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OPEN
received: 21 June 2016
accepted: 21 October 2016
Published: 09 November 2016
Establishing super-resolution
imaging for proteins in diatom
biosilica
Philip Gröger1, Nicole Poulsen1, Jennifer Klemm1, Nils Kröger1,2 & Michael Schlierf1
The intricate, genetically controlled biosilica nano- and micropatterns produced by diatoms are a
testimony for biology’s ability to control mineral formation (biomineralization) at the nanoscale and
regarded as paradigm for nanotechnology. Previously, several protein families involved in diatom
biosilica formation have been identified, and many of them remain tightly associated with the final
biosilica structure. Determining the locations of biosilica-associated proteins with high precision
is, therefore expected to provide clues to their roles in biosilica morphogenesis. To achieve this,
we introduce here single-molecule localization microscopy to diatoms based on photo-activated
light microscopy (PALM) to overcome the diffraction limit. We identified six photo-convertible
fluorescent proteins (FPs) that can be utilized for PALM in the cytoplasm of model diatom Thalassiosira
pseudonana. However, only three FPs were also functional when embedded in diatom biosilica. These
were employed for PALM-based localization of the diatom biosilica-associated protein Silaffin-3 (tpSil3)
with a mean precision of 25 nm. This allowed for the identification of distinct accumulation areas of
Sil3 in the biosilica, which cannot be resolved by confocal fluorescence microscopy. The enhanced
microscopy technique introduced here for diatoms will aid in elucidating the molecular mechanism of
silica biomineralization as well as other aspects of diatom cell biology.
Diatoms are one of the most abundant unicellular microalgae found in all aquatic ecosystems across the world1.
A distinct feature of diatoms is their silica-based cell wall that exhibits an intricate multi-scale pattern with structural elements spanning from micrometers down to tens of nanometers. The morphogenesis of the diatom biosilica is a highly complex process that occurs with the help of self-assembled biosilica forming templates2. Over the
past decade, biochemical studies have identified several organic components (i.e. silaffins, cingulins, silacidins,
SiMat proteins, long-chain polyamines, chitin) that are tightly associated with the diatom silica and believed to
be involved in formation of the species-specific silica nano- and micropatterns3–8. Live cell imaging of the peptides and proteins thought to be involved in cell wall formation has become possible over the past years due to
the development of methods for their genetic transformation9. Through the expression of GFP fusion proteins in
the model diatom Thalassiosira pseudonana, cell wall associated proteins have been localized to distinct regions
of the biosilica. For example, the silaffin tpSil3 is associated with the so called valve region whereas the cingulins
are associated with the girdle band region (Fig. 1)7. However, these studies have not been able to determine the
precise localization of the GFP-fusion proteins as confocal imaging is limited by the diffraction limit of light
(≈​250 nm) (Fig. 1B). To visualize the intricate nanometer-sized silica pattern, typically electron microscopy is
used10–12. While electron microscopy offers unparalleled spatial resolution, it is very challenging to use this imaging technique for the visualization of silica embedded proteins and impossible to perform imaging with live
cells (Fig. 1A). Fluorescence microscopy, on the other hand, allows live cell imaging and direct access to simultaneous multi-color and thus multi-protein visualization. Recently, structured illumination microscopy with its
resolution of up to 150 nm has been applied to localize fusion proteins inside diatoms13. Yet, single-molecule
localization microscopy (SMLM) which extends the range of fluorescence microscopy to spatial resolutions in the
tens of nanometer regime14,15, has not been applied to diatoms. Both photo-activatable localization microscopy
(PALM) and stochastic optical reconstruction microscopy (STORM) employ temporal and spatial separation
of single-fluorophores to reconstruct a high resolution representation from a series of hundreds of individual
images16. While STORM is based on immunofluorescence localization of proteins using antibodies, PALM offers
1
B CUBE–Center for Molecular Bioengineering, TU Dresden, Arnoldstr. 18, 01307 Dresden, Germany. 2Department of
Chemistry and Food Chemistry, TU Dresden, Arnoldstr. 18, 01307 Dresden, Germany. Correspondence and requests
for materials should be addressed to M.S. (email: [email protected])
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Figure 1. The diatom T. pseudonana. (A) Left: SEM image of the T. pseudonana biosilica displaying parts of
the girdle band region and the planar valve region featuring the prominent fultoportulae and a ridge network.
Right: False coloring of the EM picture to highlight structural features of the T. pseudonana biosilica. (B)
In vivo confocal microscopy images of two T. pseudonana cells with the GFP-tagged Sil3 protein in green
(488 nm excitation) in ‘girdle view’ (left) and ‘valve view’ (right) orientation. The red fluorescence is caused by
autofluorescence of the chloroplasts (647 nm excitation). All scale bars are 1 μ​m.
the opportunity to use fusion proteins based on switchable fluorescent proteins, with the benefit of an intrinsic
high labeling density. SMLM is able to bridge the resolution gap between confocal and electron microscopy.
Therefore, super-resolution microscopy is particularly interesting for studying diatom biomineralization, since
the feature sizes of biosilica range typically between ten and several hundred nanometers.
Here, we have established SMLM for the diatom T. pseudonana with the aim of visualizing the precise localizations of protein-based templates inside the biosilica. As the silica may limit antibody access to the protein of
interest, we chose to use PALM, which is based on the expression of fusion proteins with photo-convertible fluorescent proteins. Over the past years, a large variety of switchable fluorescent proteins have been developed17,18.
We have screened six different fluorescent proteins (FPs) to identify and evaluate suitable candidates. To this end,
we compared the photo-conversion abilities of cytosolic FPs with that of biosilica-embedded FPs. For the biosilica
embedding we created fusion proteins with Silaffin-3 (tpSil3), which is thought to be involved in biosilica formation and remains permanently entrapped inside the biosilica of the valve region19,20. Unexpectedly, we have found
that only a subset of the photo-convertible proteins can be used when embedded in the biosilica. In contrast, all
FPs could be activated efficiently in the cytosol. Reconstruction microscopy on the single-molecule level allowed
for localization of Dendra2, mEOS3.2 and Dronpa fusion-proteins embedded in the silica with an average precision of 28 nm, 25 nm and 25 nm, respectively.
Results
Photo-conversion of fluorescent proteins in the cytoplasm of living diatoms. To find suitable
fluorescent proteins (FPs) as super-resolution probes for biosilica embedded fusion-proteins, we chose to screen
six different candidates, namely PATagRFP21, PAmCherry122, PA-GFP23, mEOS3.224, Dendra225 and Dronpa26
(Table 1 and SI Table S1). PATagRFP, PAmCherry1 and PA-GFP are photo-activatable FPs that undergo an activation from a non-fluorescent state to a fluorescent state, whereas mEOS3.2 and Dendra2 are photo-convertible
FPs that can be converted from one fluorescent to another fluorescent state with a red-shifted excitation and
emission spectrum. Dronpa is a photo-switchable FP that can be repeatedly switched between a non-fluorescent
and a fluorescent state.
In particular, we focused on a range of different emission wavelengths, since diatom chloroplasts exhibit a
broad autofluorescent background that could interfere with imaging of the FPs. The chloroplast absorbance of
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Cytosolic expression
Silica embedded
Photo-conversion
mechanism
Before
activation
After
activation
Before
activation
After
activation
Suitable for silica
embedded imaging
PATagRFP
PA
Dark
Orange
Dark
Dark
No
PAmCherry1
PA
Dark
Orange
Dark
Dark
No
PA-GFP
PA
Dark
Green
Dark
Dark
No
mEOS3.2
PC
Green
Orange
Green
Orange
Yes
Dendra2
PC
Green
Orange
Green
Orange
Yes
Dronpa
PS
Dark
Green
Dark
Green
Yes
FP
Table 1. Photo-conversion behavior of FPs in diatoms. All FPs used for SMLM in diatoms together with the
respective screening results are listed. Abbreviations for the conversion mechanism are: photo-activatable (PA),
photo-convertible (PC) or photo-switchable (PS). Color names represent the excitation wavelengths 488 nm
(green) and 561 nm (orange).
T. pseudonana peaks at λ​exc ≈​ 450 nm (chlorophyll c and fucoxanthin) and at λ​exc ≈​ 670 nm (chlorophyll a) with
an emission ranging roughly from 650 nm to 750 nm (SI Fig. S1) preventing imaging of FPs emitting in those
wavelength windows. Another important selection criterion was a strict monomericity of the FPs, to avoid unnatural aggregation of the silaffin fusion proteins during silica formation. Thus, we excluded previously reported
multimeric FPs like Kaede27, KFP128, or IrisFP29.
In a first set of experiments, we tested the photo-activation of all candidate proteins expressed in the cytosol of
diatoms. We inserted the FP of choice into the expression vector pTpfcp30 for constitutive cytosolic expression in
T. pseudonana (for a scheme of all vectors used for cloning see SI Fig. S7). Successful transformants (at least 25%
of the screened clones showed fluorescence) were identified by fluorescence microscopy and further tested for an
activatable fluorescence signal in the cytosol after brief illumination with λ​act =​ 405 nm. All six FPs (PATagRFP,
PAmCherry1, PA-GFP, mEOS3.2, Dendra2 and Dronpa) could be activated, converted or switched to their second state (Table 1 and SI Fig. S2). As examples, the successful activation of PA-GFP from an initial dark state to
its green state, conversion of mEOS3.2 from the initial green state to the orange state and switching of Dronpa
from the dark to the green state after a 1 s illumination at λ​act =​ 405 nm are shown in Fig. 2A-C (see SI Fig. S2 for
an overview of all FPs used in this study).
Photo-conversion of biosilica-embedded proteins in live diatoms. In a second set of experiments,
we tested the activation of the FPs when embedded in the diatom biosilica. For that purpose, recombinant genes
encoding C-terminal fusion proteins of the FPs with tpSil3 were constructed, stably incorporated into the T.
pseudonana genome, and expressed under control of the constitutive fcp promoter (see SI Fig. S7). When tpSil3
is expressed under the control of the fcp regulatory elements, it is incorporated into all regions of the biosilica31.
In contrast to cytosolic FP expression, only three of the six FPs that were tested were convertible when fused to
tpSil3 (Table 1, SI Figs S2 and S3). In particular, the three FPs that require photo-activation from a dark state (i.e.
PATagRFP, PAmCherry1 and PA-GFP) were not activatable–even after multiple seconds of λ​act =​ 405 nm exposure (see Fig. 2C showing PA-GFP as an example). By screening at least 24 clones and multiple cells per clone,
it is very likely that the absence of fluorescence originates from a hindered photo-activation rather than a failed
genetic transformation.
In contrast, all the FPs that are either photo-convertible or photo-switchable (i.e. mEOS3.2, Dendra2, Dronpa)
were successfully converted from the green emission state or dark state to the orange emission state. Examples of
a successful photo-conversion of mEOS3.2 and photo-switching of Dronpa are shown in Fig. 2E,F, respectively,
where tpSil3-mEOS3.2 was converted and tpSil3-Dronpa was switched with a 1 sec activation with λ​act =​ 405 nm.
As in the cytosolic construct, the conversion happens during a single frame of UV illumination and seems not
to be hindered by the silica-embedment. A time course analysis of photo-conversion over multiple cycles of UV
illumination showed a rapid saturation of converted fluorescence within a few cycles for all three fusion proteins
(SI Fig. S3).
The observed difference in photo-convertibility might originate from the underlying molecular mechanisms. Indeed, the conversion mechanism of PA-GFP, PATagRFP and PAmCherry1 relies on a decarboxylation
of Glu22232–34. This glutamic acid sits in the β​-barrel of the protein and is most likely partly exposed to the silica environment. Thus, it is conceivable that the surrounding silica environment impairs the decarboxylation.
Interestingly, the three FPs that were converted inside the biosilica do not require this decarboxylation–they rely
on a breakage of the polypeptide backbone near the chromophore (mEOS3.2, Dendra2)35 or a cis-trans isomerization of the chromophore (Dronpa)36. These three conversions occur in the center of the β​-barrel, which is most
likely protected from the surrounding biosilica.
Super-resolution microcopy of silica embedded fusion proteins. After identifying suitable FPs for
biosilica embedded super-resolution imaging, we moved forward to perform PALM imaging using Dendra2,
mEOS3.2 and Dronpa fused to tpSil3 (SI Movie S1). The high chloroplast background fluorescence impaired
single-molecule detection in vivo, and thus high resolution imaging (see SI Fig. S1). Additionally, the chloroplasts
changed their autofluorescence during 405 nm illumination, which further challenged the identification of single
photo-converted molecules (Fig. S3). Therefore, the chloroplasts were removed by extraction of the cells with a
detergent-based buffer yielding pure biosilica. The tpSil3 fusion proteins in diatom silica were imaged using a low
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Figure 2. Epifluorescence images of photo-conversion. The red channel (647 nm excitation) always shows
the chloroplast emission. (A) Photo-activation of cytosolic PA-GFP. The Green channel (488 nm excitation)
shows activated PA-GFP after UV exposure. (B) Photo-conversion of cytosolic mEOS3.2. The Green channel
shows unconverted mEOS3.2, which is converted via λ​act ≈​ 405 nm to its red shifted form that can be excited
at 561 nm (yellow channel). (C) Photo-switching of cytosolic Dronpa. The green channel shows switching
of Dronpa between its dark and active state. (D) TpSil3-PA-GFP is embedded in biosilica and cannot be
activated with UV light. (E) TpSil3-mEOS3.2 embedded in biosilica (green). Embedded tpSil3-mEOS3.2 can
be efficiently converted (yellow). (F) TpSil3-Dronpa can be converted. All scale bars are 1 μ​m.
conversion power and a high imaging power to localize individual Dendra2, mEOS3.2 and Dronpa molecules.
After a relatively short recording time of approximately two minutes (1000 frames at 10 frames per second),
the movie was analyzed using the super-resolution software ThunderSTORM37 and the resulting localizations
visualized using a custom-written MATLAB code. Figure 3A shows a reconstructed single-molecule localization
image of Dendra2 with the T. pseudonana biosilica in valve view and the focal plane in the girdle band region
(see Fig. 1B right). By performing multiple line scans perpendicular to the biosilica cylinder, we determined an
average full width at half maximum (FWHM) of 76.0 nm ±​ 4.6 nm (±​S.D.). One of those line scans is depicted in
Fig. 3B. Taking into account the localization precision per fluorophore of 28 nm (this would be a FWHM of 66 nm
assuming a Gaussian distribution) and a linker length of 3 nm (the distance between the chromophore of the FP
and tpSil3), the 76 nm can be deconvolved to an underlying thickness of 53 nm ±​ 3 nm (see SI Fig. S8 for details).
This value is in very good agreement with the average thickness of the valve of 60 nm determined by scanning
electron microscopy38. In comparison, the line scans of the epifluorescence image, which is limited in its resolution by the light diffraction limit, yielded a FWHM of 510 nm ±​ 55 nm (Fig. 3A,B).
The intricate biosilica pattern of diatoms is most planar in the valve region and can thus be best observed
there. We used TIRF imaging to determine the locations of tpSil3-Dendra2 and tpSil3–mEOS3.2 embedded in
the biosilica pattern. In epifluorescence images (Fig. 3D,E left), a continuous fluorescence distribution with inhomogeneous intensities over the valve region was observed. In the PALM image (Fig. 3D,E right and Fig. S5),
clear fluorescent and non-fluorescent regions can be identified. Interestingly, the large circular non-fluorescent
gaps correlate in number and position with the structural elements of fultoportulae observed in wildtype in
SEM images (see Fig. 1A and SEM images in Fig. 3D,E). This suggests that tpSil3-Dendra2 and tpSil3-mEOS3.2
are not present within the external tubes of the fultoportulae which are approximately 100 nm in diameter, but
rather correlate to the outer region of the fultoportulae basal chamber–a thicker and larger structure (SI Fig. S4).
Furthermore, we could not observe a distinct ridge-like pattern in the reconstructed super-resolution images, but
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Figure 3. PALM on tpSil3. (A) Comparison of an epifluorescence image and the reconstructed superresolution image of tpSil3-Dendra2 with z-focus on the girdle band area of the diatom. The position of the line
scan is highlighted. (B) Line scan through the silica cell wall showing the fluorescence intensity profile for both
imaging modalities. The FWHM of the PALM image is denoted. (C) Fourier ring correlation for the superresolution image shown in A reveals an effective resolution estimate of 74.7 nm. Comparison of epifluorescence
images and the reconstructed super-resolution image of Dendra2 (D), mEOS3.2 (E) and Dronpa (F) fused to
tpSil3 with z-focus on the valve region of the diatom. Enlarged details of the fultoportulae to the right. An SEM
image in the same scaling that corresponds to the enlarged detail of the fluorescence image is displayed for
comparison. Scale bars are 1 μ​m, and 100 nm for the zoomed images.
a rather sparse localization on the central area of the valve, except for a high density around a central pore like
structure. The position and size of this pore-like structure correlates well with the central fultoportulae observed
in electron micrographs. We hypothesize that tpSil3 is a major component in the basal chamber of the fultoportulae and plays only a minor role in ridge formation.
We further localized the tpSil3-Dronpa fusion protein, although with a lower localization count per frame
(5 versus 14 for mEOS3.2 and Dendra2, SI Fig. S6). This leads to a longer acquisition time (2500 frames) until
a similar super-resolution image is formed (Fig. 3F). The lower localization count might originate from a lower
expression or bleaching during acquisition due to the UV activation laser.
In the valve regions, 17763 fluorophores (tpSil3-Dendra2) and 14327 fluorophores (tpSil3- mEOS3.2) were
localized to form the corresponding super-resolution images. Dendra2 and mEOS3.2 could be localized with
an average localization precision39 of 28 nm and 25 nm, respectively (SI Fig. S6A). This is in good agreement
with previously reported localization precisions for these fluorophores in different biological environments14,40.
Although there have been reports of light trapping41 and lensing42 effects of the nanostructured diatom frustules
we did not experience a pronounced distortion of the far-field image leading to inferior fluorophore localization which indicates that silica-embedding does not affect imaging resolution with these fluorophores. The high
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amount of localized fluorophores (with a mean of 14 localizations per frame, see SI Fig. S6B) and the localization
precision support our hypothesis that the non-fluorescent areas are indeed true gaps in the tpSil3 protein arrangement rather than an artifact generated by sparse image reconstruction. Further analysis of the nearest neighbor
distribution clearly peaks under 20 nm for all SMLM images, thus indicating a dense protein pattern (SI Fig. S6C)
that satisfies the Nyquist Criterion43. Additionally, a Fourier ring correlation was calculated to estimate the resolution44. For the super-resolution image in Fig. 3A we achieve according to the Fourier ring correlation criteria a
resolution of 74.7 nm (Fig. 3C), which corroborates the observed biosilica FWHM of 76 nm.
Conclusions
Currently, the high background of chloroplasts hinders in vivo single-molecule localization microscopy, however
recently developed super-resolution techniques based on fluctuation analysis45 are promising approaches for a
suppression of the chloroplast background. Despite this limitation, with extracted cells, we achieved localization
precisions of 28 nm, 25 nm and 25 nm for silica-embedded tpSil3-Dendra2, tpSil3-mEOS3.2 and tpSil3-Dronpa,
respectively. This resolution is well beyond the diffraction limit and allows the investigation of the intricate biosilica pattern observed in EM and if said pattern originates from an underlying protein template for silica biogenesis. Recently reported approaches of correlative super-resolution and electron microscopy might allow additional
correlation within a single cell46,47. Moreover, newly developed fluorescent proteins compatible with a biosilica
embedded photo-conversion mechanism may further increase the resolution and thus allow more insights in the
underlying protein pattern. Structured illumination microscopy studies showed that biarsenic Cyanine fluorophores (AsCy3 and AsCy3e) can be further used for silica embedded fusion proteins and are due to their blinking
nature also potentially suited for single-molecule localization microscopy13,48. We further anticipate that the use
of multicolor PALM and colocalization studies49 will allow deeper insights in protein-protein interactions during
silica biogenesis. Such insight should be instrumental for understanding the molecular mechanisms that control
morphogenesis of the species-specific diatom silica structures with precise nanoscale control.
Methods
Cell Cultivation. Thalassiosira pseudonana (Hustedt) Hasle et Heimdal clone CCMP1335 was cultured in
artificial seawater medium according to the North East Pacific Culture Collection protocol at 18 °C under constant light at 5,000–10,000 lux as previously described50.
Construction of T. pseudonana expression vectors. DNA sequences of the photo-activatible FPs with
their codons optimized for expression in T. pseudonana51 were synthesized by GeneArt with restriction sites
added to allow for single step cloning into the diatom expression vectors (see SI Table S2). For cytosolic expression of the FPs the plasmid DNA was digested with EcoRV and NotI and ligated into the EcoRV and NotI sites of
pTpfcp30. For expression of the FPs as a C-terminal gene fusion with tpSil3 the plasmid DNA was digested with
EcoRV and NotI and ligated into the EcoRV and NotI sites of pTpfcp/Sil3nt31. See SI Fig. S7 for a vector scheme
including the Sil3-fusion construct.
Genetic transformation of T. pseudonana. The resulting gene constructs were introduced into
T. pseudonana cells using the Biorad PDS-1000/He particle delivery system as described previously30.
Co-transformations were performed with the pTpfcp/nat plasmid DNA30 for selection of transformed cell lines
on agar plates containing 150 μ​g/mL ClonNat (Jena Biosciences).
Biosilica isolation. Due to the intense chloroplast autofluorescence for PALM imaging of FPs fused to tpSil3
it was necessary to remove cytosolic components via SDS and EDTA treatment as described7. Cell walls were
resuspended in PBS prior to imaging.
Imaging by fluorescence microscopy. Diatoms were imaged on a Nikon N-STORM inverted microscope with a 100x Oil Immersion TIRF objective with a numerical aperture of 1.49. Four fiber-coupled lasers
are available for illumination at 405, 488, 561 and 647 nm, respectively. Corresponding filter sets from Semrock,
Chroma and AHF were used to block out all unwanted fluorescence and scattered laser light (see SI Mat/Met for a
detailed description). The image was projected onto an Andor Ixon 897 EMCCD camera. To visualize the chloroplasts, the red 647 nm laser was used at low power (1.5 mW end of fiber) for 100 ms. For screening of the FPs, the
appropriate excitation wavelength (SI Table S1) was chosen at a power of 40 mW and an exposure time of 100 ms.
The photo-conversion test was performed by illuminating the sample with 405 nm light for 1 second at 20 mW.
Super-resolution images were recorded at 20 fps with an excitation power of 80 mW. Image stacks of at least 1000
images were used (1000 for Fig. 3A,D and SI Fig. S5, 1200 for Fig. 3E, 2500 for Fig. 3F) to ensure a sufficient localization count. For mEOS3.2 and Dendra2 green excitation is sufficient to localize pre-converted FPs. Dronpa is
dark in its ground state, a continuous 405 nm activation of 8 mW in addition to the green excitation was required.
Super-resolution reconstruction. For super-resolution image reconstruction, the software thunder-
STORM37 was used (see SI for detailed parameter); one of the top performer in a quantitative software evaluation52. For visualization, a custom written MATLAB script with a GUI was developed to display super-resolution
data and confirm the correct reconstruction. The latter was performed by coloring the individual localization
events according to the localization parameter of choice (like localization precision, goodness of fit, etc.). This
allows easy identification of outliers and reconstruction quality. See SI Fig. S9 for a GUI screenshot. Scripts and
GUI are available upon request.
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Acknowledgements
The authors thank Anke Borrmann (B CUBE, TU Dresden) for expert technical assistance, and Thomas Kurth
(Electron Microscopy Facility BIOTEC/CRTD, TU Dresden) and Damian Pawolski (B CUBE, TU Dresden)
for help with electron microscopy. This work was supported by the German Federal Ministry of Education and
Research BMBF (03Z2EN11 & 03Z2ES1 to MS) and the Deutsche Forschungsgemeinschaft DFG (FOR2038
SCHL 1896/2-1 and KR 1853/5-1).
Author Contributions
P.G., N.P. and M.S. conceived the experiments. M.S and N.K. supervised the project. P.G. performed all imaging
and data analysis. J.K. created FP constructs, transformed and cultivated the diatom cell cultures. P.G. and M.S.
wrote the paper. All authors contributed through discussions and commented on the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Gröger, P. et al. Establishing super-resolution imaging for proteins in diatom biosilica.
Sci. Rep. 6, 36824; doi: 10.1038/srep36824 (2016).
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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© The Author(s) 2016
Scientific Reports | 6:36824 | DOI: 10.1038/srep36824
8
Supplementary Information for
Establishing super-resolution imaging for proteins in diatom biosilica
Philip Gröger1, Nicole Poulsen1, Jennifer Klemm1, Nils Kröger1,2, Michael Schlierf1*
1
B CUBE – Center for Molecular Bioengineering, TU Dresden, Arnoldstr. 18, 01307 Dresden,
Germany
2
Department of Chemistry and Food Chemistry, TU Dresden, Arnoldstr. 18, 01307 Dresden,
Germany
*correspondence: [email protected]
1
Index
SI Methods
3
Table S1.
Fluorescent proteins used and their core parameter
4
Figure S1.
Emission spectra of live diatom
5
Figure S2.
Screening of all fluorescent proteins and their activation capability
6
Figure S3.
Photo-conversion analysis of active tpSil3 clones
7
Figure S4.
SEM micrograph of the inside of the valve biosilica
8
Figure S5.
Additional PALM images on tpSil3–Dendra2 and tpSil3-mEOS3.2
9
Figure S6.
Localization parameter for tpSil3-Dendra2, mEOS3.2 and Dronpa
10
Figure S7.
Vector design for cloning
11
Figure S8.
Convolution to estimate tpSil3-Dendra2 filament thickness
12
Figure S9.
MATLAB GUI to visualize super-resolution data from thunderSTORM
13
Table S2.
Codon optimized sequences for the fluorescent proteins
14
Movie S1.
Raw movie of tpSil3-Dendra2, -mEOS3.2 and Dronpa
15
2
SI Methods
SEM imaging of Thalassiosira pseudonana
Diatoms were lysed (as described under Biosilica Isolation in the main methods), dehydrated with
ethanol and afterwards critically point dried in a Leica-CPD 300. The dry diatoms were spread onto a
carbon conductive adhesive tape and sputter coated with platinum in a Baltec SCD 050. Imaging was
performed using a JEOL JSM-7500F scanning electron microscope with a SE2 detector and an
acceleration voltage of 15kV.
ThunderSTORM settings
Image filtering was performed using the “à trous” undecimated wavelet transform with a B-spline
order of 3 and scale of 2. For localization approximation a local threshold (in an 8-connected
neighborhood) of 1x the standard deviation of the filtered image was chosen. The sub-pixel
localization was performed via maximum likelihood fitting of the integrated form of a symmetric 2D
Gaussian function with a radius of 3 pixel. The initial sigma was 1.6 pixel.
Super-resolution image reconstruction
The visualization of the localized molecules was performed by drawing a normalized symmetric 2D
Gaussian for each of them and summing up all the Gaussians to form a final image. As the standard
deviation, the localization precision (calculated via the Thompson-Larson-Webb formula as
mentioned in the supplementary note for ThunderSTORM) was chosen.
Filter Sets used for imaging
Excitation WL Laser bandpass
Dichroic
Emission bandpass
405
390/40 BrightLine HC (Semrock) H 405 LPXR superflat (AHF) 460/50 ET (Chroma)
488
475/35 BrightLine HC (Semrock) H 488 LPXR superflat (AHF) 525/45 BrightLine HC (Semrock)
561
555/25 ET (Chroma)
647
628/40 BrightLine HC (Semrock) H 643 LPXR superflat (AHF) 700/75 ET (Chroma)
H 560 LPXR superflat (AHF) 609/54 BrightLine HC (Semrock)
3
Table S1. Fluorescent proteins used and their core parameter
Abbreviations: λex & λem … excitation and emission spectrum peak position, tmature … maturation time at 37°C,
-1
-1
Brightness refers to the product of the extinction coefficient (in Mol cm ) and the quantum yield divided by 1000.
The first three FPs are photo-activatable FPs, meaning their non-fluorescent ground state can be activated,
irreversibly. MEOS3.2 and Dendra2 are photo-convertible FPs and can be irreversibly switched from green to
orange fluorescence. Dronpa is a photo-switchable FPs and can be reversibly switched between a fluorescent
and non-fluorescent state.
FP
Transitions
State
λex (nm)
λem (nm) Brightness tmature (min)
PATagRFP
Off à Orange, 405 nm
Orange
562
595
25.1
75
PAmCherry1 Off à Orange, 405 nm
Orange
564
595
9.3
23
PA-GFP
Off à Green, 400 nm
Green
504
517
13.7
27
Green
507
517
53.2
20
mEOS3.2
Green à Orange, 405 nm
Orange
572
593
17.7
Green
490
507
22.5
90
Dendra2
Green à Orange, 480 nm
Orange
553
573
19.3
Off à Green, 400 nm
Dronpa
On
503
518
80.7
40
Green à Off, 503 nm
4
Figure S1. Emission spectra of live diatom
Emission spectra of T. pseudonana for different excitation wavelengths. Vertical lines represent the laser lines.
The shaded area depict the detection window. The absorption spectrum is plotted in black on a separate y-axis.
Clearly visible are the absorption peaks for chlorophyll a at around 670nm and chlorophyll c and fucoxanthin
around 450nm.
5
Figure S2. Screening of all fluorescent proteins and their activation capability
Each row displays the activation of a different fluorescent protein. The activation was performed with the
appropriate laser light as specified in Table S1. The colors represent the four different excitation wavelengths
405nm (blue), 488nm (green), 561nm (yellow) and 647nm (red). Chloroplasts are always visible in the red
channel.
6
Figure S3. Photo-conversion analysis of active tpSil3 clones
Photo-conversion statistics for Dendra2 (A), mEOS3.2 (B) and Dronpa (C). The top panel show the individual
imaging channels during conversion. Each UV cycle equals one second of 405nm illumination at 20mW. To
analyze the conversion quantitatively, a region containing the biosilica cell wall and overlapping as little as
possible with chloroplasts was selected (depicted in white). The intensity in this region was summed up and the
relative intensity change with 488 nm, 561 nm and 647 nm excitation is plotted in the lower panels, showing the
clear photo-conversion or –activation of the probes.
7
Figure S4. SEM micrograph of the inside of the valve biosilica
SEM image of isolated biosilica valve of T. pseudonana. The inside of the valve shows the thick basal chamber of
the fultoportulae. A zoomed image shows a diameter estimate of the outer region of the fultoportulae basal
chamber of around 400 nm. Scale bars are 1 µm and 100 nm for the zoomed image.
8
Figure S5. Additional PALM images on tpSil3-Dendra2 and tpSil3-mEOS3.2
Comparison of epifluorescence images (left) and the reconstructed super-resolution image (right) of (A) tpSil3Dendra2 and (B) tpSil3-mEOS3.2 with z-focus on the valve region of the diatom. All scale bars are 1µm.
9
Figure S6. Localization parameter for tpSil3-Dendra2, mEOS3.2 and Dronpa
Parameter of the reconstructed super-resolution image in Figure 2D, E and F for Dendra2, mEOS3.2 and
Dronpa, respectively. (A) Localization precision of all localizations. (B) Number of localized events per frame. (C)
Nearest neighbor distribution of all localized events.
10
Figure S7. Vector design for cloning
(Top) Cytosolic expression featuring the fluorescent protein in between the fcp promoter and terminator.
(Bottom) Sil3 fusion constructs, with an N-terminal Sillafin 3 followed by the C-terminal FPs
11
Figure S8. Convolution to estimate tpSil3-Dendra2 filament thickness
Convolution process to estimate the underlying filament thickness based on a measured filament thickness of
76nm (FWHM) in the super-resolution image. The gauss profile of the measured filament is displayed in red in all
plots. (A) Underlying filament with a thickness of 53nm. (B) The first convolution contribution: Fluorophore
localization uncertainty of 28nm. (C) The second convolution contribution: Linker length between protein and
fluorophore of 3nm. Assumed as points on a sphere of 3nm radius. (D) Final convolution of A) to C) resulting in
the desired width of 76nm.
12
Figure S9. MATLAB GUI to visualize super-resolution data from thunderSTORM
Screenshot of the custom written MATLAB GUI for super-resolution data visualization with several features
annotated.
13
Table S2. Codon optimized sequences for the fluorescent proteins
DNA sequences of all the fluorescent proteins used in this study - plus their translation. Codon optimized for
expression in T. pseudonana.
PATagRFP
702 nucleotides, 234 amino acids
1
1
151
51
301
101
451
151
601
201
ATGTCCGAGTTGATCAAAGAAAACATGCAC
M S E L I K E N M H
TTGCCATTCGCATTCGACATCTTGGCCACG
L P F A F D I L A T
GTTTTGACGGCAACGCAAGACACCTCATTG
V L T A T Q D T S L
GATGGTGGATTGGAGGGAAGAGTGGATATG
D G G L E G R V D M
ATCATCAAAGAGGCCGACAAAGAGACATAC
I I K E A D K E T Y
ATGAAGTTGTACATGGAAGGTACTGTGAAC
M K L Y M E G T V N
AGCTTTATGTACGGATCCTCCACATTCATC
S F M Y G S S T F I
CAAGACGGATGCTTGATCTACAACGTGAAG
Q D G C L I Y N V K
GCATTGAAGTTGGTTGGAGGTGGACACTTG
A L K L V G G G H L
TGGGAGCAACATGAGGTGGCAGTGGCAAGA
W E Q H E V A V A R
AACCACCACTTCAAGTGCACATCAGAGGGC
N H H F K C T S E G
AACCACACGCAGGGAATCCCAGACTTCTGG
N H T Q G I P D F W
ATCAGGGGAGTGAACTTCCCATCAAACGGA
I R G V N F P S N G
ATCTGCAACTTCAAGACGACGTACCGTTCC
I C N F K T T Y R S
TACTCCGACTTGCCATCAAAGTTGGGACAC
Y S D L P S K L G H
GAGGGAAAGCCATACGAGGGAACACAAACA
E G K P Y E G T Q T
AAGCAATCATTCCCAGAGGGATTCACATGG
K Q S F P E G F T W
CCAGTGATGAAGAAAAAGACGTTGGGATGG
P V M K K K T L G W
AAGAAGCCAGCCAAGAACTTGAAGATGCCT
K K P A K N L K M P
AAGTTGAACTGA
K L N *
ATGCGTATCAAGGTGGTGGAAGGTGGACCA
M R I K V V E G G P
GAGCGTGTGACAACATACGAGGATGGTGGT
E R V T T Y E D G G
GAGCCATCCACCGAGAAGTTGAAGCCAGCA
E P S T E K L K P A
GGTGTTTACTACGTGGACCGTCGTTTGGAG
G V Y Y V D R R L E
GTGTTTGAGATTGAGGGTGAAGGCGAAGGA
V F E I E G E G E G
CCAGCAGATATTCCAGACTACTTCAAGTTG
P A D I P D Y F K L
GGAACGAACTTCCCATCCGATGGACCAGTG
G T N F P S D G P V
GAAGTCAAGACGACGTACAAGGCAAAGAAG
E V K T T Y K A K K
ATGGACGAGTTGTACAAATGA
M D E L Y K *
CGACCATACGAGGGAACACAGACAGCAAAG
R P Y E G T Q T A K
TCCTTCCCAGAGGGATTCAAGTGGGAGCGT
S F P E G F K W E R
ATGCAGAAAAAGACAATGGGATGGGAGGCC
M Q K K T M G W E A
CCAGTTCAGTTGCCTGGTGCATACAACGTG
P V Q L P G A Y N V
TCCGGCGAGGGCGAGGGCGATGCCACCTAC
S G E G E G D A T Y
CAGCACGACTTCTTCAAGTCCGCCATGCCC
Q H D F F K S A M P
TTCAAGGAGGACGGCAACATCCTGGGGCAC
F K E D G N I L G H
GACCACTACCAGCAGAACACCCCCATCGGC
D H Y Q Q N T P I G
ACTCTCGGCATGGACGAGCTGTACAAGTAA
T L G M D E L Y K *
GGCAAGCTGACCCTGAAGTTCATCTGCACC
G K L T L K F I C T
GAAGGCTACGTCCAGGAGCGCACCATCTTC
E G Y V Q E R T I F
AAGCTGGAGTACAACTACAACAGCCACAAC
K L E Y N Y N S H N
GACGGCCCCGTGCTGCTGCCCGACAACCAC
D G P V L L P D N H
GGAAAGCCATTCGAGGGAAAGCAGTCCATG
G K P F E G K Q S M
CAGTCCTTCCCAAAGGGATACTCCTGGGAG
Q S F P K G Y S W E
GTGATGCAGAAAAAGACATTGAAGTGGGAG
V M Q K K T L K W E
GAAAAGGGTGTTAAGTTGCCAGGTGCACAC
E K G V K L P G A H
GATTTGGAAGTGAAAGAGGGTGGACCATTG
D L E V K E G G P L
AGATCATTGACATTCGAGGACGGTGGAATC
R S L T F E D G G I
CCATCCACCGAGAAGATGTACGTGCGTGAT
P S T E K M Y V R D
TTCGTGGATCACTGCATCGAGATCTTGTCC
F V D H C I E I L S
GAGGGCGAGGGAAAGGGAAAACCATACGAG
E G E G K G K P Y E
CCAGACTACTTCAAGCAATCATTCCCAGAG
P D Y F K Q S F P E
CCACCAAACGGACCAGTGATGCAGAAAAAG
P P N G P V M Q K K
ACGTACAAGGCCAAGAAGGTGGTCCAATTG
T Y K A K K V V Q L
TGA
*
GGAACACAGACGGCCAACTTGACAGTGAAA
G T Q T A N L T V K
GGATACTCGTGGGAGCGTACAATGACATTC
G Y S W E R T M T F
ACATTGAAGTGGGAGCCATCCACCGAGAAG
T L K W E P S T E K
CCAGATGCACATTTCGTGGATCACCGTATC
P D A H F V D H R I
TTGGGAAAGCCATTCGAGGGAAAGCAGTCG
L G K P F E G K Q S
AAGCAGTCATTCCCAGAGGGATACTCGTGG
K Q S F P E G Y S W
CCAGTGATGCAAAAGCGTACAGTGAAGTGG
P V M Q K R T V K W
AAGAAGGTGGTCCAATTGCCAGACTACCAT
K K V V Q L P D Y H
ATGGACTTGAAAGTGAAAGAGGGTGGACCA
M D L K V K E G G P
GAGAGATCAATGAACTACGAGGACGGTGGA
E R S M N Y E D G G
GAGCCATCCACAGAGAAGTTGTACGTGCGT
E P S T E K L Y V R
TTCGTGGATCACCACATCGAGATCAAGTCC
F V D H H I E I K S
PAmCherry
711 nucleotides, 237 amino acids
1
1
151
51
301
101
451
151
601
201
ATGGTGTCAAAGGGTGAAGAGGACAACATG
M V S K G E E D N M
TTGAAAGTGACGAAGGGTGGACCATTGCCA
L K V T K G G P L P
GTGATGAAGTTTGAGGATGGTGGTGTTGTG
V M K F E D G G V V
TTGTCAGAGAGAATGTACCCAGAGGATGGT
L S E R M Y P E D G
AACCGAAAGTTGGACATCACGTCCCACAAC
N R K L D I T S H N
GCCATCATCAAAGAATTCATGCGTTTCAAG
A I I K E F M R F K
TTCACGTGGGATATTTTGTCGCCACAGTTT
F T W D I L S P Q F
ACCGTGACGCAGGATTCATCATTGCAAGAC
T V T Q D S S L Q D
GCCTTGAAGGGTGAAGTTAAGCCACGTGTG
A L K G E V K P R V
GAGGATTACACGATCGTTGAGCAATACGAG
E D Y T I V E Q Y E
GTGCACATGGAAGGATCCGTGAACGGACAT
V H M E G S V N G H
ATGTACGGATCCAACGCATACGTGAAGCAC
M Y G S N A Y V K H
GGTGAATTCATCTACAAAGTGAAGTTGCGT
G E F I Y K V K L R
AAGTTGAAAGACGGTGGACACTACGATGCA
K L K D G G H Y D A
AGAGCAGAGGGACGTCATAGTACAGGTGGA
R A E G R H S T G G
PAGFP
720 nucleotides, 240 amino acids
1
1
151
51
301
101
451
151
601
201
ATGGTGAGCAAGGGCGAGGAGCTGTTCACC
M V S K G E E L F T
ACCGGCAAGCTGCCCGTGCCCTGGCCCACC
T G K L P V P W P T
TTCAAGGACGACGGCAACTACAAGACCCGC
F K D D G N Y K T R
GTCTATATCATGGCCGACAAGCAGAAGAAC
V Y I M A D K Q K N
TACCTGAGCCACCAGTCCAAGCTGAGCAAA
Y L S H Q S K L S K
GGGGTGGTGCCCATCCTGGTCGAGCTGGAC
G V V P I L V E L D
CTCGTGACCACCTTCAGCTACGGCGTGCAG
L V T T F S Y G V Q
GCCGAGGTGAAGTTCGAGGGCGACACCCTG
A E V K F E G D T L
GGCATCAAGGCCAACTTCAAGATCCGCCAC
G I K A N F K I R H
GACCCCAACGAGAAGCGCGATCACATGGTC
D P N E K R D H M V
GGCGACGTAAACGGCCACAAGTTCAGCGTG
G D V N G H K F S V
TGCTTCAGCCGCTACCCCGACCACATGAAG
C F S R Y P D H M K
GTGAACCGCATCGAGCTGAAGGGCATCGAC
V N R I E L K G I D
AACATCGAGGACGGCAGCGTGCAGCTCGCC
N I E D G S V Q L A
CTGCTGGAGTTCGTGACCGCCGCCGGGATC
L L E F V T A A G I
mEOS3.2
681 nucleotides, 227 amino acids
1
1
151
51
301
101
451
151
601
201
ATGTCTGCAATCAAGCCAGACATGAAGATC
M S A I K P D M K I
CCATTCGCATTCGACATCTTGACAACGGCC
P F A F D I L T T A
TGCAACGCACGTAACGATATTACGATGGAA
C N A R N D I T M E
GGTGTCTTGACGGGTGATATTGAGATGGCC
G V L T G D I E M A
CACGACAAGGACTATAACAAAGTGAAGTTG
H D K D Y N K V K L
AAGTTGCGTATGGAAGGCAACGTGAACGGA
K L R M E G N V N G
TTCCATTACGGAAACCGTGTGTTCGCAAAG
F H Y G N R V F A K
GGCGACACGTTCTACAACAAAGTGCGATTC
G D T F Y N K V R F
TTGTTGTTGGAGGGAAACGCACATTACCGT
L L L E G N A H Y R
TACGAGCACGCAGTGGCACACTCAGGATTG
Y E H A V A H S G L
CACCATTTCGTGATCGACGGTGATGGAACT
H H F V I D G D G T
TACCCAGACAACATCCAGGACTACTTCAAG
Y P D N I Q D Y F K
TACGGAACGAACTTCCCAGCAAACGGACCA
Y G T N F P A N G P
TGCGATTTCCGTACGACGTACAAGGCCAAA
C D F R T T Y K A K
CCAGATAACGCAAGACGTTGA
P D N A R R *
Dendra2
693 nucleotides, 231 amino acids
1
1
151
51
301
101
451
151
601
201
ATGAACACACCTGGAATCAACTTGATCAAA
M N T P G I N L I K
GAGGGTGCACCATTGCCATTCTCGTACGAC
E G A P L P F S Y D
GAGGATAAGGGAATCTGCACGATCCGTTCC
E D K G I C T I R S
TTGCATGTTCGTGACGGTTTGTTGGTCGGA
L H V R D G L L V G
GAGATCTTGGGAAACGACTCCGACTACAAC
E I L G N D S D Y N
GAAGATATGCGAGTCAAGGTGCACATGGAA
E D M R V K V H M E
ATTTTGACAACGGCCGTGCATTACGGAAAC
I L T T A V H Y G N
GACATTTCATTGGAGGGCGACTGTTTCTTC
D I S L E G D C F F
AACATCAACATGGCCTTGTTGTTGGAGGGT
N I N M A L L L E G
AAAGTGAAGTTGTACGAGCACGCAGTGGCC
K V K L Y E H A V A
GGCAACGTGAACGGACATGCATTCGTGATT
G N V N G H A F V I
CGTGTGTTTACAAAGTACCCAGAGGACATC
R V F T K Y P E D I
CAGAACGTGCGTTTCAAGGGAACGAACTTT
Q N V R F K G T N F
GGTGGACACTACTTGTGCGATTTCAAGACG
G G H Y L C D F K T
AGATACTCTCCATTGCCATCACAAGTCTGG
R Y S P L P S Q V W
Dronpa
678 nucleotides, 226 amino acids
1
1
151
51
301
101
451
151
601
201
ATGGTGTCAGTGATTAAGCCAGACATGAAG
M V S V I K P D M K
TTGCCATTCGCCTACGATATTTTGACGACG
L P F A Y D I L T T
ATCTGCAACGCAACAAACGATATTACGTTG
I C N A T N D I T L
GATGGTGTCTTGAAGGGTGACGTGAACATG
D G V L K G D V N M
CACGACAAGGACTACTCCAACGTGAACTTG
H D K D Y S N V N L
ATCAAGTTGCGTATGGAAGGTGCAGTGAAC
I K L R M E G A V N
GTGTTCTGCTACGGAAACCGTGTGTTCGCA
V F C Y G N R V F A
GACGGTGACTGCTACATCTACGAGATCCGT
D G D C Y I Y E I R
GCATTGTCATTGGAGGGTGGTGGACACTAC
A L S L E G G G H Y
CATGAGCACGCAGAGGCACATTCAGAGTTG
H E H A E A H S E L
GGACATCCATTCGCAATTGAGGGTGTTGGA
G H P F A I E G V G
AAGTACCCAGAGAACATCGTGGACTACTTC
K Y P E N I V D Y F
TTCGATGGTGTCAACTTCCCAGCAAACGGA
F D G V N F P A N G
CGTTGCGATTTCAAGACGACGTACAAGGCA
R C D F K T T Y K A
CCACGTCAAGCCAAATGA
P R Q A K *
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(see attached movie file)
Movie S1. Raw movie of tpSil3-Dendra2, -mEOS3.2 and Dronpa
The movie contains the first 500 frames of the raw movie used to create the PALM images shown in this study
(Fig. 2). From left to right: Dendra2, mEOS3.2 and Dronpa. One pixel equals 106.7 nm.
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