Nature Protocols: doi: /nprot Supplementary Figure 1. Principle effects of LSFM illumination and detection on image quality.

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1 Supplementary Figure 1 Principle effects of LSFM illumination and detection on image quality. (A) Top view of a scheme showing the arrangement of illumination objective, sample chamber and detection objective in a light sheetbased fluorescence microscope. The inset depicts the illumination and detection of a single line in a specimen. (B) Top view of the objective arrangement in the mdslm. In the inset, image quality indicators are provided as colored arcs. The green arc marks the quarter with an expected high quality, the orange arcs the quarters with an expected medium quality and the red arc the quarter with an expected low quality. (C) Y maximum projection of a Z stack with the image quality indicators introduced in B.

2 Supplementary Figure 2 Example quantification of the serosa window area over time during serosa window closure. (A) Z maximum projection of a Tribolium embryo during gastrulation that commences serosa window closure seen from the ventral side. (B) Time lapse of single planes, 26 µm below the ventral surface of the embryo, showing the process of serosa window closure. The first row depicts the raw planes. In the second row, an entropy filter was applied to the raw planes. Then, an intensity threshold was set to obtain the binary images. The binary images were inverted and further processed by morphological operations to extract the serosa window area in each image, as shown in the third row. The fourth row illustrates the raw planes marked with the extracted serosa window area. (C) A plot of the serosa window area as a function of development time, providing evidence that the serosa window area during serosa window closure follows an exponential decay. ZP, Z maximum projection with image processing; PA, plane with intensity adjustment.

3 Supplementary Figure 3 Chronological staging table of Tribolium embryogenesis. The left column contains Z maximum projections along four orientations (0 (ventral), 90 (lateral), 180 (dorsal), and 270 (lateral)) of a Tribolium embryo at the beginning of five major embryogenetic events. In the middle column, an outline of the most important developmental processes with a temporal indication in hours (developments at 35 C and at room temperature) and stage indication in percent of full development. Please note that 00:00 h relates to our standard imaging starting point (that is after a 1 h egg laying period / 15 h of incubation at 25 C and 1 h of preparation at room temperature), whereas the stage indication in percent is provided for the whole of embryogenesis. The right column shows Z maximum projections of developmental processes during the respective embryogenetic events that have been the focus of previous research. Red arrows indicate the location of the process mentioned in the bullet points. RT, room temperature (23±1 C); ap., approximately.

4 Supplementary Figure 4 Scheme of the recommended control experiments. In addition to the imaging procedure of the fluorescent embryo (left column), we suggest two control experiments that can be run in parallel: a fluorescent control embryo (middle column) and a wild type control embryo (right column). All steps in which the control experiments have to be considered are indicated and also mentioned in the Procedure section.

5 Supplementary Figure 5 Operating principle of the digital scanned laser light sheet-based fluorescence microscope (DSLM). In contrast to other types of light sheet-based fluorescence microscopes, the DSLM does not have a static light sheet. Instead, a dynamic light sheet is generated by scanning the laser beam with a piezo-driven two-axes scanning mirror (see green annotations). The laser beam, originating from the laser source, is guided by the scanning mirror through an f-theta and a tube lens into the illumination objective. The dynamic light sheet illuminates a thin slice of the Tribolium embryo, and the emitted fluorescence is guided by a perpendicularly arranged detection objective through an appropriate filter and a tube lens into the camera. The detail on the right shows the sample holder with the embryo in front of the detection objective. Adapted and modified from ref. 18 with permission, Nature Publishing Group.

6 Supplementary Figure 6 Perfusion system. (A) Overview of mdslm with connected perfusion system. The main elements of the circulatory perfusion system are labelled. Controlled transfer of PBS from the reservoir is achieved via the inflow pump through the heating tube into the sample chamber. From the chamber, excessive PBS is transferred via the outflow pump back into the reservoir. Upper detail box shows a top view of the sample chamber. The perfusion system connects to the periphery of the sample chamber and, therefore, does not impair the imaging process. Lower detail box shows the front view of the sample chamber with connected inflow and outflow tubes. The inflow tube connects to the lower and the outflow tube to the upper joint of the sample chamber. Therefore, the PBS outflow rate is limited by the inflow rate. Directions of flow are indicated with white arrowheads. A third tube is connected to a catch basin located beneath the sample chamber in case of a leakage, as indicated with a gray arrowhead. (B) Setup and equilibrium of perfusion system. A constant temperature in the sample chamber is maintained via a combination of PBS flow rate and the temperature generated in the heating tube. Prior to the experiment, the perfusion system is given two hours to ensure a stable temperature in the sample chamber. The circulatory flow of PBS and relative warmth are indicated with colored arrowheads.

7 Supplementary Figure 7 Illustration of equipment setup. (A) Front (left) and side view (right) of the glass cover slip holder. (A ) Front (left panel) and side view (right panel) of the holder in A with attached glass cover slip. (A ) Glass cover slip holder in the LSFM sample chamber. From left to right: top view of glass cover slip holder perpendicular to Z (the detection axis), top, front and side view of the glass cover slip holder rotated by 45 around Y (the rotation axis). (B) Capillary holder (left panel) with capillary attached (right panel). (C) Syringe components with cut lines indicated in orange (left panel). Components with indicated parts cut off (right panel). (C ) Assembled syringe (top panel), with rubber hose (middle panel) and capillary (bottom panel) attached. (D) Humidity chamber. (D ) Scaffold created from serological pipette fragments without (left panel) and with object slide (right panel).

8 Supplementary Figure 8 Test recording of agarose-embedded beads column for microscope calibration. The agarose-embedded beads column is shown along four orientations as a Z (first row), X (second row) and Y (third row) maximum projections. The maximum projections illustrate correct calibration of the microscope without any imaging artifacts. The rotated Y maximum projections (fourth row) demonstrate a correctly set up recording of the bead agarose column. Individual beads can be identified along all directions as shown in the detail images (fifth row). Due to the point spread function, beads are expected to appear as bright, round dots in the Z maximum projection and elongated along X and Y maximum projections. ZP, Z maximum projections with image processing; XP, X maximum projections with image processing; YP, Y maximum projections with image processing.

9 Supplementary Figure 9 Test recording of the embryo along four directions prior to time lapse imaging. The embryo is shown in four orientations in the transmission light channel (first row) and as Z (second row), X (third row) and Y (fourth row) maximum projections. The maximum projections illustrate a correctly set up recording along four directions without any imaging artifacts. Some preparation issues and misconfigurations of the imaging setup (e.g. insufficient amount of agarose in the stability layer leading to a drift of the embryo, incorrect Z spacing, incorrect configured volume of view), might be more apparent in X and Y maximum projections than in the Z maximum projections. Indicators for a correctly set up recording are: X and Y maximum projections depict smooth object boundaries and show no motion blur; Z maximum projections and the corresponding X maximum projections exhibit high similarity; and in the Y maximum projections, the shape of the embryo should be roughly circular. ZP, Z maximum projections with image processing; XP, X maximum projections with image processing; YP, Y maximum projections with image processing.

10 Supplementary Figure 10 Qualitative coverage of the embryo. The embryo is shown along four orientations as Z and Y maximum projections. The Y maximum projections (second row) illustrate that the quality of the recording strongly depends on the amount and type of biological material passed by the illumination and emission light paths. If no biological material obstructs the light paths, the resulting image quality is high (lower left area in all orientations). However, if the amount of material that has to be passed increases, the image quality drops (upper left and lower right areas in all orientations). The image quality is worst in areas where both light paths have to pass the complete embryo (upper right areas in all orientations). The rotated Y maximum projections (third row) highlight the fact that features of the embryo might only be partly visible in some orientations. The green lines outline the clearly detectable germ band borders from the respective direction. ZP, Z maximum projections with image processing; YP, Y maximum projections with image processing.

11 Supplementary Table 1 Metadata and parameters for the three example datasets. Dataset (DS) DS0001 DS0002 DS0003 Species Line Line Genotype Stock Stock Medium Stock Conditions Egg Laying Period Egg Laying Medium Pre-imaging Incubation Tribolium castaneum (Herbst) / Arthropoda Insecta Coleoptera Tenebrionidae EFA-nGFP transgenic line 10 (background strain: vermilion white) one insert / homozygote ~300 adults, less than 4 months old full grain wheat flour ( , Demeter, Darmstadt, Germany) supplemented with 5% (wt/wt) inactive dry yeast (62-106, Flystuff, San Diego, CA, USA) 12:00 h light / 12:00 h darkness at 25 C and 70% relative humidity (DR-36VL, Percival Scientific, Perry, IA, USA) 01:00 h at 25 C and 70% relative humidity exposed to light 405 fine wheat flour ( , Demeter, Darmstadt, Germany) supplemented with 5% (wt/wt) inactive dry yeast (62-106, Flystuff, San Diego, CA, USA) 15:00 h at 25 C and 70% relative humidity in darkness LSFM Type mdslm based on DSLM 18,72 with structured illumination 19 Laser Lines Excitation Objective Emission Objective Emission Filters Camera Dataset File Type (Raw) Dechorionation Mounting Agarose Imaging Buffer 488 nm / 60 mw diode laser (PhoxX , Omicron Laserprodukte GmbH, Rodgau-Dudenhofen, Germany) 2.5x NA 0.06 EC Epiplan-Neofluar objective ( , Carl Zeiss, Göttingen, Germany) 10x NA 0.3 W N-Achroplan objective ( , Carl Zeiss, Göttingen, Germany) 525/50 single-band bandpass filter (FF03-525/50-25, Semrock/AHF Analysentechnik AG, Tübingen, Germany) High-resolution CCD (Clara, Andor, Belfast, United Kingdom) TIFF, 16 bit grayscale (planes are deposited as Z stacks, indicated as PL(ZS) in the file name) ~30 s in 10% (vol/vol) sodium hypochlorite ( ML, Sigma Adlrich, Taufkirchen, Germany) in PBS ph 7.4 ( , Gibco Life Technologies GmbH, Darmstadt, Germany) 1% (wt/vol) low-melt agarose (6351.2, Carl Roth, Karlsruhe, Germany) in PBS ph 7.4 ( , Gibco Life Technologies GmbH, Darmstadt, Germany) PBS ph 7.4 ( , Gibco Life Technologies GmbH, Darmstadt, Germany) Imaging Temperature 35 C (perfusion system) room temperature (23±1 C) room temperature (23±1 C) 92 Gigabyte (raw) 642 Gigabyte (raw) 193 Gigabyte (raw) Dataset Size 37 Gigabyte (ZIP) 273 Gigabyte (ZIP) 77 Gigabyte (ZIP) 0.47 Gigabyte (JPEG2000) 5.54 Gigabyte (JPEG2000) 1.62 Gigabyte (JPEG2000) Retrieval survived, developed to healthy adult, produced fertile progeny survived, developed to healthy adult, produced fertile progeny survived, developed to healthy adult, produced fertile progeny Figures 10 (upper row) 4D-F; 7; 8; 9; 10 (lower row); 12 (upper row) 11 Supplementary Figures - 2; 3; 9; 10 - Supplementary Videos - 3; 4; 5 6

12 Time Points (TP) 49 (TP0001-TP0049) 241 (TP0001-TP0241) 49 (TP0001-TP0049) TP Interval 00:30 h 00:30 h 00:30 h Total Time (TP TP Interval) 24:00 h 120:00 h 24:00 h Directions (DR) 4 (DR0001-DR0004) 4 (DR0001-DR0004) 4 (DR0001-DR0004) DR Orientations 0, 90, 180, 270 0, 90, 180, 270 0, 90, 180, 270 Channels (CH) 1 (CH0001) 1 (CH0001) 2 (CH0001-CH0002) CH0001 Excitation 488 nm 488 nm 488 nm CH0001 Power 135 µw (close to the embryo) 135 µw (close to the embryo) 135 µw (close to the embryo) CH0001 Exposure Time 50 ms 50 ms 50 ms CH0001 Emission Filter 525/50 single-band bandpass filter 525/50 single-band bandpass filter 525/50 single-band bandpass filter CH0002 Excitation nm, structured illumination 50 Hz CH0002 Power µw (close to the embryo) CH0002 Exposure Time ms CH0002 Emission Filter /50 single-band bandpass filter Planes (PL) 175 (PL0001-PL0175) 198 (PL0001-PL0198) 183 (PL0001-PL0183) Z Spacing 2.58 µm 2.58 µm 2.58 µm Z Distance (PL Z Spacing) µm µm µm X-Dimensions (XD) 1040 pixels (raw) 600 pixels (cropped) 1040 pixels (raw) 600 pixels (cropped) 1040 pixels (raw) 600 pixels (cropped) X Spacing µm µm µm X Length (XD X Spacing) µm (raw) µm (cropped) µm (raw) µm (cropped) µm (raw) µm (cropped) Y-Dimensions (YD) 1392 pixels (raw) 1000 pixels (cropped) 1392 pixels (raw) 1000 pixels (cropped) 1392 pixels (raw) 1000 pixels (cropped) Y Spacing µm µm µm µm (raw) µm (raw) µm (raw) Y Length (YD Y Spacing) µm (cropped) µm (cropped) µm (cropped) The original datasets, associated figures and videos in full quality are available at

13 Supplementary Note Troubleshooting of common problems during LSFM calibration and subsequent imaging processing. Please note that in some figures the problem and in others the reason is shown. A green checkmark in the upper right corner indicates the correct execution, whereas a red cross indicates an incorrect execution. (A-A ) Bright dot not visible when glass cover slip holder is placed in microscope chamber (Step 9). (A) The correct glass cover slip holder position reflects the laser light directly into the detection objective. (A -A ) Glass cover slip holders that are positioned offset either left (A ) or right (A ) cause the reflected laser light to miss the detection objective. Also ensure that the glass cover slip holder is positioned at the correct height (front view in detail box in A). (B-B ) Light sheet does not span straight across the complete field of view along Y (Step 11). (B) The light sheet should extend approximately 20% over the field of view along Y, so that the visible part of the light sheet is homogeneous. (B ) If the scanning range of the activated scanning mirror is too small, the light sheet does not span across the complete field of view along Y. (B ) If the scanning mirror is not adjusted correctly, the light sheet is tilted. (C-C ) Light sheet does not appear focused (Step 11). (C) The light sheet should be a straight focused line with minimal width highlighting surface irregularities on the inserted glass cover slip. (C ) The light sheet appears broader when the waist is not positioned in the field of view. (C ) It appears blurry when the focal plane is not in the center of the light sheet.

14 Supplementary Note (continued) (D-D ) Beads are missing or appear blurry on either end of Y (Step 14). (D) The beads should appear focused and homogeneously distributed along the vertical direction across the full height of the acquired image. (D ) If beads are missing on either end or on both ends of the image, the light sheet height has to be readjusted, the glass capillary is in the field of view, or the agarose-embedded beads column is too short. (D ) If beads appear focused in the image center and blurry on the outer edges in the vertical direction, the light sheet is tilted. The light sheet orientation needs to be readjusted using the glass cover slip holder. (E-E ) Beads appear blurry across the complete field of view (Step 14). (E) A circle of homogenously distributed beads should appear and beads should be focused across the full Z distance of the Y maximum projection. (E ) If beads appear dim and elongated, the light sheet waist is not positioned correctly and the position of the illumination objective needs to be readjusted using the piezo nano-positioners. (E ) If beads appear blurry, the light sheet does not overlap with the focal plane. The position of the detection objective needs to be readjusted using the piezo nano-positioners.

15 Supplementary Note (continued) (F-F ) Difficulties in mounting the embryo on the agarose hemisphere (Step 28 ). (F) The semi axes of the agarose hemisphere should be at best identical (see also Figure 3G). (F -F ) If the pole radius is smaller (F ) or greater (F ), the embryo tends to tip over during the mounting procedure. (G-G ) The embryo moves after being placed on the pole of the agarose hemisphere (Step 28). (G-G ) During the mounting process, the posterior end of the embryo should be attached to the brush tip, while the anterior end remains accessible. (G ) If the embryo is attached vice versa, either reposition the embryo with a second brush, or release the embryo into the PBS and restart the picking process. (H-H ) Embryo detaches when sample holder is inserted into sample chamber (Step 31). (H) The drop of liquid agarose to fix the embryo has to be correctly placed on the agarose hemisphere. (H ) If it is placed on the steel pipe, the agarose does not reach the embryo. (H ) If the drop is positioned directly on the embryo, the embryo might detach. (I-I ) Incorrect amount of agarose for the mounting and stability layers (Steps 31 / 40 / 44). (I) The mounting and stability layers should cover approximately between a quarter and half of the embryo surface at maximum and the agarose flanks should be as steep as possible. The embryo shown here survived the imaging process and developed into a fertile adult. (I ) If too little agarose is applied, the embryo detaches during the insertion into the sample chamber. (I ) If too much agarose is applied, the survival chance of the embryo and the imaging quality are decreased. The embryo shown here did not survive the imaging process.

16 Supplementary Note (continued) (J-J ) Blurred or dark regions at anterior or posterior regions of the embryo (Step 36). (J) The embryo should be visible and in focus in the complete acquired image. (J ) If the light sheet height is insufficient, the embryo is only partially visible. (J ) A tilted light sheet results in blurry regions at the top and bottom along Y. (K-K ) During long-term imaging, the whole embryo appears blurred or certain regions appear dark after some time (Step 36). (K) The embryo should remain focused in the image volume, even during long-term imaging. (K ) Insufficient amount of agarose in the stability layer might lead to a drift of the embryo out of the image volume. Besides, the Z stack should be large enough to cover slight movements of the embryo over time. (K ) If the chamber sealing for example generates excessive force on the detection objective, a focus drift may occur over time.