were fixed and subjected to indirect immunofluorescence using goat anti-Nup358 (c, e, g) and rabbit anti-Lap2β (d, f, h) as primary antibodies. Goat anti-rabbit A488 (to detect Lap2β) and donkey anti-goat A633 (to detect Nup358) were used as secondary antibodies. a – schematic of the positions of Lap2β and Nup358 in the INM and the NPC, respectively. b – diagram depicting the height differences of individual pixels in g and h. c, d – confocal images of nuclei. Note that the laser focus was placed in the middle of the nuclei. e, f – fluorescence intensity images of an individual nucleus with the laser focus on the gold surface. g, h – height image of the same nucleus as in e, f, as analyzed by MIET. The measured height is the distance from the silica spacer.


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Figure 3. Three-dimensional height profiles of the inner (top) and outer (bottom) nuclear membrane of a typical HeLa cell nucleus, as determined by MIET imaging of (a) A633-labeled Lap2β and A488-labeled Nup358 (sample type I) and (b) A488-labeled Lap2β and A633-labeled Nup358 (sample type II). The measured height is the distance from the silica spacer. The outer nuclear membrane roughly follows the profile of the inner nuclear membrane.


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Figure 4. Histograms of the distance between Lap2β and Nup358 in all pixels belonging to the nuclei of six different cells for sample type I (a) and sample type II (b). Each histogram was normalized by the total number of pixels used to compile it. Red circles denote the mean values of the histograms. Together with the standard deviations of the single histograms (not shown), they were used to calculate the weighted arithmetic mean for each sample type (red dashed lines). The red rectangles show the standard deviation of the weighted mean. For sample type I (a), the average distance between ONM and INM is (35 ± 19) nm, for sample type II (b) (31 ± 16) nm.


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Figure 5. Schematic of three different situations used to calculate MIET calibration curves. Red dots represent the dye molecules at different heights h above the SiO2 spacer. In c), the upper row of dye molecules was used for the label of the INM, while the lower row of dye molecules was used for the label of the ONM.


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Figure 6. MIET calibration curves for A633 on the outer nuclear membrane (red solid line) or inner nuclear membrane (red dashed line) and A488 on the inner nuclear membrane (green solid line) or outer nuclear membrane (green dashed line). The height is measured from the interface between silica spacer and cell medium.


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Figure 7. FRET between A488 and A633. A and B show the lifetime of A488 before and after bleaching of A633, respectively, in a typical nucleus. For clusters of 4-by-4 pixels, average lifetimes $% and $ were determined (see histograms in D) and converted into distances as explained in the text. These distances are visualized in the false-color image shown in E and the histogram C. In this analysis, A488 was coupled to Lap2β (sample type II)


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For TOC graphics only


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