SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction

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1 Nature Neuroscience Supplementary Information SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction Meng-Tsen Ke, Satoshi Fujimoto, and Takeshi Imai. Correspondence should be addressed to T.I. (imai@cdb.riken.jp). Supplementary Table 1-5. Supplementary Figure Supplementary Note: Supplementary s 1-14 are available on the Nature Neuroscience website. S1

2 Supplementary Table 1. Refractive indices of various optical clearing agents. Although the refractive index is an important parameter, a high-index solution does not necessarily clear the tissue samples. For example, ZnI 2 solution was not effective. Reagent Refractive index Water ScaleB ScaleU ScaleA ProLong Gold (Invitrogen) % (w/v) sucrose SlowFade Gold (Invitrogen) FocusClear (CelExplorer Labs) % glycerol % (v/v) 2,2 -thiodiethanol (TDE) SeeDB (80.2 % w/w fructose) SeeDB37 (86.7 % w/w fructose) BABB Saturated ZnI S2

3 Supplementary Table 2. Quantitative data for experiment in Figure 6a on the lateral dendrites of sister mitral cells. Mitral cell numbers are indicated in Figure 6c. Tortuosity indexes for all dendrite segments were calculated as in Figure 2b. # dendrite total dendrite length average dendrite node end number ( m) length ( m) number number (D) (L) L/D average tortuosity S3

4 Supplementary Table 3. Coverage area of 13 sister mitral cells in Figure 6a were determined based on z-projected images and are shown in Supplementary Figure 13. Percentages of shared coverage areas were determined for all ps of sister mitral cells and were % (mean s.d., n = 144 ps). Overlapping mitral cell (% overlapping area) Mitral cell number S4

5 Supplementary Table 4. Advantages and disadvantages of optical clearing agents, SeeDB, ScaleA2, and BABB (or dibenzyl ether). We have not tested CLARITY 33 and Clear T (Kuwajima et al., Development 140, (2013)), which were reported after the initial submission of our manuscript. CLARITY appears to clear whole adult mouse-brain samples more efficiently than SeeDB and ScaleA2, and also facilitates whole-mount immunohistochemistry. However, CLARITY requires more complicated and time-consuming procedures (3 days for acrylamide perfusion, 3 days for washout, 8 days for electrophoresis, 2 days for washout, and 2 days for index matching to prepare cleared whole mouse brain samples for imaging) 33. Clear T is compatible with DiI tracing, but causes mild sample expansion. 2,2 - thiodiethanol 8,9 maintains sample size, but is incompatible with fluorescent proteins (Supplementary Fig. 6). SeeDB ScaleA2 BABB, dibenzyl ether Transparency Sample size (linear expansion) Constant 150% expansion 50% shrinkage Preservation of morphology Agarose embedding Sample solidity soft fragile firm Speed of clearing 3 days 3 weeks 3 days Reversibility (for IHC) Long-term storage Confocal microscopy Two-photon microscopy Light-sheet microscopy Fluorescent proteins (short term) Fluorescent dextran dyes Lipophilic dyes (DiI) , impossible; +, difficult; ++, f; +++, excellent. S5

6 Supplementary Table 5. Summary of imaging conditions. Depths (z) are not calibrated., confocal microscopy;, two-photon microscopy; A-488/647, Alexa Fluor 488/647; TDE, 2,2 -thiodiethanol. Fig. number Fig. 2a left Fig. 2a middle Fig. 2a right Fig. 2c left Fig. 2c middle Fig. 2c right Fig. 3a Fig. 3b Fig. 3c Fig. 3d Fig. 3e Fig. 4a Fig. 4b Suppl & age P21-28 adult P70 P72 P7 P7 Fig. 5b P21-28 Fig. 5c P21-28 Fig. 6a 14 P21-28 Fig. S1b adult Fig. S7a Fig. S7b Fig. S7c P21-28 Fig. S8a P7 Fig. S8b P7 Fig. S8c P5 Fig. S9 P70 Fig. S10a Fig. S10b Fig. S11a Fig. S11b Fig. S11 Fig. S & P72 P21 P21 P7 sample type, protocol in vivo SeeDB ScaleA2 whole brain unfixed SeeDB ScaleA2 SeeDB ScaleA2 SeeDB37 SeeDB37 SeeDB37 whole brain SeeDBp whole brain SeeDBp SeeDB SeeDB SeeDB SeeDB cryostat section cryostat section SeeDB whole brain SeeDBp whole brain SeeDBp whole brain SeeDBp SeeDB SeeDB37 SeeDB37 SeeDB37 SeeDB37 whole brain SeeDB37 SeeDB whole brain SeeDBp Image acquisition condition objective lens information (all from Olympus) Volume (x y z) dye objective lens ( m) type specifications / Tiles, Step size ( m) immersion A x water XLPLN25XWMP immersion NA=1.05 WD=2mm water A-647 UPLSAPO20X 20x NA=0.75 WD=0.6mm A-647 UPLSAPO20X 20x NA=0.75 WD=0.6mm EYFP 25x scale XLPLN25XWMP immersion NA=1.0, WD=4mm PBS EYFP 25x scale XLPLN25XSVMP immersion NA=1.0, WD=4mm 30% glycerol EYFP 25x scale XLPLN25XSVMP immersion NA=1.0, WD=4mm Sca/eA2 EYFP 1,270 1, m UPLSAPO10X2 10x 10 m NA=0.4, WD=3.1mm EYFP 1,270 1, m UPLSAPO10X2 10x 10 m NA=0.4, WD=3.1mm EYFP 7, ,000 m 25x customized 14 1, 25 m customized NA=0.9, WD=8mm 90% TDE EYFP 17,752 6,593 7,000 m 25x customized 35 13, 500 m customized NA=0.9, WD=8mm 90% TDE EYFP ,000 m 25x customized 25 m customized NA=0.9, WD=8mm 90% TDE EGFP 2,539 2, m 25x scale XLPLN25XSVMP 5 4, 5 m immersion NA=0.9, WD=8mm 30% glycerol tdtomato 4,564 2, m 25x customized 9 4, 20 m customized NA=0.9, WD=8mm 80% TDE EYFP; A-647 UPLSAPO10X2 10x NA=0.4, WD=3.1mm EYFP; A-647 UPLSAPO10X2 10x NA=0.4, WD=3.1mm EGFP; A-647 2,462 1, m UPLSAPO10X2 10x 2 1, 4 m NA=0.4, WD=3.1mm EYFP 1,270 1, m UPLSAPO10X2 10x 5 m NA=0.4, WD=3.1mm EYFP; A-647; DAPI UPLSAPO20X 20x NA=0.75 WD=0.6mm EYFP; A-647; DAPI UPLSAPO20X 20x NA=0.75 WD=0.6mm EGFP; A-647 UPLSAPO10X2 10x NA=0.4, WD=3.1mm DiI UPLSAPO10X2 10x NA=0.4, WD=3.1mm DiI UPLSAPO10X2 10x NA=0.4, WD=3.1mm DiI UPLSAPO4X 4x NA=0.16 WD=13mm EYFP ,800 m 25x scale XLPLN25XSVMP 5 m immersion NA=0.9, WD=8mm 30% glycerol EYFP 8, ,000 m 25x customized 16 1, 25 m customized NA=0.9, WD=8mm 90% TDE EYFP 16,145 7,842 6,000 m 25x customized 35 17, 300 m customized NA=0.9, WD=8mm 90% TDE EYFP 12, ,000 m 25x scale XLSLPLN25XSVMP 29 1, 5 m 8mm NA=0.9, WD=8mm 30% glycerol EYFP 11,159 14,202 m 25x scale XLSLPLN25XSVMP 21 26, single plane 8mm NA=0.9, WD=8mm 30% glycerol EYFP ,000 m 25x scale XLSLPLN25XSVMP 5 m 8mm NA=0.9, WD=8mm 30% glycerol EYFP 2,540 2,540 2,106 m 10x water UMPLFLN 10XW 13 m immersion NA=0.3, WD=3.5mm water EGFP, tdtomato 6,594 9,637 3,000 m 25x customized 13 19, 15 m customized NA=0.9, WD=8mm 80% TDE S6

7 Supplementary Figure 1 Effects of -thioglycerol. (a) Hemi-forebrain samples of a Thy1-YFP-H mouse (P21) were cleared with SeeDB, with or without -thioglycerol (0.5%). After a three-day clearing protocol at room temperature, the samples were further incubated at 37 C for 5 days. Browning was prevented by addition of 0.5% -thioglycerol. Grids are mm. (b) The same samples shown in (a) were imaged from the surface of the cerebral cortex using confocal microscopy. The reconstructed x-z images of the cerebral cortices are shown. Accumulation of autofluorescence in the surface area (*) was prevented by addition of 0.5% -thioglycerol. Scale bar, 200 m. S7

8 Supplementary Figure 2 Transmission images of P21 mouse hemi-forebrain samples cleared with various optical clearing agents. Grids are mm. S8

9 Supplementary Figure 3 Transmission images of adult (P84) mouse hemi-forebrain samples cleared with various optical clearing agents. Grids are mm. S9

10 Supplementary Figure 4 Optimized clearing conditions for neonatal brain samples (SeeDBp). When we used embryonic and neonatal brain samples, they showed moderate expansion during optical clearing with SeeDB (up to 125% linear expansion). Therefore, we optimized the condition for these samples and found that addition of 0.1 PBS in the 20-80% fructose solutions minimizes the sample volume changes without compromising optical clarity. Sample expansion/shrinkage during the optical clearing was examined using P3 mouse forebrain samples. Grids are mm. Data are mean s.e.m. (n = 3 each). S10

11 Supplementary Figure 5 Experimental procedures and imaging setup. (a) Schematic diagram of experimental procedure. The detailed protocol is described in Methods. SeeDB37 was incubated at 37 C. SeeDB37ht was performed at 50 C. Other procedures were performed at room temperature (25 C). For the clearing of neonatal mouse brains, 20-80% (w/v) fructose was prepared with 0.1 PBS instead of H 2 O. Addition of 0.5% -thioglycerol is recommended for all the fructose solutions. (b-d) Imaging setup. For some imaging experiments, samples were S11

12 embedded in agarose before clearing. Cleared samples were trimmed and placed in Petri dishes. A coverslip (b, for short-wd objectives) or a handmade glass-bottomed Petri dish (c, for long-wd objectives) was placed on top of the block, and H 2 O (for the waterimmersion objective lens), 30% glycerol (refractive index 1.38, for the Scale-immersion objective lens), or 80% 2,2 -thiodiethanol (refractive index 1.49, for our customized objective lens) was used for immersion. Agarose embedding was useful not only to maintain the integrity of fragile samples, but also to minimize movement artifacts when imaging using upright microscopes. When a sample was not embedded in agarose, a sample with excess volume of SeeDB was placed in a handmade chamber (made of 6 mm-thick silicone rubber sheet) and sealed with a handmade glass-bottomed Petri dish (d). No bubbles should be left in the chamber. Because SeeDB is viscous, it should not be used for immersion. For imaging with the inverted microscope, samples in SeeDB were placed on a glass-bottomed chamber. Samples in SeeDB were imaged at room temperature (25 C) and those in SeeDB37 were imaged at 37 C with a thermo plate. S12

13 Supplementary Figure 6 Stability of fluorescent proteins in tissues. Samples were cleared with various clearing agents. Cryosections of mouse brains electroporated with various fluorescent proteins (ECFP, EGFP, EYFP, and tdtomato) were analyzed. Quantification of fluorescence in tissue sections after optical clearing for 72 hrs (% PBS samples) is shown. Data are mean s.e.m. for 5 ROIs. A representative result out of 3 independent experiments is shown. S13

14 Supplementary Figure 7 Compatibility with immunohistochemistry. (a, b) Immunohistochemistry after optical clearing. Brain samples (P21) cleared with SeeDB or ScaleA2 were recovered in PBS and then made into cryosections. Brain samples cleared with ScaleA2 did not fully recover, as they remained ~120% of the original size and fragile, which made the sectioning very difficult. Cryosections were stained with rabbit anti-gephyrin (Abcam, ab25784) (a), which is an inhibitory post-synaptic marker, or with mouse anti-map2 (Sigma, M9942) (b), which is a dendrite marker. Whereas antigenicity was unchanged after clearing with SeeDB and retrieval, no specific signals were observed after clearing with ScaleA2 and retrieval, for these two antibodies. Another anti-gephyrin (Synaptic Systems, mouse mab7a) stained S14

15 both SeeDB-treated and ScaleA2-treated samples (data not shown). These results suggest that conformations of cellular proteins are altered after ScaleA2 treatment. Because EYFP signals were quenched in ScaleA2 samples, laser power was set stronger to image EYFP in this sample. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer. (c) Optical clearing after wholemount immunostaining. OMP-GFP knock-in mouse, which express GFP in olfactory sensory neurons, were used for immunostaining. Dopaminergic short axon cells were stained with anti-tyrosine Hydroxylase. After immunostaining, the whole-mount sample was cleared with SeeDB and imaged using confocal microscopy. A single confocal image at a depth of 75 m from the surface. Scale bars, 100 m. S15

16 Supplementary Figure 8 Confocal images of DiI labeled neurons. Whole-brain samples were cleared using SeeDBp protocol. (a) Axons of olfactory sensory neurons (P7). (b) Mitral cell axons in the lateral olfactory tract (P7). (c) Axons from the anterior olfactory nucleus traveling along the anterior commissure (indicated by arrowheads, P5). Whole-mount samples were used for imaging. Scale bars, 100 m. S16

17 Supplementary Figure 9 Adult samples (P70) cleared with SeeDB were imaged using a commercially available objective lens with long WD. A 25 objective lens (Olympus, XLPLN25XSVMP, NA = 1.0, WD = 4.0 mm, designed for refractive index 1.38) was used for two-photon microscopy. Imaging depth was limited by the WD and the spherical aberration caused by refractive index mismatch (1.38 vs. 1.48). See Figure 3g for spatial resolution with this objective lens. Scale bars, 100 m. S17

18 Supplementary Figure 10 Two-photon images of adult Thy1-YFP-H (P72) taken from the medial side. The sample was cleared with SeeDB37. (a) An optical section along a coronal plane (indicated in schema) was reconstructed from 16 blocks. Volume rendering is available in Supplementary 6. (b) Images along a sagittal plane (indicated in schema, z = 2,100 m from the medial surface) were tiled and stitched (16 34 tiles). Serial optical sections are available in Supplementary 7. Scale bars, 1 mm. A, anterior; P, posterior; D, dorsal; V, ventral; M, medial; L, lateral. S18

19 Supplementary Figure 11 Two-photon imaging of P21 mouse forebrain. Thy1-YFP-H mouse was cleared with SeeDB37 and imaged using a commercially available long WD objective lens (Olympus, XLSLPLN25XSVMP, NA = 0.9, WD = 8.0 mm, designed for refractive index 1.38). Serial optical sections and volume rendering are available in Supplementary 8 and 9, respectively. Scale bars are 1 mm (a, b) and 100 m (c). A, anterior; P, posterior; D, dorsal; V, ventral. S19

20 Supplementary Figure 12 Imaging of the entire olfactory bulb using two-photon microscopy. We used Thy1-YFP-G mouse (P21) brain cleared with SeeDB. Using a 10 objective lens (Olympus, UMPLFLN 10XW, NA = 0.30, WD = 3.3 mm, water immersion), the entire olfactory bulb was imaged in 2 2 quadratic prisms. The images were then tiled for each z-plane to reconstruct the entire olfactory bulb. Scale bars, 500 m. The serial optical sections are available in Supplementary 10. A, anterior; P, posterior; M, medial; L, lateral. S20

21 Supplementary Figure 13 Coverage areas of sister mitral cells. (a) Coverage areas of mitral cells in Figure 6a are shown. The coverage area was defined as the area within 100- m distance from the traced lateral dendrites of a mitral cell. Scale bar, 500 m. S21

22 Supplementary 1. Adult Thy1-YFP-H line (P70) imaged using confocal microscopy. Serial x-y images were taken at an interval of 10 m. Depths (z) indicated in the movie are non-calibrated values. The real depths are 1.49 larger. See legend for Figure 3a. Supplementary 2. Volume rendering of adult Thy1-YFP-H mouse (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side, and 14 1 blocks were tiled. See legend for Figure 3c. Supplementary 3. Serial horizontal optical sections of Thy1-YFP-H line (P72). Hemi-brain sample was cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side every 500 m. See legend for Figure 3d. Supplementary 4. Thy1-YFP-H mouse (P72) imaged using two-photon microscopy. Serial x-y images were taken at an interval of 25 m. See legend for Figure 3e. Supplementary 5. Volume rendering of serial optical sections shown in Supplementary 2. See legend for Figure 3e. Supplementary 6. S22

23 Volume rendering of adult Thy1-YFP-H line (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from medial side, and 14 1 blocks were tiled. See legend for Suplementary Figure 10a. Supplementary 7. Serial optical sections of Thy1-YFP-H line (P72) imaged from medial face using two-photon microscopy. Images were taken from the medial side every 300 m, and blocks were tiled. See legend for Supplementary Figure 10b. Supplementary 8. Thy1-YFP-H line (P21) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from the dorsal side, and serial x-y images are shown. See legend for Supplementary Figure 11c. Supplementary 9. Volume rendering of serial optical sections shown in Supplementary 8. See legend for Supplementary Figure 11c. Supplementary 10. The entire olfactory bulb of a Thy1-YFP-G mouse (P21) imaged using two-photon microscopy. Serial tiled x-y images were taken from the medial side. See legend for Supplementary Figure 12. Supplementary 11. Callosal axons imaged using two-photon microscopy. Serial x-y images were taken at an interval of 10 m. See legend for Figure 4. Left, anterior; Right, posterior. S23

24 Supplementary 12. Topographic organization of corpus callosum imaged using two-photon microscopy. Anterior and posterior regions of the cerebral cortex layer II-III neurons were labeled with tdtomato (magenta) and EGFP (green), respectively, using in utero electroporation. Serial x-y images were taken at an interval of 15 m. InSight DeepSee Dual (Spectra Physics) was used to excite tdtomato and EGFP at 1,040 nm and 920 nm, respectively. Our customized 25 objective lens was used. Left, anterior; Right, posterior. Supplementary 13. Reconstruction of individual callosal axons sparsely labeled by Cre-loxP system. See legend for Figure 4b. Supplementary 14. Tracing lateral dendrites of sister mitral cells. Serial confocal images to reconstruct Figure 6a. Depths (z) in the movie are non-calibrated values. The real depths are 1.49 larger. Only presumptive mitral cells were reconstructed in Figure 6a. See legend for Figure 6. Green, OMP-GFP; magenta, Alexa-647 dextran. S24