A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Summary

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Abstract

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (–), and ScaleS4 solution, for a total of 10.5–14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

Introduction

Tissue clearing methods have improved depth-independent imaging of biological and clinical samples with light microscopy, allowing for extraction of structural information on intact tissues^1,2. Optical clearing techniques could also potentially speed up, and reduce the cost for histological analysis. Currently, three major clearing approaches are available: hydrophilic, hydrophobic, and hydrogel-based methods^1,2. Hydrophilic approaches surpass in preserving fluorescence signals and tissue integrity and are less toxic compared to the other two approaches^3,4.

A hydrophilic clearing method, ScaleS, holds a distinctive position with its preservation of structural and molecular integrity as well as potent clearing capability (clearing-preservation spectrum)⁵. In a previous study, we developed a rapid and isometric clearing protocol, ScaleSF, for tissue slices (~1-mm thickness) by modifying the clearing procedure of ScaleS⁶. This clearing protocol requires sequential incubations of brain slices in three solutions for 10.5-14.5 h. The method is featured with a high clearing-preservation spectrum, which is compatible even with electron microscopy (EM) analysis (Supplementary Figure 1), allowing for multi-scale high-resolution three dimensional (3D) imaging with accurate signal reconstruction⁶. Thus, ScaleSF should be effective especially in the brain, where neuronal cells elaborate exuberant processes of tremendous length, and arrange specialized fine subcellular structures for transmitting and receiving information. Extracting structural information with scales from circuit to subcellular levels on neuronal cells is quite useful toward better understanding of brain functions.

Here, we provide a detailed protocol to visualize neuronal structures with scales from the mesoscopic/circuit to microscopic/subcellular level using ScaleSF. The protocol includes tissue preparation, tissue clarification, handling of cleared tissues, and confocal laser scanning microscopy (CLSM) imaging of cleared tissues. Our protocol focuses on interrogating neuronal structures from circuit to subcellular component scales. For a detailed procedure for preparation of the solutions and stereotaxic injection of adeno-associated virus (AAV) vectors into mouse brains, refer to Miyawaki et al. 2016⁷ and Okamoto et al. 2021⁸, respectively.

Protocol

All the experiments were approved by the Institutional Animal Care and Use Committees of Juntendo University (Approval No. 2021245, 2021246) and performed in accordance with Fundamental Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan (2006). Here, male C57BL/6J mice injected with AAV vector carrying enhanced green fluorescent protein (EGFP) gene and parvalbumin (PV)/myristoylation-EGFP-low-density lipoprotein receptor C-terminal bacterial artificial chromosome (BAC) transgenic mice (PV-FGL mice)⁹ were used. PV-FGL mice were maintained in C57BL/6J background. No sex-based differences were found with regard to this study.

1. Tissue preparation

1. Perfusion fixation
   NOTE: Perform steps 1.1.1 through 1.1.3 in a fume hood to limit the exposure to paraformaldehyde (PFA).
   1. Anesthetize adult male mice (8–16 weeks old) by an intraperitoneal injection of overdose of sodium pentobarbital (200 mg/kg). Confirm adequacy of anesthesia by the absence of toe-pinch withdrawal and eye-blink reflexes.
   2. Open the thoracic cavity and cut the right atrial appendage with surgical scissors. Perfuse the mice with 20 mL of ice-cold phosphate buffer saline (PBS) using a 23 G needle attached to a 20 mL syringe, followed by perfusion of 20 mL of ice-cold 4% PFA in 0.1 M phosphate buffer (PB) using another 20 mL syringe.
      CAUTION: PFA is toxic and teratogenic. Avoid inhalation or contact with skin, eyes, and mucous membrane.
   3. Remove brain tissues from the skull with tweezers. Transfer the brain tissues to a 15 mL tube containing 4% PFA in 0.1 M PB, protect the samples from light, and gently rock overnight at 4 °C on a shaker at 50–100 rpm.
   4. NOTE: The harvested brain tissues can be stored for several weeks in 0.02% sodium azide (NaN₃) in PBS at 4 °C.
      CAUTION: NaN₃ is toxic. Avoid inhalation or contact with skin, eyes, and mucous membrane. Handle it inside a fume hood.
2. Brain slice preparation
   1. Prepare 4% agar in PBS by adding 2 g of agar to 50 mL of PBS. Microwave the mixture until the agar is fully dissolved. Let the solution cool to 40-45 °C.
      NOTE: The viscous property provided by agaropectin, a major component of agar, improves ease of cutting of tissue slices.
   2. Add 10 mL of the agar solution to a 6-well culture plate. Submerge the brain tissue in the agar solution using forceps. Let the agar solidify on ice.
   3. Remove the embedded brain tissue from the well and trim the agar with a razor blade. Secure the agar block onto the bottom of the vibratome bath with superglue and pour 0.1 M PB in the buffer tray.
   4. Clean another razor blade using a lint-free tissue paper soaked in ethanol and attach the blade to the blade holder of the vibrating tissue slicer.
   5. Set the sectioning speed to 0.14 mm/s with 1.4 mm amplitude and the frequency to 75–77 Hz. Cut the brain tissue into 1-mm-thick slices and collect the slices in a 6-well cell culture plate containing PBS.
      NOTE: The brain slices can be stored for several weeks in 0.02% NaN₃ in PBS at 4 °C.

2. Tissue clarification

NOTE: The compositions of ScaleS solutions used are listed in Table 1. Samples should be protected from light by covering with a foil. The clearing steps is shown in Figure 1A.

1. Add 8 mL of ScaleS0 solution to one well of a 6-well cell culture plate and add 8 mL of ScaleS4 solution to another well of the plate and pre-warm to 37 °C in an incubator.
2. Transfer the brain slices to the pre-warmed ScaleS0 solution with a spatula and incubate for 2 h at 37 °C in a shaking incubator at 90 rpm.
3. Transfer the permeabilized brain slices in 8 mL of PBS(–) in a 6-well cell culture plate with a spatula and wash for 15 min by keeping in an orbital shaker at 40–60 rpm. Repeat this twice.
4. Transfer the brain slices in the pre-warmed 8 mL of ScaleS4 solution with a spatula and clear them by incubating in a shaking incubator at 90 rpm for 8–12 h at 37 °C. A cleared brain slices can be seen in Figure 1.

3. Brain slice mounting

NOTE: A customizable imaging chamber is used for reliable mounting of cleared brain slices (Figure 2)⁶. The chamber consists of the chamber frame and bottom coverslip. The microscope stage adaptors are also designed to mount the imaging chamber on microscope stages directly (Figure 2A,B). The chamber frame and microscope stage adaptors can be 3D-printed using in-house or outsourced 3D-printing services. 3D computer-aided design (CAD) data of the imaging chamber are provided in Furuta et al. 2022⁶.

1. For chamber preparation, attach the chamber frame to a coverslip using a pressure-sensitive adhesive.
2. Prepare 1.5% agarose in ScaleS4D25(0) solution (ScaleS4 gel) by adding 1.5 g of agarose to 100 mL of the solution in a bottle. Mix the solution well by stirring and microwave the solution until the agarose is fully dissolved. Once done, allow the solution to cool to 37 °C.
3. Mount the cleared brain slice onto the bottom coverslip of the imaging chamber with a spatula. Wipe away the excess solution from the cleared slice using a clean lint-free tissue paper.
4. Add the ScaleS4 gel on the brain slice using a micropipette to fill the imaging chamber. Place another coverslip on top with forceps and place a piece of lint-free tissue paper and a glass slide on the coverslip in this order.
5. Transfer the imaging chamber to a refrigerator at 4 °C. Place metal weights on the glass slide and leave them for 30 min.
6. Remove the metal weights, glass slide, lint-free tissue paper, and coverslip from the imaging chamber, and wipe the excess gel away (Figure 2A,B).
7. Place the imaging chamber in a 60 mm glass Petri dish and attach the rim of the imaging chamber to the dish with a putty-like pressure sensitive adhesive. Attach the chamber at multiple points to the Petri dish.
8. Pour ScaleS4 solution in the dish and shake gently for 1 h at 20-25 °C on an orbital shaker at 40-60 rpm. Substitute with fresh solution and remove air bubbles on the gel surface by gently scraping the surface using a 200 µL pipette tip. Mount the immersed imaging chamber on a microscope stage (Figure 2C).

4. CLSM imaging

1. Acquire images using a CLSM equipped with a multi-immersion objective lens of a long working distance (WD) (16x/0.60 numerical aperture [NA], WD = 2.5 mm).
   NOTE: High NA objective lenses can provide high diffraction-limited resolution.
2. Turn on all the relevant imaging equipment (workstation, microscope, scanner, lasers, and mercury lamp) and launch a CLSM imaging software.
3. Set the correction collar of the multi-immersion objective lens to 1.47. ScaleS4 solution has a refractive index (RI) of around 1.47^5,7. RI mismatch-induced aberrations can disturb the image formation (Figure 3).
4. Immerse the objective lens in the solution, and let it approach the slice slowly. Remove any air bubbles trapped on the tip of the objective lens. Find regions of interest (ROIs) in the cleared tissues using epifluorescence.
5. Set image acquisition parameters by testing appropriate settings.
   1. Determine the bit depth for image acquisition. The data size of the image increases with the bit depth.
   2. Set the detection wavelength. Adjust the appropriate gate for the detector according to the emission spectrum. Ensure that the detection wavelength does not cover any laser lines.
   3. Set the xy resolution. Larger formats provide better xy resolutions, but it takes longer time to collect the images.
   4. Set the scan speed. A slower scan speed provides a high signal-to-noise ratio. However, it also increases the pixel dwell time and the risk of photobleaching. Choose accordingly.
   5. Adjust the pinhole size. The pinhole size controls optical section thickness. A smaller pinhole size creates a thinner optical section, and thus better z resolution, but reduces fluorescence signal. Making the pinhole size larger provides a thicker optical section with stronger fluorescence signal.
   6. Set the laser power, the detector/amplifier gain, and offset. Gradually increase the laser power and detector/amplifier gain until a suitable image is obtained. A high laser power carries the risk of photobleaching. Adjust the offset (contrast) appropriately to obtain a high signal-to-noise ratio.
   7. Determine the tilling area needed based on the size of ROI. Ensure that the entire length and width of the ROI is captured.
   8. Navigate the cleared tissues in all planes, and set the start and end points of the stack. Set the z-step size according to the desired z-resolution.
6. Collect images when satisfied with the image acquisition settings, and record captured images. Process the images using an image analysis software.

Results

Optical clearing of a mouse brain slice of 1-mm thickness was achieved using this protocol. Figure 1B represents transmission images of a mouse brain slice before and after the clearing treatment. The tissue clearing method rendered a 1-mm-thick mouse brain slice transparent. A slight expansion in final sizes of brain slices was found after the incubation in the clearing solution for 12 h (linear expansion: 102.5% ± 1.3%). The preservation of fluorescence and structural integrity of the tissues...

Discussion

Critical steps within the protocol
There are a few critical steps in the protocol that should be conducted with utmost caution to obtain meaningful results. Uniform fixation of samples is imperative for 3D imaging within large-scale tissues. The objective lens, sample, and immersion fluid should have matching RI. RI-mismatch among them will lead to highly disturbed imaging of EGFP-expressing cells within the cleared brain slices (Figure 3). The correction collar adjustment of the o...

Materials

Name	Company	Catalog Number	Comments
16x multi-immersion objective lens	Leica Microsystems	HC FLUOTAR 16x/0.60 IMM CORR VISIR
Agar	Nacalai Tesque	01028-85
Agarose	TaKaRa Bio	L03
Dimethyl sulfoxide	Nacalai Tesque	13407-45
D-Sorbitol	Nacalai Tesque	06286-55
γ-cyclodextrin	Wako Pure Chemical Industries	037-10643
Glycerol	Sigma-Aldrich	G9012
Huygens Essential	Scientific Volume Imaging	ver. 18.10.0p8/21.10.1p0 64b
Imaris	Bitplane	ver. 9.0.0
Leica Application Suite X	Leica Microsystems	LAS X, ver. 3.5.5.19976
Methyl-β-cyclodextrin	Tokyo Chemical Industry	M1356
Paraformaldehyde	Merck Millipore	1.04005.1000
Phosphate Buffered Saline (10x; pH 7.4)	Nacalai Tesque	27575-31	10x PBS(–)
Sodium azide	Nacalai Tesque	31233-55
Sodium pentobarbital	Kyoritsu Seiyaku	N/A
TCS SP8	Leica Microsystems	N/A
Triton X-100	Nacalai Tesque	35501-15
Urea	Nacalai Tesque	35940-65
Vibrating tissue slicer	Dosaka EM	PRO7N