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. 2025 Dec 15;12:1945. doi: 10.1038/s41597-025-06406-6

Fifty high-content light sheet fluorescence microscopy datasets of Tribolium castaneum embryogenesis

Frederic Strobl 1,2,✉, Pinelopi Goumenaki 1, Kristina Mirkes 1, Henrik Tonner 1, Mariia Golden 1, Julia Ratke 1, Franziska Krämer 1, Stefan Münster 2, Ernst H K Stelzer 1
PMCID: PMC12705995  PMID: 41398413

Abstract

The red flour beetle (Tribolium castaneum) is a key model organism in developmental biology, genetics, and agricultural research. To address the limited availability of high-quality microscopy data documenting its embryonic morphogenesis, we assembled the first Systematic Live Imaging Collection of Embryogenesis (SLICE-1), a large open-access resource for Tribolium embryogenesis. Using light sheet fluorescence microscopy, we acquired fifty high-content datasets totaling 200 days of recording time, 5.7 million images, and 2.7 Terabytes of data. Imaging employed five novel transgenic lines expressing mEmerald-labeled nanobodies that target distinct intracellular structures under control of the consecutively and ubiquitously active tubulin alpha 1-like protein promoter. Each line encompasses multiple sublines representing independent genomic insertions of the same transgene. Embryos were imaged in triplicates under standardized conditions, and all but three specimens developed into healthy, fertile adults, confirming the minimal invasiveness of the experimental procedure. SLICE-1 enables quantitative and comparative analyses of embryonic development, provides an overview of nanobody functionality in insects, and serves as a training resource for image processing, machine learning, and neural networks.

Subject terms: Embryogenesis, Morphogenesis, Transgenic organisms, Model invertebrates

Background & Summary

Developmental biology seeks to understand how a single cell gives rise to a fully functional multicellular organism, uncovering fundamental principles that orchestrate cell migration, proliferation, and differentiation1. Three-dimensional imaging techniques have become a cornerstone methodology for examining embryonic morphogenesis from the subcellular to the in-toto level2. In particular, light sheet fluorescence microscopy (LSFM) is well-suited for long-term live imaging due to its high signal-to-noise ratio, fast recording speed, deep tissue penetration, and low photobleaching/phototoxicity3. Microscopy-based experiments typically follow one of two strategies: (i) an on-demand approach, which addresses specific scientific questions, or (ii) a systematic approach, which generates comprehensive, multi-dimensional datasets intended as reusable resources for large, interdisciplinary research communities4. In particular, systematically acquired morphogenetic data serve both as convergence points that enable the chronological alignment of on-demand data across a universal developmental framework, and as divergence points that provide the foundation for uncovering previously unrecognized morphogenetic processes or formulating hypotheses about underlying biological mechanisms. Beyond developmental biology, such data are valuable to a range of adjacent fields. Biophysicists use image data to infer biomechanical forces during morphogenesis5,6, while laboratories focused on a single model organism employ such information to embed their findings in a broader evolutionary context7. Additionally, the performance of machine learning- and artificial intelligence-based image analyses improves with increasing volume and diversity of training data8.

While ample imaging data are available for the zebrafish (Danio rerio) and the fruit fly (Drosophila melanogaster) in open-access databases such as Zenodo9, Image Data Resource (IDR)10 and BioImage Archive11, comparable resources remain scarce for emerging model organisms like the red flour beetle (Tribolium castaneum). Recognized as a valuable complement for studying both conserved and divergent aspects of insect development12, Tribolium offers several advantages: genetic tractability13,14, a key phylogenic position within the Holometabola15, and a slower, seemingly more ancestral mode of embryogenesis16. However, the limited availability of microscopy data restricts its broader use in quantitative and comparative research. Expanding the number of openly accessible datasets is therefore essential to complement its well-established genetic toolkit17–20 with comprehensive morphogenetic context. This will not only strengthen the interface between the molecular, cellular, tissue and organismal levels, but also introduce a second analytical axis for comparison, thereby fostering integrative and evolutionary informed analyses.

In this data descriptor, we present the first Systematic Live Imaging Collection of Embryogenesis (SLICE-1) comprising fifty high-content fluorescence microscopy datasets of Tribolium castaneum embryonic development, totaling 200 days of recording time, 5.7 million images, and 2.7 Terabytes of data (Supplementary Table 1). Datasets were acquired using LSFM in conjunction with five novel transgenic lines specifically designed for long-term fluorescence live imaging. Each line encompasses multiple sublines representing distinct genomic insertions of the same transgene. To specifically label intracellular structures, we assayed the functionality of five nanobodies21, which are derived from vertebrate antigens, in Tribolium. The nanobodies target (i) cytokeratin-8 (i.e. the non-binding nanobody control), (ii) histone H2A/H2B, (iii) proliferating cell nuclear antigen (PCNA), (iv) lamin A/C, and (v) actin. Imaging began at the end of blastoderm formation and continued for either ~24 hours (covering gastrulation and part of germband elongation) or ~120 hours (covering gastrulation, germband elongation, germband retraction, dorsal closure, and part of muscular movement). Each specimen was recorded along four orientations spaced 90° apart, ensuring the availability of ventral, lateral, and dorsal (or ventrolateral and dorsolateral) views, and enabling central downstream processing approaches such as multiview fusion for the generation of superior representations of the data22. All but one embryo hatched morphologically intact, all remaining but one larva developed into healthy adults, and all remaining but one adult were fertile. Raw data were pre-processed (alignment of the anterior-posterior axes of the embryos with the y axis of the images followed by cropping in all three spatial dimensions to remove non-informative background and reduce data volume) and z maximum projections were calculated. Five of the assayed sublines displayed atypical fluorescence signal, likely attributable to position effects arising from variation in transgene insertion sites.

Methods

Tribolium castaneum strains and rearing

For the creation of novel transgenic lines, the Tribolium castaneum Plain-White-As-Snow background strain23 was used. Tribolium cultures were kept in sizes of about 200–500 individuals on growth medium (full grain wheat flour (SP061036, Demeter) supplemented with 5% (wt/wt) inactive dry yeast (62–106, Flystuff)) in one-liter glass bottles in a 12:00 h light / 12:00 h darkness cycle either at 32 °C or at 25 °C and 70% relative humidity (DR-36VL, Percival Scientific). Single pair crossings were performed in small glass vials filled with 1.5–2.5 g growth medium. All animal-related experiments were approved by the institutional ethics committee of the Goethe University (Tierschutzkommission der Goethe-Universität, file number BB-20250515-St) and conducted in agreement with the German Animal Welfare Act (Tierschutzgesetz/Tierschutz-Versuchstierordnung, based on ETS No.12324 and EU Directive 2010/63/EU25) as well as the ARRIVE 2.0 guidelines26.

Plasmids, germline transformation and transgenic lines / sublines

Based on commercially available nanobody-encoding plasmids (cat. nos. tcg/ccr/lcg/acg, ChromoTek/Proteintech), Tribolium-optimized coding sequences for nanobodies against (i) cytokeratin-821, (ii) histone H2A/H2B27, (iii) PCNA21, (iv) lamin A/C21, and (v) actin28 were de-novo synthetized (GeneArt, Thermo Fisher Scientific), amplified by PCR and inserted into the #O slot of pAGOC{#P’#O(LA)-mEmerald}23, replacing the default Lifeact (LA) coding sequence. Next, for all five intermediates, the endogenous tubulin alpha 1-like protein promoter29, which is constitutively and ubiquitously active, was inserted scarlessly into the #P slot. The five resulting transformation-ready plasmids (Table 1, ‘Plasmid’ column) were separately injected into F1 pre-blastoderm embryos to achieve germline transformation using the previously described pATub’piggyBac helper plasmid23 as the piggyBac transposase source. All F1 survivors were mated with wild types, and in (i) three, (ii) four, (iii) two, (iv) five, and (v) six crossings (twenty in total), at least one transgenic F2 individual was found among the descendants. All transgenic individuals were mated with wild-type individuals to estimate the number of insertions and, for each F1 crossing that resulted in transgenic progeny, one of the F2 crossing setups that resulted in ~50% transgenic F3 progeny was chosen to found a subline (Table 1, ‘Transgenic line’ and ‘Subline’ columns, and Table 2). Among the F3 progeny, one female per subline was used to start the four-generation AGOC mating procedure for a systematic creation of homozygous cultures as described previously23 (Supplementary Table 2). Homozygous F7 individuals were obtained for (i) three, (ii) four, (iii) two, (iv) five, and (v) one of the sublines (fifteen in total, cf. Supplementary Table 2, ‘F6’ row) and used to establish F7+ continuative homozygous cultures. For one (#2) of the five homozygous lethal sublines (all five were actin nanobody-expressing sublines), a mixed culture was established and regularly curated (i.e. wild-type individuals were removed during every culture clean-up routine). In addition to the newly created sublines, the previously described EFA-nGFP line30 as well as the AGOC{ATub’H2B-mEmerald} #3 and AGOC{ATub’#O(LA)-mEmerald} #1 sublines23 were used.

Table 1.

Plasmids as well as transgenic Tribolium lines and sublines created for or used during this study. In the homozygous viability (‘HzVb’) column, green shading indicates homozygous viable sublines, while red shading indicates homozygous lethal sublines. In the systematic imaging (‘SysImg’) column, green shading indicates sublines for which systematic live imaging was performed in triplicates, yellow shading indicates sublines for which only one dataset was acquired, and red shading indicates that no live imaging data was collected. In the position effect (‘PE’) column, green shading indicates expression-as-expected sublines, while red shading indicates position-effect sublines.

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Table 2.

Insert number estimation assays for all newly created transgenic sublines.

Transgenic line Subline F3 marker distribution
graphic file with name 41597_2025_6406_Taba_HTML.gif graphic file with name 41597_2025_6406_Tabb_HTML.gif total
AGOC{ATub’Cyto8°NB-mEmerald} #1 43.7% (31) 56.3% (40) 71
#2 49.5% (54) 50.5% (55) 109
#3 49.4% (78) 50.6% (80) 158
AGOC{ATub’H2A/H2B°NB-mEmerald} #1 46.7% (43) 53.3% (49) 92
#2 42.9% (39) 57.1% (52) 91
#3 50.0% (56) 50.0% (56) 112
#4 47.6% (49) 52.4% (54) 103
AGOC{ATub’PCNA°NB-mEmerald} #1 45.0% (36) 55.0% (44) 80
#2 57.5% (61) 43.5% (47) 108
AGOC{ATub’Lamin°NB-mEmerald} #1 56.6% (64) 43.3% (49) 113
#2 56.1% (64) 43.9% (50) 114
#3 49.6% (56) 50.4% (57) 113
#4 55.4% (67) 44.6% (54) 121
#5 49.5% (51) 50.5% (52) 103
AGOC{ATub’Actin°NB-mEmerald} #1 49.6% (62) 50.4% (63) 125
#2 51.7% (62) 48.3% (58) 120
#3 47.4% (46) 52.6% (51) 97
#4 45.5% (50) 54.5% (60) 110
#5 39.5% (64) 60.5% (98) 162
#6 45.5% (51) 54.5% (61) 112
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For a detailed description of the insert number estimation assay, please refer to the respective study23. Note that only the results from the crossings used to establish sublines are shown, data from discarded crossings are not included.

Light sheet fluorescence microscopy

Light sheet fluorescence microscopy was implemented using a sample chamber-based digital scanned laser light sheet fluorescence microscope (DSLM)31, which generates a dynamic light sheet by rapidly scanning a collimated Gaussian laser beam with a single two-axes piezo-driven scanning mirror (M-116.DG, Physik Instrumente GmbH & Co KG). A 488 nm / 20 mW diode laser (PhoxX 488-20, Omicron Laserprodukte GmbH) with a 488 nm cleanup filter (xX.F488, Omicron Laserprodukte GmbH) served as the illumination light source. Excitation was performed through a 2.5× NA 0.06 EC Epiplan-Neofluar objective (422320-9900-000, Carl Zeiss AG) while the emission was collected through a 10× NA 0.3 W N-Achroplan objective (420947-9900-000, Carl Zeiss AG). A 525/50 single-band bandpass filter (FF03-525/50-25, Semrock/AHF Analysentechnik AG) and a high-resolution charge-coupled device camera (Clara, Andor) were used for detection. Conventionally3, the illumination axis is defined as x, the rotation axis as y, and the detection axis as z, with y being collinear with Earth’s gravity. Three micro-translation stages (M-111.2DG, Physik Instrumente GmbH & Co KG) and one precision rotation stage (M-116.DG, Physik Instrumente GmbH & Co KG) were used for sample translation along x, y, and z as well as rotation around y, respectively. The effective laser power was regularly measured with an optical power and wavelength meter (OMM-6810B and OMH-6703B, Newport).

Embryo collection, preparation, mounting, live imaging and retrieval

In principle, embryo collection, preparation and mounting were performed as described previously32,33. To establish imaging cultures for homozygous viable sublines (cf. Table 1, ‘HzVb’ column), ~300 individuals were taken from the F7+ continuative (mC/mC) homozygous cultures. To establish transient (i.e. one-generation) imaging cultures of a second actin nanobody-expressing subline, ~150 (mC/-) hemizygous virgin females from the AGOC{ATub’Actin°NB-mEmerald} #2 subline were mated with ~150 wild-type males. Between collection and preparation, embryos were incubated at room temperature (23 °C ± 1 °C) for 15 h to reach the end of blastoderm formation. Pre-selection of transgenic (i.e. fluorescent) embryos was not necessary for specimens derived from imaging cultures of homozygous viable sublines, since respective descendants are always homozygous. For embryos derived from transient imaging cultures, which may be wild-type or heterozygous, pre-selection was not performed to avoid harmful light exposure.

In the DSLM, axial image (z) stacks of embryos were recorded in one fluorescence channel along four directions in 90° steps and at up to 301 time points with an interval of 30 min (corresponding to a total recording time of up to 150 h). Embryos were illuminated with a laser power of 135 µW during a 50 ms exposure time window of the camera. All z stacks have a lateral voxel pitch of 0.645 µm and an axial voxel pitch of 2.58 µm (i.e. a pitch ratio of 1:4). Once imaging was completed, embryos were retrieved from the microscope chamber as described previously34.

Image processing

Image processing was performed using Fiji35 (based on ImageJ36, Version 1.53f) and Mathematica (Version 13.3.0.0)37. Firstly, in Fiji, z maximum projections were calculated for all z stacks and concatenated to time (t) stacks. Secondly, using a custom Mathematica program (cf. Code Availability section), z and t stacks of directions in which the embryo was tilted were rotated around z to align the anterior-posterior axes of the embryos with the y axis of the images. The applied rotations typically ranged between 1° and 5°, with pixels resampled using a Gaussian kernel. During this step, the images were also cropped to a size of 600 × 1000 × 150 voxels, or in the case of large embryos, 600 × 1100 × 150 voxels. Thirdly, in Fiji, the t stacks for all directions of a dataset were combined to horizontal t stack montages, which were manually inspected to classify sublines as either expression-as-expected sublines or as position-effect sublines, i.e. sublines that exhibit atypical fluorescence patterns caused by the local regulatory landscape of the transgene insertion site (cf. Table 1, ‘PE’ and ‘Comment’ columns). Fourthly, for datasets from expression-as-expected sublines, t stack montages were subjected to histogram matching in Fiji (Bleach Correction → Histogram Matching) to equalize the signal intensity over the entire time course and then adjusted in brightness and contrast. Since histogram matching may obscure position effect signals, t stack montages for datasets from position-effect sublines were adjusted only in brightness and contrast. Fifthly, the processed t stack montages were split up into single direction t stacks as well as re-combined to vertical t stack montages. Finally, all images were ZIP-compressed based on the TIFF-intrinsic deflate option.

Data Record

The SLICE-1 datasets are available for download at Zenodo38 as ZIP-compressed TIFF files. Depending on dataset volume, files were compiled into a single or split into two or four ZIP archives (cf. Supplementary Table 1, ‘Access’ column). In cases where datasets are split into multiple archives, the first part always contains all t stack files. Each dataset encompasses six acquisition dimensions: the first (x) and second (y) spatial dimensions are obtained simultaneously during one camera acquisition period. The third spatial dimension (z) is represented by the optical sections that are recorded while the embryos are moved stepwise through the light sheet. The resulting z stacks are stored as single files using the TIFF-intrinsic multi-page function (indicated as PL(ZS) within the file name). Together, the three spatial dimensions define the volume of view. The further dimensions are the fluorescence channel (one), the direction (four) and the time point (up to 301), which are stored as separate files (B2 folder, indicated as CH, DR or TP in the file name, respectively).

For convenience, z maximum projections are provided for each dataset. These are simplifications of the z stacks in which one spatial dimension (z) is collapsed. Projections are provided as t stacks in two versions per dataset, (i) as raw z maximum projections (B3 folder, indicated as PL(ZM) within the file name) and (ii) as processed z maximum projections, either with histogram matching followed by brightness/contrast adjustments (for datasets from expression-as-expected sublines, indicated as PL(ZH) within the file name) or alternatively z maximum projections with only brightness/contrast adjustments (for datasets from position-effect sublines, indicated as PL(ZA) within the file name). Respective t stacks are stored as single TIFF multi-page files (B3 and B4 folders, indicated as TP(TS) within the file name), the processed versions are further provided as horizontal and vertical direction montages (B5 folder, indicated as DR(AX) or DR(AY) within the file name, respectively). A comparative overview of all transgenic lines, based on image data from expression-as-expected sublines, is shown in Fig. 1. Each dataset includes comprehensive metadata in form of a human- and machine-readable XLSX file (typically located in the B1 folder, but extracted from the archives for convenient separate download). In addition, each dataset is accompanied by two movie files: a TIFF multi-page file at native resolution (denoted as FQ for ‘full quality’) viewable in e.g. Fiji, and a PNG-compressed AVI downscaled by a factor of two (denoted as HD for ‘high definition-like’) viewable in e.g. the VLC Media Player. These movies are based on the horizontal direction montages and include orientation, time and scale labels.

Fig. 1.

Fig. 1

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Comparative overview of all five transgenic lines based on image data from expression-as-expected sublines. Shown are z maximum projections derived from z stacks of embryos from sublines in which the tubulin alpha 1-like protein promoter drives the expected ubiquitous and constitutive expression pattern. All embryos are shown in ventrolateral view.

Data Overview

For each dataset, depending on the total recording duration, the onset time points of two to five key embryogenetic events were annotated (Table 3): early gastrulation (defined as the initial period of gastrulation lasting until the onset of germband elongation), germband elongation, germband retraction, dorsal closure, and muscular movement (defined as the period from the end of dorsal closure until hatching). These annotations are also listed in the metadata files.

Table 3.

Embryogenetic events starting time point annotation.

Subline Dataset Starting time point
EG GE GR DC MM
Datasets from sublines expressing mEmerald-labeled nanobodies against cytokeratin-8
AGOC{ATub’Cyto8°NB-mEmerald} #1 0001 0010 0026 — — —
0002 0012 0029 — — —
0003 0011 0027 0079 0203 0232
AGOC{ATub’Cyto8°NB-mEmerald} #2 0004 0011 0024 — — —
0005 0011 0028 — — —
0006 0010 0025 0078 0202 0228
AGOC{ATub’Cyto8°NB-mEmerald} #3 0007 0007 0028 — — —
0008 0010 0026 — — —
0009 0009 0025 0073 0191 0220
Datasets from sublines expressing mEmerald-labeled nanobodies against histone H2A/H2B
AGOC{ATub’H2A/H2B°NB-mEmerald} #1 0010 0001 0014 0066 0203 0233
0011 0001 0015 0073 0218 —
0012 0004 0019 0062 0185 0211
AGOC{ATub’H2A/H2B°NB-mEmerald} #2 0013 0002 0019 0088 0256 0282
0014 0001 0016 0069 0216 —
0015 0001 0011 0065 0195 0231
AGOC{ATub’H2A/H2B°NB-mEmerald} #3 0016 0004 0020 0086 0255 —
0017 0004 0023 0086 0239 0276
0018 0002 0020 0084 0237 0277
Datasets from sublines expressing mEmerald-labeled nanobodies against PCNA
AGOC{ATub’PCNA°NB-mEmerald} #1 0019 0007 0022 — — —
0020 0009 0023 — — —
0021 0002 0018 0077 0188 0213
AGOC{ATub’PCNA°NB-mEmerald} #2 0022 0010 0024 0090 — —
0023 0009 0026 — — —
0024 0007 0020 0058 0184 0211
Datasets from sublines expressing mEmerald-labeled nanobodies against lamin A/C
AGOC{ATub’Lamin°NB-mEmerald} #1 0025 0013 0026 0070 0174 0200
0026 0014 0028 0073 0175 0199
0027 0013 0028 0078 0180 0207
AGOC{ATub’Lamin°NB-mEmerald} #2 0028 0008 0026 — — —
0029 0009 0021 0078 0235 —
0030 0016 0031 0096 0236 —
AGOC{ATub’Lamin°NB-mEmerald} #3 0031 0011 0028 — — —
0032 0009 0031 unknown1 0244 —
0033 0003 0018 0065 0169 0201
AGOC{ATub’Lamin°NB-mEmerald} #4 0034 0014 0028 — — —
0035 0014 0033 — — —
0036 0013 0034 0107 0226 0257
AGOC{ATub’Lamin°NB-mEmerald} #5 0037 0010 0024 — — —
0038 0011 0027 — — —
0039 0008 0025 0095 0217 —
Datasets from sublines expressing mEmerald-labeled nanobodies against actin
AGOC{ATub’Actin°NB-mEmerald} #2 0040 0010 0028 0086 0232 —
0041 0003 0016 0069 0212 0249
0042 0004 0023 0106 0251 —
AGOC{ATub’Actin°NB-mEmerald} #5 0043 0014 0031 0082 0201 0234
0044 0010 0025 0080 0207 0241
0045 0008 0019 0061 0155 0183
Additional datasets from various lines/sublines (controls and peculiarities of development)
EFA-nGFP 0046 0004 0018 0083 0230 0262
AGOC{ATub’H2B-mEmerald} #3 0047 0011 0025 0080 0180 0212
AGOC{ATub’#O(LA)-mEmerald} #1 0048 0001 0014 0071 0227 0259
AGOC{ATub’H2A/H2B°NB-mEmerald} #1 0049 unknown2 0038 0120 — —
AGOC{ATub’H2A/H2B°NB-mEmerald} #3 0050 0004 0017 0067 0173 —
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Starting time points were identified by the following morphogenetic criteria: early gastrulation (EG), rearrangement of the uniform blastoderm; germband elongation (GE), closure of the serosa window on the ventral side; germband retraction (GR), first sign of movement of the posterior tip of the germband towards the posterior pole; dorsal closure (DC), rupture of the serosa in the anterior-ventral region; muscular movement (MM), completion of germband flank closure.

1Due to a slight germband torsion in this embryo (cf. Technical Validation section), the precise starting time point of germband retraction could not be determined. 2Due to an atypical proliferation pattern during the 12th synchronous division wave of blastoderm formation in this embryo (cf. Technical Validation section), the precise starting time point of early gastrulation could not be determined.

Technical Validation

During the entire project and each experiment, particular attention was focused on a systematic approach and dataset comparability. Important technical considerations are:

  • Transgene design. All newly created sublines are based on highly similar transgenes and all nanobody expression cassettes employ identical promoter and fluorescent protein coding sequences. Further, all transgenes carry the same mOrange- and mCherry-based eye markers. The differences stem from a few nucleotides within the nanobody coding sequences that determine antigen-binding specificity. In consequence, the nanobody-mEmerald fusion proteins are highly similar in terms of amino acid number and sequence. For instance, the histone H2A/H2B and actin nanobodies consist of 125 and 120 amino acids, respectively, and have a pair-wise identity of 68.5%.

  • Nanobody choice. Nanobodies were chosen according to anticipated utility and availability. Since all nanobodies used in this study were raised against vertebrate antigens, their cross-reactivity with respective Tribolium analogues was uncertain. Hence, the cytokeratin-8 nanobody was used as a non-binding nanobody control, since ‘bona fide’ cytoplasmatic intermediate filaments39 are not present in Tribolium according to genome sequencing and analysis studies40–42. Given that future nanobodies will also likely be raised against vertebrate proteins, imaging data from cytokeratin-8 nanobody-expressing sublines provide a valuable reference for assessing binding specificity. For instance, comparison with other sublines (cf. Fig. 1) suggests that nanobodies against histone H2A/H2B and actin bind their respective Tribolium orthologues, whereas those against PCNA and lamin A/C do not.

  • Insert number estimation. Transgenesis via the piggyBac transposase system may result in multiple insertions of the transgene within a single F1 individual43,44. In consequence, respective F2 descendants may inherit two or more inserts, leading to variable fluorescence intensity and expression patterns across embryos. To avoid such inconsistencies, the insert number was estimated for each transgenic F2 individual, and only those scoring as single-insert carriers were used to establish sublines (cf. Table 2).

  • Homozygous imaging cultures. In each newly founded subline, the transgenic F2 individuals as well as their F3 descendants are hemizygous for the transgene. Imaging cultures established using these individuals (e.g. by simply pooling all transgenic F3 descendants) would produce a mixture of wild-type, hemizygous and homozygous embryos, with the latter two likely differing in fluorescence intensity. Hence, for the fifteen sublines confirmed to be homozygous viable, continuative homozygous cultures were established (cf. Supplementary Table 2). These cultures, from which imaging cultures were subsequently drawn, produce exclusively homozygous embryos.

  • Microscope calibration and effective laser power. Prior to each recording, the DSLM was calibrated using a two-step routine34 to avoid common issues (e.g. offset and/or tilt between the light sheet and the focal plane of the detection objective). The effective laser power (i.e. the laser power at the precise location where the embryos were positioned during imaging, as opposed to the nominal output of the laser module) was measured regularly. If deviations were detected, the laser output was adjusted accordingly to ensure consistent illumination light density across all dataset acquisitions.

  • Multiple sublines per transgenic line. To robustly evaluate the functionality of each of the five nanobodies in Tribolium and assess potential inter-subline variability, at least two sublines were created per transgenic line and included in the imaging schedule. This strategy prevents common bottlenecks associated with relying on a single subline such as position effects leading to highly specific expression patterns that may hinder proper evaluation of nanobody functionality (cf. Table 1, ‘PE’ column).

  • Exclusion of sublines from systematic imaging. Among the six AGOC{ATub’Actin°NB-mEmerald} sublines, only #5 was homozygous viable. In consequence, four sublines (#1, #3, #4, and #6) were completely excluded from imaging, while one subline (#2) was retained to provide morphogenetic data from a second actin nanobody-expressing subline due to a lack of alternatives. Notably, all embryos imaged from this subline were hemizygous for the transgene.

  • Acquisition parameters for systematic imaging. Embryo collection, preparation, and mounting were standardized across all sublines to ensure consistent imaging conditions. In consequence, recordings were always started at a comparable developmental stage (end of blastoderm formation) and were conducted under identical acquisition parameters (e.g. laser power, exposure time, or temporal interval). Notably, all datasets cover gastrulation (as one of the most dynamic morphogenetic events), and for each subline, at least one dataset was acquired that covers all embryogenetic events from gastrulation to dorsal closure. All recordings were performed on the same microscope, and no major hardware components (e.g. laser module, fluorescence filter, or camera) were replaced during the study period. All embryos were imaged along four directions, capturing ventral, dorsal, and both lateral views (or alternatively both ventrolateral and dorsolateral views). This multi-view approach is particularly important for Tribolium embryos, as the germband can rotate around the anterior-posterior axis during development45 (cf. Supplementary Table 1, ‘Views’ column).

  • Imaging in triplicates. To evaluate subline performance and establish a stochastically robust dataset collection, at least three embryos from separate embryo collections were recorded for all sixteen remaining sublines to allow assessment of intra-subline variability. For convenience, replicates were assigned consecutive dataset numbers.

  • Control lines / sublines. To provide ‘positive control’ data for the histone H2A/H2B and actin nanobody-expressing lines (i.e. those confirmed to bind the respective Tribolium orthologue, cf. Figure 1), embryos from three control lines were also imaged. These include the frequently used EFA-nGFP line30 (DS0046), which expresses nuclear-localized GFP(S65T) under control of the elongation factor 1-alpha promoter, as well as the previously described AGOC{ATub’H2B-mEmerald} #3 and AGOC{ATub’#O(LA)-mEmerald} #1 sublines23 (DS0047 and DS0048), which express mEmerald-labeled histone 2B and Lifeact46, respectively, under control of the tubulin alpha 1-like protein promoter.

  • Identification of position-effect sublines and developmental irregularities. Comprehensive visual inspection of imaging data revealed that five out of sixteen remaining newly created sublines exhibited position effects (cf. Table 1, ‘PE’ column). A brief overview regarding the expression patterns is provided in Fig. 2. However, for each of the five transgenic lines, at least one subline was obtained that displayed expression as expected, allowing proper evaluation of nanobody performance. Further, three developmental irregularities were observed: Firstly, one embryo from the AGOC{ATub’Lamin°NB-mEmerald} #3 subline exhibited a slight germband torsion, resulting in reduced anteriad (i.e. toward the anterior region of the embryo) movement of the abdominal tip along the dorsal side during germband elongation. Nonetheless, the embryo completed development without further complications (DS0032). Secondly, one embryo from the AGOC{ATub’H2A/H2B°NB-mEmerald} #1 subline exhibited an atypical proliferation pattern during the 12th synchronous division wave of blastoderm formation, but otherwise developed normally (DS0049). As data from this expression-as-expected subline are considered particularly valuable, this dataset was retained in the collection but not counted among the three technical replicates. Finally, embryos from the AGOC{ATub’H2A/H2B°NB-mEmerald} #3 subline frequently displayed developmental aberrations. Hence, only a single exemplary dataset is provided that shows an embryo undergoing self-constriction during dorsal closure (DS0050).

  • Confirmation of non-invasive imaging. To verify that the imaging procedure (e.g. the light exposure) did not induce post-embryonic aberrations, all embryos were retrieved after recording, raised to adulthood, and assessed for fertility. All embryos hatched morphologically intact, except the one from dataset DS0050. Of the resulting larvae, all but the one from DS0026 developed into healthy adults. Among the adults, all except the individual from DS0038 were fertile.

Fig. 2.

Fig. 2

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Overview of the expression patterns of the five position-effect sublines. Shown are z maximum projections with adjusted brightness and contrast. (a) The AGOC{ATub’H2A/H2B°NB-mEmerald} #1 subline shows a weak signal in the serosa during late development, particularly evident during dorsal closure when the amnion and serosa form the dorsal organ. (b) The AGOC{ATub’H2A/H2B°NB-mEmerald} #4 subline exhibits strong stripe-like expression in the germband during early development and strong expression in the brain and ventral nerve cord during late development. (c) The AGOC{ATub’PCNA°NB-mEmerald} #1 subline displays increasing expression in the head appendages and tarsi during germband retraction, which becomes particularly pronounced after dorsal closure. (d) The AGOC{ATub’Lamin°NB-mEmerald} #1 subline shows strong signal in the abdominal tip during germband retraction, which persists through dorsal closure. (e) The AGOC{ATub’Lamin°NB-mEmerald} #2 subline exhibits strong, homogeneous signal throughout the embryonic tissue, but only weak signal in the serosa.

Usage Notes

The data structure (cf. Data Records section) is compatible with any software capable of handling three-dimensional multi-view dynamic image data. ImageJ36 or its derivate Fiji35 are recommended as the primary platforms for image processing and analysis. Both are open-source frameworks widely used for working with multi-dimensional microscopy data. In addition, modern community-driven tools such as napari47 offer interactive visualization and plugin-based extensibility.

Please note that the file names contain parentheses, which are not part of the Portable Operating System Interface (POSIX) portable filename character set. Therefore, some downstream processing software may require escaping these characters or enclosing the file name in quotation marks. Furthermore, certain operating systems may issue a ‘zip-bomb’ warning when decompressing the archives due to their large size. This can be circumvented by extracting the data using dedicated tools such as 7-Zip or WinRAR, or by temporarily disabling the zip-bomb detection in the native unzip utility by temporarily changing the respective environment variable (export UNZIP_DISABLE_ZIPBOMB_DETECTION = TRUE). The total data volume after extraction exceeds the archive size only minimally.

Supplementary information

Supplementary Table 1 and 2 (453.3KB, pdf)

Acknowledgements

We thank Sigrun Becker and Sven Plath for technical support. FS received funding from the Add-on Fellowship 2019 of the Joachim Herz Stiftung. FS and EHKS received funding from the Quantitative Structural Cell Biology Projects program (Innovations- und Strukturentwicklungsinitiative ‘Spitze aus der Breite’), the LOEWE FL5 Exploration program (2. Förderstaffel, the ‘Barquito’ project) of the State of Hessen, and the ‘RobustNature’ Cluster of Excellence Application Initiative provided by the Goethe-Universität. MG was funded by the Deutsche Forschungsgemeinschaft (DFG, Graduiertenkolleg iMOL, GRK 2566, speaker Prof. Dr. Achilleas Frangakis) through a doctoral research project awarded to EHKS. FS and MG were funded by the Deutsche Forschungsgemeinschaft (DFG, 460111950) through an Emmy Noether research project awarded to SM.

Author contributions

F.S., S.M. and E.H.K.S. conceived the research. F.S., P.G. and H.T. created the plasmid vectors and transgenic Tribolium lines. F.S., P.G., K.M. and M.G. performed the imaging experiments with support from J.R., F.K. and S.M. F.S. prepared the figures and tables, processed the data, and wrote the manuscript with input from all other authors.

Funding

Open Access funding enabled and organized by Projekt DEAL.

Data availability

The Zenodo link38 redirects to the SLICE-1 dataset collection overview page, which allows entries to be sorted by various convenient criteria. For datasets divided into multiple parts, the corresponding entries are cross-linked in the ‘Additional details’ section. Digital Object Identifiers (DOIs) for all entries are listed in Supplementary Table 1 and can be resolved via the DOI website (https://www.doi.org).

Code availability

The Mathematica program notebook file (.nb) implementing the batch rotation, cropping and zipping procedure is available on GitHub (Strobl2025A-BatchRotCropZIP_V2.5.nb, https://github.com/BugCube/BatchRotCropZIP).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s41597-025-06406-6.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Strobl, F. SLICE-1. Zenodohttps://zenodo.org/communities/slice-1 (2025).

Supplementary Materials

Supplementary Table 1 and 2 (453.3KB, pdf)

Data Availability Statement

The Zenodo link38 redirects to the SLICE-1 dataset collection overview page, which allows entries to be sorted by various convenient criteria. For datasets divided into multiple parts, the corresponding entries are cross-linked in the ‘Additional details’ section. Digital Object Identifiers (DOIs) for all entries are listed in Supplementary Table 1 and can be resolved via the DOI website (https://www.doi.org).

The Mathematica program notebook file (.nb) implementing the batch rotation, cropping and zipping procedure is available on GitHub (Strobl2025A-BatchRotCropZIP_V2.5.nb, https://github.com/BugCube/BatchRotCropZIP).

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