Protocol for multi-scale light microscopy/electron microscopy neuronal imaging in mouse brain tissue

Summary An imaging technique across multiple spatial scales is required for extracting structural information on neurons with processes of meter scale length and specialized nanoscale structures. Here, we present a protocol combining multi-scale light microscopy (LM) with electron microscopy (EM) in mouse brain tissue. We describe tissue slice preparation and LM/EM dual labeling with EGFP-APEX2 fusion protein. We then detail ScaleSF tissue clearing and successive LM/EM imaging. Our protocol allows for deciphering structural information across multiple spatial scales on neurons. For complete details on the use and execution of this protocol, please refer to Furuta et al. (2022).

14. Add 500 mL of Siliconized L-25 to 9.5 mL of chloroform, and mix well by stirring. 15. Spread the 5% Siliconized L-25 solution on glass slides and coverslips. 16. Let them dry.
CRITICAL: Chloroform is toxic and carcinogenic. Handle it inside a fume hood with appropriate protective gear. All necessary precautions should be taken when disposing.

Preparation of rocket of resin
Timing: 3 days 17. Mix well 3.8 mL of Luveak-812, 2 mL of Luveak-DDSA and 2.4 mL of Luveak-MNA by slowly stirring for 5 min. 18. Add 0.12 mL of Luveak-DMP-30 to the mixture, and mix well by slowly stirring for 30 min. 19. Put the mixture into silicon capsules and polymerize the resin in an oven at 60 C for 2 days. The polymerized resin, rocket of resin, can be stored at 20 C-25 C for several years.  The solution can be stored at 4 C for 1 week.

Reagent Final concentration Amount
Methyl-b-cyclodextrin 100 mM 1.303 g ddH 2 O n/a 10 mL Total n/a 10 mL Dispense to 1 mL each. The solution can be stored at À20 C for 3 months.

Reagent
Final concentration Amount g-cyclodextrin 100 mM 1.297 g ddH 2 O n/a 10 mL Total n/a 10 mL Dispense to 1 mL each. The solution can be stored at À20 C for 3 months.

Reagent Final concentration Amount
Triton X-100 10% (w/v) 5 g ddH 2 O n/a up to 50 mL

Total n/a 50 mL
The solution can be stored at 4 C for 3 months.

Reagent
Final concentration Amount Methyl-b-cyclodextrin 100 mM 1 mM 1 mL g-cyclodextrin 100 mM 1 mM 1 mL (Continued on next page)

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Note: The original ScaleS0 solution contains N-acetyl-L-hydroxyproline (Skin Essential Actives, Hualian, Republic of China) (Hama et al., 2015). If this amino acid derivative is difficult to obtain from the manufacturer, N-acetyl-L-hydroxyproline should be omitted from the solution. N-acetyl-L-hydroxyproline provided by other manufactures can negatively affect tissue clarification (Miyawaki et al., 2016).

Reagent Final concentration Amount
103 PBS(-) 13 10 mL ddH 2 O n/a up to 100 mL Total n/a 100 mL For a detailed procedure for preparation of this solution, refer to Miyawaki et al. (2016). The solution can be stored at 4 C for 1 month.

Reagent
Final concentration Amount CRITICAL: ABC solution should be prepared before incubation with brain sections. Add 20 mL of Reagent A to 1 mL of 2% BSA in PBS, and then add 20 mL of Reagent B to the same solution, and mix immediately. Incubate the mixture for 30 min at 20 C-25 C with gentle agitation, and chill on ice.
CRITICAL: Nickel ammonium sulfate is toxic and carcinogenic. Avoid inhalation or contact with skin, eyes and mucous membrane. Handle it inside a fume hood with appropriate protective gear.

Final concentration Amount
Glycerol The solution can be stored at 20 C-25 C for 1 month.  This step describes how to prepare mouse brain slices for the successive LM/EM imaging with Sca-leSF tissue clearing.
CRITICAL: Perform steps 2 through 3 in a fume hood to limit the exposure to PFA and GA. 4. Immerse the brain in the fixative solution and rock gently for 16 h-20 h at 4 C (50 rpm-100 rpm).
CRITICAL: Samples should not be stored for extended periods of time. Procced to the next step immediately.

Brain slice preparation.
a. Embed the brain tissues in 4% agar in PBS in a standard 6-well culture plate.
Note: The temperature of the agar solution should be between 40 C and 45 C. j. Set the vibratome frequency to 75 Hz-77 Hz. k. Cut 1-mm-thick brain slices. l. Collect the slices in ice-cold PBS.
Pause point: The slices can be stored in PBS for 16 h-20 h at 4 C.
CRITICAL: NaN 3 as a preservative should be avoided. NaN 3 inactivates the enzymatic activity of APEX2.

Timing: 2 days
In this step, we explain the procedure for APEX2/BT-GO reaction, where biotin molecules are deposited with tyramide signal amplification (TSA) reaction using the peroxidase activity of APEX2 ( Figure 2) (Furuta et al., 2022). This step is essential for LM/EM dual labeling resistance for ScaleSF tissue clearing. All incubations are performed in a standard 6-well culture plate at 20 C-25 C, except where noted.
CRITICAL: APEX2/BT-GO should be performed prior to tissue clearing with ScaleSF. Peroxidase activity of APEX2 markedly declines following ScaleSF treatment ( Figure S1). CRITICAL: Samples should be protected from light.

Permeabilization.
a. Immerse the slices in 10 mL of permeabilization buffer in a 13 mL centrifuge tube. b. Rock gently for 4 h (50 rpm-100 rpm).
CRITICAL: Brain tissues should be shaken thoroughly.
7. Wash. a. Transfer the slices in 8 mL of 0.1 M PB. b. Wash the slices with 8 mL of 0.1 M PB for 5 min three times with gentle rocking (50 rpm-100 rpm). 8. BT penetration.
a. Transfer the slices in 1200 mL of BT-GO reaction mixture in a 2 mL safe-lock microcentrifuge tube. b. Rock gently for 4 h (50 rpm-100 rpm).
CRITICAL: Brain tissues should be shaken thoroughly. CRITICAL: Samples should be protected from light.
13. ScaleSF tissue clearing ( Figure 3B). a. Warm ScaleS0 and ScaleS4 solutions to 37 C. b. Immerse the brain slices in 8 mL of ScaleS0 solution, and rock gently for 2 h at 37 C (90 rpm). c. Transfer the slices in 8 mL of 13 PBS (-), and wash twice for 15 min with gentle rocking at 20 C-25 C (50 rpm-100 rpm). d. Transfer the slices in 8 mL of ScaleS4 solution, and rock gently for 8 h-12 h at 37 C (90 rpm).

Brain slices mounting
Timing: $2 h This step describes how to mount the cleared brain slices on the imaging chamber. Figure 2. Schematic diagram of APEX2/BT-GO reaction APEX2/BT-GO is a TSA system that utilizes peroxidase activity of APEX2 and H 2 O 2 produced during oxidation of glucose by glucose oxidase (GO reaction) to deposit biotinylated tyramine (BT) onto tissues. APEX2 reacts with the H 2 O 2 and oxidizes the phenolic part of BT to produce highly reactive intermediates, which in turn covalently bind to electron-rich moieties such as tyrosine residues at or near the APEX2. POD: peroxidase. a. Drop ScaleS4 gel on the cleared slice to fill the imaging chamber.
Note: The temperature of the ScaleS4 gel should be kept at 37 C.
b. Mount a coverslip on the imaging chamber, and place a piece of Kimwipe paper and a slide glass on the coverslip in this order. c. Transfer the imaging chamber in a refrigerator at 4 C. d. Place metal weights on the slide glass, and allow the gel to solidify for 30 min. e. Remove the metal weights, slide glass, Kimwipe paper and coverslip from the imaging chamber, and wipe away excess ScaleS4 gel. 16. Tissue equilibration.
a. Attach the imaging chamber to the bottom of a 60-mm glass petri dish with Blu-Tackâ. Adhesion at multiple points is required. b. Pour ScaleS4 solution to the dish, and incubate the embedded slice in the solution for 60 min at 20 C-25 C with gentle agitation (40 rpm-60 rpm). c. Substitute with fresh ScaleS4 solution.
CRITICAL: Air bubbles on the sample surface should be removed.
Optional: Using microscope stage adaptors, the imaging chambers can be mount on a microscope stage directly. In this case, the coverslip should remain attached on the imaging chamber. CRITICAL: Refractive index (RI) mismatch-induced aberrations can highly degrade the image formation. ScaleS4 solution has a RI of around 1.47 (Hama et al., 2015;Miyawaki et al., 2016).

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18. Mount the imaging chamber on a microscope stage. 19. Immerse the objective lens in ScaleS4 solution, and make it approach to the cleared slice slowly.
CRITICAL: Air bubbles trapped on the tip of the objective lens should be removed.
20. Adjust imaging acquisition settings such as laser power, scan speed, pinhole diameter, detector gain, amplifier offset/gain, xy and z-axis resolution and bit intensity resolution. 21. Collect images by a CLSM, and record captured images. 22. Image processing is done with Leica Application Suite X and ImageJ.

Timing: 2 days
Here, we explain the procedure for re-sectioning from the slices imaged with CLSM. All reactions are performed in a standard 6-well culture plate at 20 C-25 C, except where noted.
a. Transfer the slices in 8 mL of 13 PBS (-), and wash the slices with 8 mL of 13 PBS (-) for 5 min twice with gentle shaking (40 rpm-60 rpm).
Pause point: The slices can be stored in 0.02% NaN 3 in PBS at 4 C for 1 week.
26. Cryoprotection. a. Immerse the slices in 8 mL of 30% sucrose in 0.1 M PB at 4 C until sunk (a sign of complete immersion).
CRITICAL: Permeate brain slices with the cryoprotectant agent adequately. Adequate cryoprotection is of importance to reduce ice crystal formation and preserve cellular structure.
Pause point: The slices can be stored in 30% sucrose in 0.1 M PB for 16 h-20 h at 4 C.
27. Freezing brain slices. a. Immerse the slices in OCT compound for 15 min. b. Place the slice in a cryomold containing OCT compound. c. Cool isopentane in a metal container on liquid nitrogen.
CRITICAL: Cool isopentane almost to its freezing temperature. Rapid freezing is of importance to reduce ice crystal formation and preserve cellular structure.
d. Immerse the cryomold in the cooled isopentane to freeze. e. Transfer the cryomold in a freezer at À20 C.
Pause point: The blocks can be stored at À20 C for a week. 28. Cryosectioning. a. Mount the block onto the stage of a freezing microtome with OCT compound. b. Quickly freeze the OCT compound using dry ice powder and leave the block in place for 5 min. c. Cut the slice into 40-mm-thick sections and collect in ice-cold 0.1 M PB.
Pause point: The sections can be stored in 0.02% NaN 3 in PBS at 4 C for 1 week.
Optional: Imaged slices can be embedded in agar or gelatin solution, and re-sectioned with a vibratome.

CLSM imaging in re-sections
Timing: 1 day Here, we explain the procedure for CLSM imaging in re-sections with a high NA objective lens, which allows for high-resolution imaging of targeted structures.
CRITICAL: Be sure to keep re-sections wet during this step.
a. Place a re-section on a slide with a brush. b. Apply 75% glycerol in PBS to the re-section. c. Lower a coverslip onto 75% glycerol in PBS to avoid trapping any air bubbles. 30. CLSM imaging.
a. Mount the slide on a microscope stage. b. Adjust imaging acquisition settings such as laser power, scan speed, pinhole diameter, detector gain, amplifier offset/gain, xy and z-axis resolution and bit intensity resolution. d. Collect images by a CLSM equipped with high NA objective lenses, such as a 253/NA 0.95 water-immersion and 633/NA 1.40 oil-immersion objective lenses. e. Record captured images. f. Image processing is done with Leica Application Suite X and ImageJ. 31. Wash. a. Remove the coverslip from the slide. b. Immerse the re-section in 8 mL of PBS in a standard 6-well culture plate, and wash twice for 15 min with gentle rocking at 20 C-25 C (40 rpm-60 rpm).
Pause point: The sections can be stored in 0.02% NaN 3 in PBS at 4 C for 1 week.

ABC/DAB-Ni 2+ reaction
Timing: 2 days This step describes the procedure for ABC/DAB-Ni 2+ reaction. All incubations are performed in a standard 12-well culture plate at 4 C, except where noted.
a. Immerse the sections in 500 mL of ABC solution in a standard 24-well plate, and rock gently for 24 h (50 rpm-100 rpm). 34. Wash.

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a. Immerse the sections in 3 mL of PBS, and wash twice for 10 min with gentle rocking (50 rpm-100 rpm). 35. DAB-Ni 2+ reaction.
a. Immerse the sections in 1 mL of DAB-Ni 2+ solution in a standard 24-well plate on ice, and incubate for 10 min on ice with gentle agitation (40 rpm-60 rpm). b. Add 3 mL of 0.1% H 2 O 2 into DAB-Ni 2+ solution, and incubate on ice with gentle agitation until color development (40 rpm-60 rpm). c. Immerse the sections in 3 mL of 2% NaN 3 in PBS, and incubate for 15 min at 20 C-25 C with gentle agitation (40 rpm-60 rpm). 36. Wash.
a. Immerse the sections in 3 mL of PBS at 4 C and wash twice for 10 min with gentle rocking (50 rpm-100 rpm).
Pause point: The sections can be stored in 1% PFA in 0.1 M PB at 4 C for 1 month.

Processing sections for EM
Timing: 3 days a. Immerse the sections in 5 mL of Epoxy resin/propylene oxide mixture and incubate for 1 h. b. Immerse the sections in Epoxy resin and incubate for 16 h-20 h. c. Place the sections onto silicone-coated slide glass, and mount a silicone-coated coverslip on the slide. d. Place metal weights on the coverslip to make the sections flat. e. Remove the resin oozing from the slide. f. Polymerize the resin by heating in an oven at 60 C for 2 days.
Pause point: The flat-embedded sections can be stored at 20 C-25 C in a desiccator for several months.

Ultra-thin sectioning
Timing: 1 day Here, we describe how to prepare ultra-thin sections.
43. Remove the coverslip from the slide with a razor blade. The embedded sections stick to either the coverslip or the slide. 44. Identify the region of interest (ROI) with a brightfield microscope. 45. Trim the ROI using a razor blade under a stereo microscope. 46. Mount the trimmed resin block on a flat-tipped rocket of the resin with a glue. 47. Trim away excess resin with a razor blade. 48. Mount the rocket on the stage of an ultramicrotome, and align the block face with a synthetic diamond knife. 49. Remove the empty resin from the top of the sample using the synthetic diamond knife to expose the surface of the tissue. 50. Make ultrathin sections (60-80-nm thickness) with a diamond knife. 51. Collect short ribbons of ultrathin sections on TEM grids of copper, and let them dry. 52. Drop 1% UA solution on Parafilmâ, and place the grids on the droplet for 20 min. 53. Wash the grids three times by dipping them into 10 mL of ddH 2 O. 54. Place the grids on the droplet of lead citrate solution on Parafilmâ for 2 min. 55. Wash the grids three times by dipping into 10 mL of ddH 2 O, and let them dry.
Pause point: The ultrathin sections can be stored in a grid box at 20 C-25 C for several years.

Transmission electron microscopy (TEM) imaging
Timing: 1 day This step describes a procedure for TEM imaging. We usually acquire images at a resolution of 1.

EXPECTED OUTCOMES
By following the clearing protocol of ScaleSF, one can make mouse brain slices of 1-mm thickness transparent within 14.5 h after transient shrinkage and expansion (Figure 3). A multi-scale neuronal imaging in the mouse striatofugal projection system is shown in Figure 4. The mouse striatofugal projection is labeled by an injection of a LM/EM dual labeling vector, AAV2/1 SynTetOff EGFP-APEX2, into the caudate-putamen (CPu). Following tissue slice preparation, APEX2/BT-GO reaction and ScaleSF tissue clearing, the cleared brain slice is subjected to neural circuit mapping using a CLSM equipped with a multi-immersion objective lens of long WD ( Figure 4A). EGFP-labeled axons arising from the CPu extended caudally to the brainstem, forming dense terminal fields in the external segment of the globus pallidus (GPe) and substantia nigra (SN). After re-sectioning ll OPEN ACCESS the imaged brain slice perpendicularly, high-resolution image stacks collected with a high NA objective lens show varicose axon arborizations of the EGFP-labeled neurons in the SN (Figure 4C 1 ). Then, the imaged re-section is processed for ABC/DAB-Ni 2+ reaction using biotin molecules deposited by APEX2/BT-GO reaction. One should see DAB-Ni 2+ precipitates in the re-section. Following epoxy resin embedding ( Figure 4C 2 ) and ultra-thin sectioning, an axon terminal filled with DAB-Ni 2+ precipitates is imaged with TEM ( Figures 4D 1 and 4D 2 ). TEM imaging shows a symmetric synapse, which is characterized by the absence of PSD and the narrow synaptic cleft, on a dendrite of a SN neuron ( Figure 4D 2 ). For accurately tracking targets across multiple spatial scales, we took advantage of GFP fluorescence, DAB-Ni 2+ labeling and/or endogenous landmarks such as shape of brain structure and blood vessels. Unambiguous correlation of LM and EM datasets was achieved by LM/EM dual labeling with a correlative light and electron microscopy (CLEM) probe, EGFP-APEX2.

LIMITATIONS
In the present protocol, we provide a detailed procedure for successive LM/EM for mouse neuronal cells with ScaleSF tissue clearing. ScaleSF is an isometric and rapid tissue clearing method and archives a high-level of preservation of ultrastructure and fluorescence signals as well as potent clearing capability. However, two limitations remain in ScaleSF tissue clearing. The first is the ultrastructural preservation. A lipid-extracting detergent in ScaleS4 solution and/or incubations of brain tissues at 37 C for a total of 10-14 h can potentially damage the ultrastructural integrity. Indeed, we found a slight but statistically significant degradation of the ultrastructure accompanied by ScaleSF tissue clearing (Furuta et al., 2022). The second is the clearing capability of ScaleSF: brain slices of 1-mm thickness, but not a whole brain, can be cleared with the clearing protocol of ScaleSF. Although 1-mm-thick brain slices can provide good knowledge of dendritic and local axonal arbors, information about long-range projections is fragmentary and incomplete in these slices (Stepanyants et al., 2009).
In the protocol described here, LM/EM dual labeling is implemented by coupling a genetically encoded CLEM probe, EGFP-APEX2, with APEX2/BT-GO reaction. Despite its potent LM/EM dual labeling and resistance for ScaleSF tissue clearing, APEX2/BT-GO reaction itself and/or permeabilization for intracellular staining with a lipid-extracting detergent can potentially negatively affect the cellular ultrastructure. The LM/EM dual labeling gave strong EM contrast that was introduced in the form of osmiophilic polymers throughout the cytoplasm (Figure 3). Although the cytoplasmic labeling facilitates the identification of targeted structures, the labeling may interfere with interrogation of ultrastructural features of synapses such as active zones, PSDs and synaptic vesicle morphologies.

TROUBLESHOOTING
Problem 1 Brain tissues look opaque after clearing (step 13).

Potential solution
Use ScaleS solutions that are freshly prepared. ScaleS solutions can be stored up to 1 month at 4 C. Consider prolonged incubation in ScaleS4 solution.

Problem 2
Weak or very light fluorescent signal (step 21).

Potential solution
Use ScaleS solutions that are freshly prepared. Fluorescent proteins are sensitive to certain environmental conditions, such as pH, ions, and redox state.
Problem 3 3D reconstruction of CLSM images is not acceptable (step 22).

Potential solution
Set the correction collar of the multi-immersion objective lens to 1.47. ScaleS4 solution has a RI of 1.47 (Hama et al., 2015;Miyawaki et al., 2016). RI mismatch-induced aberrations can highly disturb the image formation.

Problem 4
Ice crystal damage is evident in re-sections (step 28).

Potential solution
Cool isopentane almost to its freezing temperature. Permeate brain tissues with the cryoprotectant agent adequately. This problem is caused by too slow a freezing rate and/or inadequate cryoprotection of tissues.