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Direct PNAS a is article This interest. of conflict H.J. no and declare paper. J.U.H., authors the The I.d.S.O., wrote F.P. M.M., and research; I.d.S.O., performed M.M., and B.G. data; and analyzed K.A., M.D., L.H., A.F., K.M., uhrcnrbtos ..adFP eindrsac;MM,IdSO,SA,SF,P.B., S.F., S.A., I.d.S.O., M.M., research; designed F.P. and G.M. contributions: Author [email protected]. owo orsodnemyb drse.Eal akmelrp.u.eor [email protected] Email: addressed. be may correspondence whom To aaieaaye forC aawt hs bandfrom setup. nanoCT obtained Com- the those of and worm. advantages applicability with the future velvet highlight data methods a CT imaging popular of our other of appendage analyses walking demon- parative as a samples, radia- for biological conducting small strated synchrotron for very relevant of requiring especially analysis is detailed without setup Our nm) facilities. tion (≈100 nanometer high-resolution data generate processing low efficiently data 3D and and the routinely device to CT the on pipeline laboratory a hampers features present still We of level. nm) visualization (≥500 conventional three-detailed systems of CT resolution and limited laboratory-based become the nondestructively However, has various of specimens. structures imaging internal investigating, and external both dimensionally, (CT) for tomography popular computed X-ray Significance odmntaeteptnilapiain ftenanoCT the of applications potential the demonstrate To pro- to ability their with combined power resolving high Their b uihSho fBoniern,TcnclUiest fMunich, of University Technical Bioengineering, of School Munich a,b a,b e nttt fMtrasRsac,Helmholtz-Zentrum Research, Materials of Institute enadGleich Bernhard , NSlicense. 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EVOLUTION APPLIED PHYSICAL SCIENCES Onychophora, or velvet worms—a small group of terrestrial A BC invertebrates pivotal for understanding animal evolution (17). Despite their relevance, there are still many gaps in our knowl- edge of these animals. A recent discussion regarding the evolu- tion of their locomotory system, for example, has brought them back into the scientific spotlight (18). In particular, researchers try to understand the evolution of the limb musculature from their ancestors—marine Cambrian animals called lobopodians— to exclusively terrestrial modern onychophorans (18). Neverthe- less, it is still difficult to draw any major conclusions about this DE issue, as the complex limb musculature of extant onychophorans remains poorly understood. In this study we scanned an onychophoran limb using the nanoCT setup and explored its external and internal anatomy in detail. The onychophoran limb has previously been investigated, to a certain extent, by widely available imaging methods such as light microscopy, histology, SEM, and confocal laser scanning microscopy (CLSM) (e.g., refs. 19–21). Therefore, we also gen- erated corresponding SEM and CLSM data and performed a comparative study to assess the reliability and usefulness of the nanoCT technique for studying complex biological samples. FG

Results Resolution of the NanoCT. The 2D resolution of the nanoCT setup has been tested by acquiring images of a standard resolution test pattern (JIMA RT RC-04), in which the width of the dark lines and bright spaces is exactly 200 nm (Fig. 1 A–C). The background-corrected projection image initially obtained shows identifiable lines and spaces but the edges are not well-defined and the image is noisy (Fig. 1A). This situation could be substan- tially improved by subsequently applying different image pro- Fig. 1. Resolution of the nanoCT setup. (A–C) Projection images of a reso- cessing operations to the original image. Using a Gaussian filter lution pattern with 200-nm lines and spaces. (D and F) Transversal CT slices and a Richardson–Lucy deconvolution (22–24) clearly enhances through the center of a silicon sphere (diameter ≈30 µm) with 98-nm voxel the definition of the lines and spaces, although the overall size. (E and G) Analysis of the edge profiles corresponding to the orange lines through the border of the sphere and the surrounding air. Error func- image still exhibits a substantial amount of noise, evidenced by tions with a free width parameter (blue lines) were fitted to the data points its granulated appearance (Fig. 1B). This image noise can be of the respective edge (orange dots). The 10% MTF measure of the corre- further improved by applying a dictionary denoising algorithm sponding MTF and the rmse of the regression are given in the lower box in (25, 26), which reduces the noise distribution while preserv- each plot. (A) The background was corrected with a flat-field image; no ing the edges of the structures. The result is an image with a further processing steps were applied. (B) A 2D Gaussian filter followed lower noise level and sharper edges (Fig. 1C) compared with the by Richardson–Lucy deconvolution were used to process the background unprocessed image. corrected image in A. (C) Dictionary denoising applied to the image in B. To assess the resolution of our setup for 3D imaging we The training image for the dictionary creation was the image shown in A. analyzed nanoCT data of a highly refracting glass microsphere (D and E) FBP reconstruction without preprocessing of the projection images. ( and ) Richardson–Lucy deconvolution applied to the projection µ F G (diameter ≈30 m; Corpuscular Inc.) imaged with an effective images before FBP reconstruction. voxel size of 98 nm (Fig. 1 D and F). When reconstructing the unprocessed projections with a filtered backprojection (FBP), a transversal CT slice through the center region of the sample (Fig. Optimization for Biological Samples: Onychophoran Limb as an 1D) revealed that the sample was not perfectly round and fea- Example. An ∼350-µm-long walking appendage of the ony- tured some imperfections. We fitted a Gaussian error function to chophoran species Euperipatoides rowelli (Fig. 2A) was imaged the profile of the border between the sphere and the surrounding using the nanoCT setup. When reconstructing nanoCT data from air in a single slice in the center of the sphere. The correspond- unprocessed projection images the resulting contrast modality ing modulation transfer function (MTF) was derived in form of a is a combination of the conventional attenuation contrast and Gaussian function and the 10% MTF value was chosen as a mea- phase effects caused by Fresnel diffraction. Analysis of different sure for the resolution (Fig. 1 E and G). To estimate the good- processing steps applied to nanoCT data of the onychophoran ness of the fit of the edge profile, we also calculated the root- limb (Fig. S2) showed that the soft-tissue contrast and the image mean-square error (rmse) of the regression, which describes the sharpness could be optimized by using a combination of decon- deviation of the original data points from the fit function. Ana- volution and Paganin’s single-distance phase-retrieval algorithm lyzing the marked edge profile (Fig. 1E) demonstrated that the (27). This processing approach was used for the onychophoran raw FBP data show a very high in-plane resolution (10% MTF limb data presented in this paper and is described in detail in = 4,767 line pairs (lp) per mm; resolution ≈105 nm). By apply- Materials and Methods section and Supporting Information. ing a Richardson–Lucy deconvolution to the projections before Comparison with SEM Data. To assess the usefulness of the FBP reconstruction (Fig. 1F), the resolving power could be fur- nanoCT device for investigating the external morphology of bio- ther improved (10% MTF = 5,102 lp/mm; resolution ≈100 nm). logical samples we reconstructed the limb anatomy of the ony- Unfortunately, the noise was also amplified by this processing chophoran E. rowelli based on nanoCT data and compared it step, consequently leading to a decreased goodness of the fit (Fig. with corresponding SEM images obtained from the same species. 1G). A detailed description of the analysis steps and supplemen- Given that the limbs in adult onychophorans are larger than the tary results are presented in Supporting Information. FOV of the setup at the desired voxel sizes of approximately

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EVOLUTION APPLIED PHYSICAL SCIENCES of our setup. The images of the resolution pattern show that nanoCT particularly suitable for examining biological and medi- we can clearly visualize 200-nm structures in two dimensions. cal samples. Furthermore, we demonstrate that processing our data with In this study we demonstrate the potential of the nanoCT deconvolution and dictionary denoising techniques can consid- setup for biological samples by investigating a representative erably improve the visibility of the 200-nm lines. Unfortunately, of Onychophora, or velvet worms. This choice was motivated a smaller source-to-sample distance could not be tested due to by the evolutionary importance of these animals (17) as well the shape of the resolution pattern housing. Therefore, we were as the many gaps in our knowledge of their musculature and unable to overcome the limitations posed by Fresnel diffrac- locomotory system (18, 20, 21). Furthermore, the size of their tion effects and resolve structures below 200 nm, even though limbs allows comparative analyses between the nanoCT device our nanofocus source can create X-ray spots that allow features and other imaging techniques popular in the biological sciences smaller than this to be visualized in projection images (28). (e.g., SEM and CLSM). Since the latter are the foremost tech- Nevertheless, the resolution achieved in projection images niques applied in onychophoran research (e.g., refs. 21 and 29), does not necessarily reflect that of the CT data obtained with the results presented here also contribute relevant information the same setup. In addition to the image quality of each projec- to the field. tion image, various factors affect the resolution of the resulting Indeed, our comparative analyses revealed a set of previously CT slices, namely the tomographic acquisition parameters, the obscure features of the onychophoran limbs, mainly associated reconstruction and processing of the projections, and technical with the foot and leg musculature. The putative presence of cir- conditions such as the mechanical stability of the setup and the cular muscles in the onychophoran foot, although mentioned X-ray spot stability. The results obtained from edge analysis of twice in the literature (20, 30), remained uncertain, as these the sphere clearly show that our system is stable enough to per- structures had not been demonstrated. Our data confirm the form CT scans with resolutions down to 100 nm in the transversal existence of these muscles and reveal details of their position, plane. Deconvolution with a suitable point spread function (PSF) arrangement, and size. Given their characteristics, the circular increased the effective resolution of our CT data considerably, muscles of the foot are most likely responsible for the protrac- which was also demonstrated with the velvet worm data. Due tion of the onychophoran claws. It has been previously suggested to technical constraints we were unable to reliably determine that the claws in these animals are protracted by the hydrostatic the resolution along the rotation axis with the presented sample. action of the hemolymph on an eversible sac situated dorsal Nevertheless, when taking into account the isotropic resolution to the claws (20, 21), but the muscles involved in this process of the presented projection image and the fact that the positional remained unknown. It is reasonable to assume, however, that drifts of our setup are randomly distributed in all directions, we when the circular muscles of the foot contract they increase the conclude that the nanoCT is capable of achieving resolutions hydrostatic pressure within the foot and pump the haemolymph in the direction of the rotation axis comparable to those in the into the eversible sac, which everts and externally protracts transversal plane. the claws. In comparison with other nanoCT setups with nanofocus Our data further suggest that the function of the so-called sources and conventional CCD or flat panel detectors our foot retractor muscle might have been misinterpreted in a recent approach with a single-photon counting detector presents addi- study, in which the authors assumed that this muscle is responsi- tional benefits. First, the absence of readout and dark-current ble for elevating the foot (21). The size, position, and attachment noise in our detector greatly increases the data quality when sites of this muscle revealed in this study instead indicate that the imaging with low photon statistics. Consequently, the acquisi- foot retractor most likely plays the role of a protractor. This is tion time per projection image can still be relatively short, even in line with previous studies (19, 31), which propose an antago- for the very low photon flux characteristic of X-ray spots in the nistic relationship between the foot retractor and the prominent nanometer range. Second, the direct conversion principle of our claw retractor muscles. In other words, while the claw retractor is camera and its very sharp PSF with an FWHM of one detec- responsible for contracting the claws and elevating the foot, the tor pixel does not suffer from the usual decrease in resolution foot retractor may be responsible for depressing the foot back caused by signal spread in scintillation detectors. In summary, the to the ground. It is important to highlight, however, that the single-photon counting detector can produce sharper and less- functional morphology of most onychophoran muscles remains noisy images than conventional integrating X-ray cameras and unexplored and the nomenclature hitherto used will require a its superiority is particularly pronounced for imaging techniques thorough revision once the function of these muscles has been with relatively small numbers of detected photons, as is the case clarified. Further investigating the onychophoran myoanatomy for laboratory-based nanoCT imaging. will eventually shed light on the open question regarding Compared with quasi-monochromatic CT imaging systems the evolution of their locomotory system from a marine an- using Fresnel zone plates (5–7), our nanoCT offers several cestor (18). advantages, for example an infinite depth of focus that allows The present study used a nanoCT device to investigate the a larger and more adaptable FOV. Moreover, the full polychro- onychophoran limb anatomy. On the one hand, we demonstrated matic spectrum of the source, which reaches up to 60 kVp, broad- that this method is able to retrieve data that are congruent ens the bandwidth available for potential applications to include with commonly used methods, such as CLSM and SEM. On the more strongly absorbing samples that cannot be penetrated by other hand, the comparison of these different techniques sug- X-rays with energies below 10 keV. Furthermore, shorter acqui- gests that the nanoCT setup presents advantages that could open sition times are possible for comparable voxel sizes, reducing new doors for investigating biological samples. First, the sample the overall acquisition times for full CT datasets and, conse- preparation that precedes the imaging proved to be less expen- quently, favoring an effective workflow in research groups with sive and time-consuming for nanoCT than for SEM and CLSM, multiple users. in particular for the latter, which requires sectioning techniques The small X-ray spot of the source and the sharp system PSF and expensive fluorescent markers for specific labeling of the tar- introduce phase effects, such as edge enhancement, into the raw get structure/tissue. Second, the nanoCT dataset generated from contrast of the nanoCT setup. Those effects can be exploited a single onychophoran limb provided high-resolution details of by applying phase-retrieval techniques that strongly enhance the both its internal and external anatomy, whereas investigating soft-tissue contrast within the sample. The feasibility of phase- the corresponding internal and external features requires both contrast imaging combined with the still relatively low effective CLSM and SEM analyses, respectively. Moreover, investigating energy of the X-ray tube spectrum (20 keV at 60 kVp) render the the complete anatomy of the onychophoran limb by CLSM or

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1710742114 Muller¨ et al. 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Processing 404 NanoCT: was size voxel effective source- The a mm. and 400 mm, time of 0.94 distance exposure of all to-detector distance 2-s were source-to-rotation-axis namely, datasets a parameters, These projection, acquisition article. com- per same this subsequently in worm the and velvet presented with acquired the data acquired were For volume datasets nm. the 10 98 to limb, of bined entire size the voxel effective of source-to- was The an data h. distance in 4 source-to-detector resulting of the mm, time and 700 acquisition mm, total 0.4 a was background and distance projection exposure isotropic rotation-axis an per an with s acquired 5 achieve was of sphere to time silicon the field of flat dataset CT a The intensity. by normalized was jection and n h ebr pcmn of specimens newborn the ing Preparation. Sample laser- NanoCT GmbH). confocal MicroImaging the Zeiss (Carl with Mount- META 510 Vectashield analyzed LSM in Zeiss and microscope slides Laboratories) scanning glass (Vector on for Medium mounted labeled ing were were vibratome-sectioned (38). sections previously labeled sections described and as The Vibratome (Invitrogen) embedded, rhodamine (21). phalloidin fixed, using Mayer f-actin pieces, and Oliveira small following into cut were CLSM. and Sectioning Vibratome reproduced 37. was and 32, features 20, morphological refs. their from Europe for High-Technologies terminology (Hitachi The scan- S4000 GmbH). emission Hitachi field microscope a using electron examined ning were Samples 36). (32, described ously SEM. experiments. the before specimens camera living D7000 The Nikon 35). a 34, with (32, photographed elsewhere were peripatid described the as of kept specimens and obtained The logs. rotten and litter species leaf as such habitats, Specimens. 2.1. MAX Amira VGStudio and using Group) segmented Sciences were Visualization (FEI structures 6.0.1 highlighted The GmbH). Graphics rnsM 1711 -,ad31(oGM) n osloNcoa eDesen- de Nacional I.d.S.O.). DFG Conselho Initiative, and G.M.), Excellence Cient Insti- (to German 3-1 volvimento Munich and the 2-1, of by 4147/1-1, Ma funded University Grants Study, Technical Advanced Got- the for DFG (DFG) program, tute the Foundation Leibniz Photonics, Research Advanced Wilhelm German for tfried the Munich-Center by Excellence cul- of supported animal Cluster with was help work their for This group ture. research GM’s of members and Sweden ACKNOWLEDGMENTS. nanoCT. the an and for (32), in holders described dehydrated sample previously overnight as onto PBS, dryer postfixed mounted in a critical-point were a GmbH) and in legs Services dried leg series, separated (Science one ethanol The tetroxide containing osmium wall. pieces 1% body smaller in the into of washed cut were part and specimens small PBS Selected in weeks. times several for several water distilled in hyde .BunocxP ao ,PuesB(00 oad u-0-mXrymcocp for microscopy X-ray sub-100-nm SEM. Towards an (2010) B in Pauwels microscopy A, X-ray Sasov P, phase-contrast Bruyndonckx of 9. Applications (2003) al. et https://www.zeiss.com/microscopy/ SC, at Mayo Available Ultra. 8. 810 Xradia ZEISS (2017) Zeiss 7. methods: tomography computed X-ray Advanced (2015) C Heinzl B, Plank J, Kastner 6. oorpi applications. tomographic France IV JPhys 2017. 7, September Accessed int/products/x-ray-microscopy/xradia-810-ultra.html. 120–132. pp Belgium) Gent, informatie, Conference istratieve CT. Tomography contrast Computed phase and Radiology and Industrial 4DCT Digital CT, quantitative CT, resolution High E h pcmn eefie,peevd n rprdfrSMa previ- as SEM for prepared and preserved, fixed, were specimens The n in and .hitoyensis P. w pce fOyhpoawr olce rmtpclmicro- typical from collected were Onychophora of species Two h ouerneig nFg 2 Fig. in renderings volume The Information. Supporting oi S1 Movie 104:543–546. fioeTecnol e ´ ıfico 3)adteprptpi species peripatopsid the and (32) etakteeteeyhlflta tEclu AB, Excillum at team helpful extremely the thank We eegnrtdwt GtdoMX21(Volume 2.1 MAX VGStudio with generated were odrDiffr Powder eoesml rprto o aoTimag- nanoCT for preparation sample Before gc CP rzl rn 90921- (to 290029/2010-4 Grant Brazil) (CNPq ogico ´ .rowelli E. pcmn of Specimens 25:157–160. o h eovlto rcdr,a procedure, deconvolution the For a ensoe n4 formalde- 4% in stored been had .rowelli E. NSEryEdition Early PNAS .rowelli E. and rceig fthe of Proceedings Gn CAdmin- BC (Gent .hitoyensis P. 3)were (33) | f6 of 5 B, D,

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