Compact portable higher harmonic generation microscopy for the real time assessment of unprocessed thyroid tissue

During thyroid surgery fast and reliable intra‐operative pathological feedback has the potential to avoid a two‐stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). We explored higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) for this purpose. With a compact, portable HHG microscope, images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma) were recorded. The images were generated on unprocessed tissue within minutes and show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue.


| INTRODUCTION
Thyroid nodules are common and their incidence has increased due to incidental findings on imaging modalities [1].Prevalence of thyroid nodules is estimated as high as 5% for nodules found by palpation, and about 10 times higher when the thyroid is examined during autopsy, by ultrasonography, or during surgery [2], with higher frequencies in women and the elderly [3].The clinical importance of thyroid nodules however, rests in the need to exclude thyroid cancer, with a reported incidence of approximately 5% [4].The most common thyroid cancer is papillary carcinoma, comprising approximately 80% of all primary thyroid malignancies [5], followed by follicular carcinoma, which comprises roughly 15% of the cases [5].The work up for thyroid cancer diagnosis includes neck ultrasonography and, in case of a nodule with suspicious sonographic features, a fine-needle aspiration (FNA) biopsy.In case of uncertain diagnosis after FNA (Bethesda (III-IV-V) [6], usually, a diagnostic hemithyroidectomy (HT) is performed [7].Subsequently, in case of malignancy, a completing thyroidectomy (CT) is performed in patients with a >1 cm thyroid cancer followed by radioactive iodine (RAI) therapy [7].In the Netherlands, 32% of patients with thyroid cancer receive a CT after a diagnostic HT and thus undergo two surgical procedures [8].Ideally, the diagnostic and therapeutic procedures are combined into one surgical procedure to reduce surgical morbidity and costs.However, the gold standard for diagnosing thyroid cancer remains histopathology, which routinely takes more than 16 h for definite diagnosis (and with more advanced techniques still more than 4 h) and so, extensive histological examination is not feasible during thyroid surgery.Currently, an available technique to provide pathological feedback during a diagnostic HT is frozen section (FS) examination.However, FS comes with its limitations, as it is not reliable in evaluating certain crucial nuclear features for the diagnosis of papillary cancer, or in determining capsular or vascular invasion, features essential for follicular thyroid cancer diagnosis [9].Therefore, reliable distinguishing between a follicular adenoma, a follicular carcinoma, and a follicular variant of papillary carcinoma is not possible on FS [7,9].Thus, an alternative to histopathology and FS is required to achieve reliable intraoperative pathological feedback, to facilitate intraoperative therapeutic decision-making and to avoid a two-stage procedure in patients with thyroid cancer.
A new promising clinical imaging tool for rapid histopathology is higher harmonic generation (HHG) microscopy [10][11][12][13][14][15][16].HHG microscopy provides a non-invasive, label-free, 3D modality for imaging unprocessed tissue with high resolution within seconds to minutes, using second and third harmonic generation (SHG, THG) and multiphoton excited autofluorescence (MPEF).Because of intrinsic depth sectioning, this imaging technique has the capacity to obtain information of cell and tissue structures in three dimensions (3D).So far, only SHG, visualizing collagen, has been used to image thyroid tissue.Using polarized SHG microscopy on fixated thyroid tissue, collagen structure was found to be more ordered with malignancy, with straighter and more aligned collagen fibers in the malignant nodule capsule.[17][18][19][20][21] Huang et al used the combination of SHG and MPEF and observed the microstructure of follicle and collagen structure in thyroid tissue, and observed morphologic changes between normal, nodular goiter, and papillary cancerous thyroid tissue [22].However, they reported difficulties in the distinguishment between nodular goiter and papillary thyroid cancer via the collagen matrix.Here, we add third harmonic generation microscopy to the SHG and MPEF imaging modalities as THG has been successful in the imaging of tumor cells and nuclei in unprocessed tissue [10][11][12][13][14][15][16].THG provides contrast through the nonlinear susceptibility or index of refraction change at interfaces and inhomogeneities [23][24][25] and has been shown to have the capacity to generate high quality histopathological images of freshly excised tissue in less than 1 min, in case of brain, breast, skin and lung tumor tissue [11, 13-16, 26, 27].The combination of THG, SHG and MPEF, denoted here as HHGM, therefore seems to be a promising tool for real-time assessment of freshly excised tissue during surgery and could be promising for thyroid tissue as well.
In this pilot study, the aim is to investigate the added value of this new imaging technique using a mobile HHG microscope, by imaging healthy, non-malignant and malignant unprocessed thyroid tissue immediately after excision and compare these images with standard histology, in order to create an overview of the morphological features of thyroid that can be observed with HHGM.

| MATERIALS AND METHODS
After approval by both the medical ethical committee of the Amsterdam University Medical Centers, Location VUmc, Cancer Center Amsterdam (METC number 2021.0688), and the Biobank Pathology Unit of the Amsterdam UMC, freshly excised thyroid tissue of patients undergoing thyroid surgery (HT or TT) for any thyroid disease at Amsterdam UMC was collected for HHGM imaging.After imaging freshly excised tissue, the tissue underwent further standard procedures for histopathological diagnosis.The HHGM images were compared with FS and histopathology.Images were reviewed by three researchers (LD, SK, PV) including a pathologist specialized in thyroid diseases.Definite diagnosis of thyroid nodules (malignant or nonmalignant) was done by histopathology, as gold standard.This research project followed the Netherlands Code of Conduct for Research Integrity and the Declaration of Helsinki.

| Tissue handling
After resection, the thyroid tissue samples were placed in a tissue tray and transported to the pathology department.Tissue samples were not inked, as is often clinical practice, because the ink was found to interfere with the HHG/MPEF imaging (Figure S1).Tissue was carefully sliced to provide a flat sample necessary for imaging.Samples (maximum size of 1 Â 1 Â 1 cm 3 ) were put in a sample holder (IBIDI, 35 mm diameter μ-Dish with 0.17 mm glass thickness) and placed in the microscope.After imaging, the samples were prepared for FS and processed according to standard histopathological procedure.For frozen section analysis, the samples were sliced at the HHGM imaging side using a cryomicrotome.For standard histopathology, the samples were fixed with 4% formaldehyde, embedded in paraffin, sliced in 3 μm thick histological sections at the same HHGM imaging side, and routinely stained with hematoxylin and eosin (HE) for microscopic analysis.

| Image acquisition
Images of freshly excised unprocessed tissue samples were acquired with a mobile FD1070 microscope (co-developed with Femto Diagnostics B.V., now Flash Pathology B.V.), as previously described [14].Figure 1 shows a schematic overview of the HHGM set-up.A 50-fs laser source, centered at 1070 nm (Fidelity 2, Coherent equipped with precompensation) was used to generate the nonlinear signals.The microscope was equipped with an acousto-optic modulator (AOM) to retain sufficient peak power to generate nonlinear optical signals but at the same time avoid damaging of the tissue.Bunches of 5-10 pulses at a repetition rate of 1 MHz out of the 70 MHz pulse train were selected, to achieve a low average power of 5 mW, with a pulse peak energy of $5 nJ).Signals were collected in backscatter direction (Figure 1B,C).Analogue photo-multiplier tubes (H10720 and H7422, Hamamatsu) collected the third harmonic generation (THG), second harmonic generation (SHG), and two-photon excited autofluorescence (2PEF) signals.Dichroic mirrors and interference filters were used to separate the three signals, resulting in the following detection bandwidths: THG 350-360 nm, SHG 530-540 nm, and 2PEF 562-665 nm (Figure 1B).The laser power on the sample was $5 mW, no effects of HHGM imaging were noticed in the frozen sections or processed histological slides.
A sub-micrometer focus was achieved with the use of an oil-immersion objective with a high numerical aperture (40Â/1.3NA, Nikon), resulting in an optical resolution of $0.4 Â 0.4 Â 2.4 μm 3 [14].The THG/SHG/2PEF images were acquired with a LabVIEW program (Femto Diagnostics B.V.).Compared to Van Huizen et al [14], bidirectional scanning of the galvomirrors was implemented to increase the image acquisition speed.This resulted in an acquisition time of 0.5 s per image in a fast 'inspection' mode (1 pixel/μm) and 2.3 s per image in a high-quality mode (5 pixel/μm).The overview mosaic images were made in 'inspection mode' in which an area of 10 Â 10 mm 2 was acquired within 10 min.In inspection mode the tiles had a larger field of view than in the high-quality mode, resulting in some vignetting on the edges.Areas of interest were re-scanned with the high-quality mode.In addition to 2D images, depth scans of regions of interest were acquired to provide 3D information.In all images, THG signals are displayed in green, SHG signals in red and MPEF signals in blue.The THG and SHG signals had a dynamic range of 12 bits, the 2PEF signal was typically weaker, but the total information content in 1 pixel exceeded the possibilities of the RGB coding of 8 bits per colour.Choices were made in the contrast settings per colour to highlight specific features in the image.

| RESULTS
We imaged freshly excised, unprocessed thyroid tissue samples from eight patients undergoing thyroid surgery with HHGM microscopy, and we compared the acquired SHG/THG/2PEF images with the corresponding histopathology images.Figure 2 is an illustration of how the three imaging modalities yield complementary information: the THG signal (depicted in green) visualizes all optical interfaces in the tissue, SHG (depicted in red) mainly visualizes collagen only, and a significant MPEF signal is observed (depicted in blue), of which we discuss the origin below.Using only 5 mW of laser power yielded sufficiently large SHG and THG signals for detection at a rate of 1 Mpixel/s.Signals range from 0 to 10 000 (adc counts) in arbitrary units.In the histogram (Figure 2E) the most frequently observed value for the harmonic signals is typically the lowest bin, indicating that the signal is background free.This is not true for the MPEF signal which has a small background with the histogram peaking at 130 counts (this is typical of the thyroid samples).between the follicles can be identified.A weak MPEF signal (indicated in blue) is present within some of the follicles.Thyroid follicles are filled with colloid containing thyroglobulin, which emits fluorescence from 600 to 700 nm [28], which partially coincides with the MPEF detection range.In addition, in the epithelium layer speckles of THG signal can be seen, that sometimes coincide with the blue MPEF signal (see Figure 4).The speckles are present in the cytoplasm of the epithelial cells and are smaller than the cell nucleus, with a diameter of $1-5 and $6-7 μm respectively.The larger speckles do not have a uniform shape, but appear to be accumulations of smaller granules, whereas the cell nuclei show as smooth spheres.As the speckles emit both THG and MPEF signals, a hypothesis is that these are vesicles containing thyroglobulin or thyroid hormones T3 or T4.

| Follicular adenoma
Two follicular adenomas (FA), benign nodules, are shown in Figures 4 and 5. Figure 4 shows a comparison with frozen section H&E images.In the HHG image, the healthy tissue area appears bluer compared to the healthy tissue in Figure 3, partially due to an increased contrast setting of the MPEF image modality and partially because of unidentified structures emitting high amounts of fluorescence.In Figure 4A, a large nodule can be seen in the top right corner, with the THG signal revealing dense tissue, clearly different from the more blue and follicular structure of the healthy tissue.The nodule is surrounded by a thick collagen layer, which creates a clear division, as can also be seen in the frozen section H&E image (Figure 4B).The follicles are larger in the healthy tissue than in the nodule, and there is a dense wide epithelium that forms the walls of the follicles (Figure 4C-J).Figure 4K,L shows HHGM images of the other, non-cut side of the nodule.The non-cut side image shows an even denser tissue, with all follicles collapsed, and no 'holes' are visible.MPEF signals and SHG signals from collagen were very weak in the images from the non-cut side.
The second case of follicular adenoma in Figure 5 shows larger follicles compared to both the first FA case and the healthy tissue.Like the healthy tissue, large welldefined follicles are present, however there is no collagen between the follicles and no MPEF signals from within the colloid.The lack of collagen is in correspondence with the H&E-stained images.From the H&E is clearly visible that a lot more cell nuclei are present compared to the healthy tissue.In the HHGM the individual cells can be recognized better than in Figure 4, probably partially due to the absence of the intense green/blue speckles.

| Papillary thyroid carcinoma
To determine whether we can distinguish healthy/benign thyroid tissue from malignant tissue, we next imaged five malignant samples.Figure 6 shows two cases of papillary thyroid carcinoma (PTC), both determined to be Bethesda 6 from the FNA biopsy.Papillary thyroid carcinomas are histopathologically characterized by an unencapsulated tumor, overlapping cell nuclei with a ground-glass appearance and longitudinal grooves, with cytoplasm invagination into the nuclei [29].The HHGM images show dense collapsed follicles, with in between the dense tissue cell nuclei visible as groups of green spheres.These features are comparable to those observed in the H&E images.Both HHGM and histology images show in the first case thick collagen layers (Figure 6A-D), which are absent in the second case (Figure 6E-H).
Two more PTC cases are shown in Figure 7, preoperatively classified as Bethesda 4. These cases show less dense tissue with less collapsed follicles as compared to the PTC Bethesda 6 cases from Figure 6.Both Bethesda 4 cases show a closer resemblance to the follicular adenoma (benign) case in Figure 4, with dense tissue between follicles without surrounding collagen.In contrast to the follicular adenoma from Figure 4, these cases show more and larger follicle openings.The first case, Figure 7A-D, shows more cells and cell nuclei than the second case, Figure 7E-H, where cells can be recognized also in the HHG but not as many and not as clearly.
The THG signal in the Bethesda 6 PTC cases reveal a distinct papillary growth pattern, but some important pathological hallmarks were not recognizable compared to the histology images (Figure 6).The ground-glass appearance (yellow arrows, Figure 6), an important finding in the diagnosis of papillary carcinoma of the thyroid, only appears in paraffin embedded histological sections [30].Therefore, it is to be expected that this fixation artifact will not be recognizable in the HHGM images.The papillary growth pattern is less visible in the Bethesda 4 PTC cases (Figure 7), but these cases do show a large amount of cell nuclei.

| Follicular thyroid carcinoma
Two examples of follicular thyroid carcinomas (FTC), which are rarer than the PTC cases, are shown in Figure 8. Follicular thyroid carcinomas are histopathologically characterized by encapsulated tumors.In addition, a key feature to distinguish follicular carcinomas from follicular adenomas is capsular or vascular invasion [29].The first case was found as an FTC metastasis, so it was known that FTC was present in the thyroid, and therefore rating of the biopsy with a Bethesda score was not performed.The second case was classified as Bethesda 4. The two FTC cases show different features.
For the first case, the images are filled with cell nuclei and follicular structures are absent, while in the images of the second case the follicular structures are present.Both cases lack the SHG signals from collagen and the autofluorescence signals.The features observed in the HHG images compare well to the features identified in the H&E images.

| Spindle cell variant of papillary thyroid carcinoma
The last cancer case we discuss is the rare tumor type spindle cell papillary thyroid carcinoma (SCPTC), which is shown in Figure 9.The morphology of this SCPTC case is very different from the PTC and FTV cases in Figures 6-8.The SCPTC images contain elongated cells, growing in an almost wavelike structure, clearly visible in both HHGM and H&E images.Note that in the HHGM images almost no collagen is present, in agreement with the H&E image, and also no MPEF signal.The Bethesda score on cytology was 1, meaning nondiagnostic, which happens more often with spindle cell tumors.Because the biopsy did not result in a diagnosis, a hemithyroidectomy was performed after which the nodule was found to be a spindle cell variant of a papillary thyroid tumor.

| DISCUSSION
In this pilot study, we examined the feasibility of the HHGM technique for thyroid tissue.We show that HHG microscopy reveals several relevant pathological hallmarks.The SHG/THG/MPEF images visualize the thyroid follicle architecture, cells, cell nuclei, collagen organization and the distribution of thyroglobulin and/or thyroid hormones T3 or T4.Tumor could be distinguished from healthy tissue based on tumor cell density and growth pattern, and the presence or absence of the normal follicle architecture.Earlier nonlinear microscopy studies did not include THG as imaging modality, but used (polarization resolved) SHG and MPEF [17][18][19][20][21][22], to look in detail at the structure of the capsule around a nodule, comparing follicular adenoma with carcinoma [20,21].The added value of THG is clearly the visualization of cells and cell nuclei, important to discriminate healthy tissue from tumor tissue, and to diagnose the tumor.Though the P-SHG experiments yielded promising results for the differentiation of FA from FC, measurements of the polarization signals required long acquisition times, limiting the field of view that can be assessed within reasonable time.Not only the addition of THG, but also the MPEF signal appeared to give relevant information on the state of the tissue, as the MPEF signal, most likely from the T3 thyroglobulin or thyroid hormones T3 or T4, was absent in the tumor areas.
The HHG microscope as employed in this study enables the scanning of relatively large areas in a limited time.However, the thyroid tissue sections that need inspection are large, which made it challenging to image a full section within a time relevant for the intra-operative application.Finding the sometimes rare capsular or vascular invasions required scanning multiple sections, to be done in a limited amount of time.Therefore, a further increase in acquisition speed, field-of-view per tile, or smarter acquisition management, possibly supported by AI, is mandated.We expect, by realizing a factor 2 in improvement in each of these factors, that feedback on the nature of the thyroid tissue should be realizable within 10 min with HHGM.An alternative would be to apply exogeneous labels to the tissue, and to record their fluorescence through either confocal (Histolog Scanner, Samantree, Switzerland) or nonlinear microscopy [31], for which very fast acquisition times have been reported.With fluorescent labels, these techniques yield images similar to classical H&E-stained images, but they do require additional time to apply the labels and wash them off after imaging.The advantages of HHGM are that no preparation is required and that three complementary signals are available that visualize not only cells and cell nuclei, but also the connective tissue and extra cellular matrix.Studies on brain and lung tumor tissue earlier revealed that pathologists quickly get used to the HHGM images and are able to diagnose tissue based on HHGM [14,27,32], the information-rich HHGM images presented here suggest that this is also the case for thyroid tissue.
The HHG/MPEF images for the different tumor types show similar histological hallmarks compared to histology images and enabled the recognition of different patterns in agreement with the different grades and tumor types.Therefore, distinguishing between a follicular adenoma, a follicular carcinoma, and a follicular variant of papillary carcinoma, appears to be possible with HHGM.However, more cases per tumor type need to be imaged to determine whether the HHG observations allow indeed for a reliable discrimination between the different tumor types.The follow up study should also focus on the question whether HHGM can detect capsular or vascular invasion, as this is in histopathology a key observable to discriminate follicular carcinoma from follicular adenoma.

| CONCLUSION
This study demonstrates the feasibility of imaging thyroid tissue with the portable higher harmonic generation microscope.The results indicate that the HHGM technique may potentially be of significant clinical use in the treatment of patients with thyroid cancer.HHGM imaging provides excellent structural contrast using only a single imaging modality, without the need for externally applied contrast agents.The image quality in combination with the relatively short time in which the HHGM can generate images, makes this technique a promising tool.The clinical importance of applying HHG microscopy for thyroid tissue lies in the option to combine the diagnostic and therapeutic surgical procedure (two-stage procedure) into a one-stage procedure, by distinguishing tumorous from non-tumorous tissue intraoperatively, thus limiting operation time, hospital admission and perioperative complications and morbidity.

3. 1 |
Non-malignant thyroid tissue 3.1.1| Healthy thyroid tissue HHGM images with corresponding histology images of healthy thyroid tissue are shown in Figure 3.The tissue was removed in a completion thyroidectomy surgery, but no cancer was found in the resected lobe.The HHGM images show a good comparability to the H&E-stained images.In both images the many large follicles formed by thin epithelial layers (THG) and the collagen (SHG) F I G U R E 2 Images of the separate (A-C) and combined (D) channels of the HHGM.(E) Signal intensity histogram of a single tile for each of the three channels.Signals range from 0 to 10 000 (adc counts) in arbitrary units.In the histogram they are binned with a bin size of 10 counts.The signal measured with laser off is subtracted from the signal, here laser off signals were 1.8 for the THG, 22.2 for SHG, 3.0 for MPEF.

F I G U R E 3
Images of healthy thyroid (HT) tissue, with follicles (FO) and collagen (CO).A, C) HHGM images.B, D) H&E-stained images of comparable structures.White arrows show examples of cell nuclei.(A, B) Overview images, the HHGM mosaic consists of 10 Â 10 tiles, with a tile size 250 Â 250 μm and 1000 Â 1000 pixels.The scan time was 7 min.(C, D) Zoomed in for more detail.F I G U R E 4 HHGM and histology images of follicular adenoma nodule (FA) with a collagen (CO) capsule in healthy thyroid tissue (HT).(A, B) Large overview.(A) is the combination of three mosaics, one 25 Â 25 and two 25 Â 15 tiles, with tiles of 400 Â 400 μm 2 and 400 Â pixels.Total imaging time was approximately 40 min.(C, G) Detail of healthy tissue with follicles (FO) surrounded by collagen.(D-F, H-J) Healthy, transition area, and benign nodular tissue respectively.(K, L) The same nodule imaged from the other side of the sample, the un-cut side, showing denser tissue without follicle openings.

F I G U R E 5
Images of a follicular adenoma, showing large and empty follicles (FO) with cell nuclei (CN) in between.(A, B) Overview images, HHGM mosaic of 10 Â 10 tiles, with tile size 200 Â 200 μm 2 and 1000 Â 1000 pixels.The scan time was 7 min.(C, D) zoomed in for more detail.(A, C) HHGM images.(B, D) H&E-stained images of comparable structures.F I G U R E 6 Images of papillary thyroid carcinoma, rated as Bethesda 6.The upper and lower row are from different cases.(A, B, E, F) matched HHGM and H&E overview images, showing collagen (CO) and dense tissue of collapsed follicles (CF).(C, D, G, H) zoomed in for more detail showing dense tissue of collapsed follicles with cell nuclei (CN) in between.Yellow arrows indicate glasslike appearing cell nuclei, an H&E artifact that signals PTC.The HHGM mosaic in (A) consists of 10 Â 10 tiles, with tile size of 250 Â 250 μm 2 and 1000 Â 1000 pixels.The scan time was 7 min.The HHGM mosaic in (E) consists of 12.5 Â 12.5 tiles, with tile size 400 Â 400 μm 2 and 400 Â 400 pixels, scan time 15 min.

F I G U R E 7
Images of papillary thyroid carcinoma, rated as Bethesda 4. The upper and lower row are from different cases.(A, C, E, G) HHGM images.(B, D, F, H) H&E-stained images of matched structures.(A, B) Tumour tissue with follicles (FO), E, F) healthy tissue (HT) and a nodule (arrow).(C, D, G, H) zoomed in for more detail.(C,D) In between the follicles (FO) are large amounts of cell nuclei (CN).(G, H) Less cell nuclei present in comparison with the other case.Scan details: (A) HHGM mosaic of 6 Â 6 tiles, with tile size 250 Â 250 μm 2 and 500 Â 500 pixels.The scan time was less than 1 min.(E) HHGM mosaic of 32 Â 32 tiles, with tile size 400 Â 400 μm 2 and 400 Â 400 pixels.This was a selection from of a 42 Â 32 mosaic, with a total scan time of 23 min.

F
I G U R E 8 Images of follicular thyroid carcinoma.Top and bottom row are different cases.The first case, not Bethesda rated, is very rich in cell nuclei (area; CN, individual examples; arrows).The second was rated as Bethesda 4, showing follicles (FO) with open spaces and a lower cell count.(A, C, E, G) HHGM images.(B, D, F, H) H&E-stained images of matched structures.(A, B, E, F) Overview, HHGM mosaic of 10 Â 10 tiles, with tile size of 200 Â 200 μm 2 and 1000 Â 1000 pixels.The scan time was 7 min.(C, D, G, H) zoomed in for more detail.F I G U R E 9 Images of a spindle cell thyroid carcinoma, showing elongated cells in a wavy pattern.(A, B) Overview, HHGM mosaic of 10 Â 10 tiles, with tile size of 200 Â 200 μm 2 and 1000 Â 1000 pixels.The scan time was 7 min.(C, D) zoomed in for more detail.(A, C) HHGM images.(B, D) H&E-stained images of comparable structures.
AUTHOR CONTRIBUTIONS M. L. Groot and C. Dickhoff performed study concept and design; S. D. Kok, L. van Dommelen, P. M. Rodriguez Schaap, L. M. G. van Huizen, P. van der Valk performed development of methodology; S. D. Kok, L. van Dommelen, P. van der Valk performed acquisition and data interpretation; S. D. Kok, L. van Dommelen., M. L. Groot performed writing; all authors performed review and revision of the paper.All authors read and approved the final paper.