Large-scale 3D Tissue Imaging and Clearing

All the imaging experiments are summarized in Table 2. We used the passive CLARITY method (as described previously [5 (link)]) for all the tissue clarification experiments. The hydrogel monomer (HM) solution recipe consisted of 1–4% (wt/vol) acrylamide, 0.05% (wt/vol) bisacrylamide, 4% paraformaldehyde (PFA), 1× phosphate-buffered saline (PBS), deionized water, and 0.25% thermal initiation VA-044 (Wako Chemicals, Boston, MA, USA; NC0632395). All animal procedures were followed according to Institutional Animal Care and Use Committee (IACUC) guidelines. For whole brain clearing, transcardiac perfusion was performed with 20 mL HM solution, followed by overnight incubation at 4 °C. The rat brain was perfused with 4% PFA, post-fixed for 16 h, and then frozen in isopentane for storage. The frozen brain was thawed at room temperature in PBS buffer, then sliced and incubated in HM solution overnight at 4 °C. The human brain tissue was incubated in 4% PFA for ~ 2 days, followed by incubation in HM solution overnight at 4 °C. All the perfused tissues were de-gassed and then stored at 37 °C for 3–4 h for hydrogel polymerization. The tissues were cleared by incubating (with shaking) in clearing buffer (4% (wt/vol) sodium dodecyl sulfate (SDS), 0.2 M boric acid, pH 8.5) at 37 °C until clear (2–3 weeks). Afterwards, the tissues were washed with 0.2 M boric acid buffer (pH 8.5) with 0.1% Triton X-100 for up to 24 h. The cleared tissue was labeled with DAPI (1 μg/mL final concentration) and/or the blood vessel marker tomato lectin (Vector Labs, Burlingame, CA, USA; FL-1171) by incubating in the labeling solution for 3–4 days. After washing with the buffered solution (0.2 M boric acid buffer, pH 7.5, 0.1% Triton X-100), the tissue was transferred into 85–87% glycerol solution in graded fashion (i.e. 25%, 50%, 65%, and finally 87%) for final clearing and imaging. For uniform tissue expansion (4–4.5× uniformly), a Thy1-eYFP mouse brain slice (250 μm, perfused and fixed with 4% PFA and sliced with vibratome) was gelled and digested following the protein retention expansion microscopy (proExM) protocol [34 (link)]. The sample was stored in 1× PBS before changing the buffer to 65% glycerol (with 2.5 mg/mL 1,4-diazabicyclo[2.2.2]octane (DABCO)) for the LSTM imaging. All imaging experiments were performed with an effective light sheet thickness of 2–5 μm.Table 2

Summary of imaging experiments reported in this study

Samples	Fig. No.	Label	Det. objective	Illum. objective	Imaging volume dimensions	No. of images/raw data	Imaging time
Thick human brain tissue	4	DAPI	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	~ 10.5 mm × 14.1 mm × 3 mm	116,736/~ 0.97 TB	~ 2.7 h
Mouse brain with attached spinal cord	5a	Thy1-eYFP	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	11.8 mm × 27.6 mm × 5.2 mm	388,687/~ 3.3 TB	~ 9 h
Thick mouse brain slice	5b	Thy1-eYFP	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	~ 9.6 mm × 13.5 mm × 5.34 mm	256,560/~ 2.1 TB	~ 5.9 h
Thick mouse brain slice	5c	Thy1-eYFP	25×/1.0NA/8 mm/WD	4×/0.28NA/28.5WD	6 mm × 9.6 mm × 1.9 mma	211,616/~ 1.8 TB	~ 4.9 h
Thick rat brain slice	6a, b	Tomato lectin	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	~ 20 mm × 16.5 mm × 3.6 mm deepb	285,821 slices/2.4 TB	~ 6.6 h
expanded (~ 4×) mouse brain slice	6c	Thy1-eYFP	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	33.2 mm × 19.3 mm × 2 mmc	723,200/~ 6 TB	~ 22 h
Hydra live imaging	7	GCaMP6s	10×/0.6NA/8mmWD	4×/0.28NA/28.5WD	1.2 mm × 1.2 mm × 0.136 mm	23,001/~ 193 GB	~ 1 h live imaging

Summary of the datasets reported in this report.

^aThe image volume acquired was ~ 6 mm × 9.6 mm × 1.9 mm; however, due to constraints of high-quality volume rendering, a smaller (0.5-mm-thick) subset was used for the rendering shown in Fig. 5c

^bThe approximate imaging volume was ~ 20 mm × 16.5 mm × 3.6 mm, and a few ~ 5-mm-deep image stacks were acquired to demonstrate the imaging depth in Fig. 6b

^cThe imaging volume acquired was ~ 33.2 mm × 19.3 mm × 2 mm to ensure complete coverage of ~ 1-mm-thick expanded non-rigid tissue

TB terabytes, GB gigabytes, h hours