Mesoscopic Optical Imaging of Whole Mouse Heart

Summary

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Abstract

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

Introduction

Structural remodeling associated with cardiac diseases can affect electrical conduction and introduce electromechanical dysfunctions of the organ^1,2. Current approaches used to predict functional alterations commonly employ MRI and DT-MRI to obtain an overall reconstruction of fibrosis deposition, vascular tree, and fiber distribution of the heart, and they are used to model preferential action potential propagation (APP) paths across the organ^3,4. These strategies can provide a beautiful overview of the heart organization. However, their spatial resolution is insufficient to investigate the impact of structural remodeling on cardiac function at the cellular level.

Placing this framework at a different order of magnitude, where single cells can play individual roles on action potential propagation, is challenging. The main limitation is the inefficiency of standard imaging methods to perform high-resolution imaging (micrometric resolution) in massive (centimeter-sized) tissues. In fact, imaging biological tissues in 3D at high resolution is very complicated due to tissue opaqueness. The most common approach to perform 3D reconstructions in entire organs is to prepare thin sections. However, precise sectioning, assembling, and imaging require significant effort and time. An alternative approach that does not demand cutting the sample is to generate a transparent tissue. During the last years, several methodologies for clarifying tissues have been proposed^5,6,7,8⁠. The challenge to produce massive, transparent, and fluorescently-labeled tissues has been recently achieved by developing true tissue transformation approaches (CLARITY⁹⁠, SHIELD¹⁰⁠). In particular, the CLARITY method is based on the transformation of an intact tissue into a nanoporous, hydrogel-hybridized, lipid-free form that enables to confer high transparency by the selective removal of membrane lipid bilayers. Notably, this method has been found successful also in cardiac preparation^11,12,13,14⁠. However, since the heart is too fragile to be suitable for an active clearing, it must be cleared using the passive approach, which requires a long time to confer complete transparency.

In combination with advanced imaging techniques like light-sheet microscopy, CLARITY has the potential to image 3D massive heart tissues at micrometric resolution. In light-sheet microscopy, the illumination of the sample is performed with a thin sheet of light confined in the focal plane of the detection objective. The fluorescence emission is collected along an axis perpendicular to the illumination plane¹⁵. The detection architecture is similar to widefield microscopy, making the acquisition much faster than laser scanning microscopes. Moving the sample through the light sheet permits obtaining a complete tomography of big specimens, up to centimeter-sized samples. However, due to the intrinsic properties of the Gaussian beam, it is possible to obtain a very thin (of the order of a few microns) light-sheet only for a limited spatial extension, thus drastically limiting the field of view (FoV). Recently, a novel excitation scheme has been introduced to overcome this limitation and applied for brain imaging, allowing 3d reconstructions with isotropic resolution¹⁶.

In this paper, a passive clearing approach is presented, enabling a significant reduction of the clearing timing needed by the CLARITY protocol. The methodological framework described here allows reconstructing a whole mouse heart with micrometric resolution in a single tomographic scan with an acquisition time in the order of minutes.

Protocol

All animal handling and procedures were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and conformed to the principles and regulations of the Italian Ministry of Health. The experimental protocol was approved by the Italian Ministry of Health (protocol number 647/2015-PR). All the animals were provided by ENVIGO, Italy. For these experiments, 5 male C57BL/6J mice of 6 months of age were used.

1. Solution preparation

1. Prepare 4% Paraformaldehyde (PFA) in Phosphate-Buffered Saline (PBS) (pH 7.6) in a chemical hood. Store the 4% PFA aliquots at -20 °C for several months.
2. Prepare Hydrogel solution: Mix 4% Acrylamide, 0.05% Bis-acrylamide, 0.25% Initiatior AV-044 in 0.01 M PBS in a chemical hood. Keep the reagents and the solution on ice during the entire preparation. Store the hydrogel aliquots at -20 °C for several months.
3. Prepare Clearing solution: Mix 200 mM Boric acid, 4% Sodium Dodecyl-Sulfate (SDS) in deionized water; pH 8.6 in a chemical hood. Store the solution between 21-37 °C to avoid SDS precipitation.
4. Prepare fresh Tyrode solution on the day of the experiment: Add 10 mM Glucose, 10 mM HEPES, 113 mM NaCl, 1.2 mM MgCl₂, and 4.7 mM KCl; titrate to pH 7.4 using 1 M NaOH.

2. Heart isolation

1. Inject 0.1 mL of 500 I.U. Heparin subcutaneously 30 min before the heart isolation procedure.
2. Fill a 30-mL syringe and three 6-cm Petri dishes with fresh Tyrode solution. Make a small rift (3-4 mm in depth) on the border of one of the Petri dishes and place it under a stereoscopic microscope.
3. Fix a 1 mm-diameter cannula to the syringe and insert it in the rift of the Petri dish. Make sure there are no air bubbles in the syringe.
4. Fill a 20-mL syringe with 4% PFA and keep it in the chemical hood. Prepare an empty Petri dish under the hood.
5. Anesthetize the mouse with 3% Isoflurane/oxygen at a flow rate of 1.0 L/min and sacrifice it by cervical dislocation according to animal welfare rules in force.
6. After the sacrifice, remove the fur over the chest and open the chest to have full access to the heart.
7. Isolate the heart, immerse it in the Petri dish previously filled with 50 mL of Tyrode Solution. Use surgical scissors to cut the aorta immediately near the aortic arch to have the heart exposed.
8. Transfer the heart under a stereoscopic microscope and carefully perform the cannulation. Do not insert the cannula too deep into the aorta (no more than 2 mm) to avoid tissue damage.
9. Use a little clamp and a suture (size 5/0) to fix the heart to the cannula.
10. Perfuse the heart with 30 mL of the Tyrode solution with a constant pressure of 10 mL/min to remove blood from the vessels.
11. Detach the cannula from the syringe and place the heart in the Petri dish filled with Tyrode solution. Be careful not to have air bubbles in the cannula; otherwise, remove the air bubbles properly.
12. Attach the 20-mL syringe filled with cold 4% PFA to the cannula and perfuse the heart at the same constant pressure.
13. Incubate the heart in 10 mL of 4% PFA at 4 °C overnight (O/N). To avoid tissue degradation, perform steps 2.6-2.13 in the shortest time possible.

3. Heart clearing

1. The following day, wash the heart in 0.01 M PBS 3 times at 4 °C for 15 min.
   NOTE: After this step, the heart can be stored in PBS + 0.01% sodium azide (NaN3) at 4 °C for several months.
2. Incubate the heart in 30 mL of Hydrogel solution in shaking (15 rpm) at 4 °C for 3 days.
3. Degas the sample at room temperature using a dryer, a vacuum pump, and a tube system that connects the dryer to both the pump and a nitrogen pipeline.
   1. Place the sample in the dryer and open the vial, keeping the cap on it.
   2. Close the dryer and remove the oxygen from the tube by opening the nitrogen pipeline.
   3. Turn on the vacuum pump to remove the oxygen from the dryer for 10 min.
   4. Turn off the pump and use the knob of the dryer to open the nitrogen pipeline. Once the pressure is equal to the atmospheric pressure, carefully open the dryer and quickly close the vial.
4. Keep the heart in the degassed Hydrogel solution at 37 °C for 3 h at rest.
5. When the Hydrogel is properly polymerized and appears entirely gelatinous, carefully remove the heart from it and place it in the sample holder.
6. Insert the sample holder with the heart in one of the clearing chambers and close it properly to avoid leaks of the clearing solution.
7. Switch on the water bath where the clearing solution container is placed and the peristaltic pump to start the recirculation of the clearing solution.
8. Change the clearing solution in the container once a week to speed up the clarification procedure.

4. Cellular membrane staining

1. Once the heart appears completely clarified, remove it from the sample holder and wash it in 50 mL of warmed-up PBS for 24 h. Wash again in 50 mL of PBS + 1% of Triton-X (PBS-T 1x) for 24 h.
2. Incubate the sample in 0.01 mg/mL Wheat Germ Agglutinin (WGA) - Alexa Fluor 633 in 3 mL of PBS-T 1x in shaking (50 rpm) at room temperature for 7 days.
3. After the 7-day incubation, wash the sample in 50 mL of PBS-T 1x at room temperature in shaking for 24 h.
4. Incubate the sample in 4% PFA for 15 min and then wash it 3 times in PBS for 5 min each.
   NOTE: After this step, the heart can be stored in PBS + 0.01% NaN3 at 4 °C for several months.
5. Incubate the heart in increasing concentrations of 2,2′-Thiodiethanol (TDE) in 0.01 M PBS (20% and 47% TDE/PBS) for 8 h each, up to the final concentration of 68% TDE in 0.01 M PBS to provide the required refractive index (RI = 1.46). This is the RI matching medium (RI-medium) to acquire images¹⁶⁠.

5. Heart mounting and acquisition

NOTE: All the components of the optical system are listed in detail in the Table of Materials.

1. Gently fill about 80% of the external cuvette (quartz, 45 mm × 45 mm × 42.5 mm) with the RI-medium.
   NOTE: Here, it is possible to use different non-volatile solutions that guarantee a RI of 1.46.
2. Gently fill the internal cuvette (quartz, 45 mm × 12.5 mm × 12.5 mm) with the same RI-medium.
3. Immerse the sample inside the internal cuvette. The sample incubations described above allow the sample to remain stable inside the RI-medium without being held.
4. Gently move the sample to the bottom of the cuvette using thin tweezers and arrange the heart with its longitudinal axis parallel to the cuvette's main axis to minimize the excitation light path across the tissue during the scanning.
5. Gently fix the tailored plug above the internal cuvette with two screws.
6. Mount the sample to the microscope stage using the magnets.
7. Translate the vertical sample stage manually to immerse the internal cuvette into the external one.
8. Turn on the excitation light source (wavelength of 638 nm), setting a low power (in the order of 3 mW).
9. Move the sample using the motorized translator to illuminate an inner plane of the tissue.
10. Turn on the imaging software (HCImageLive) and set the camera Trigger on External (light-sheet) mode to drive the acquisition trigger of the camera by the custom software controlling the entire setup.
11. Enable Autosave in the Scan Settings panel and set the output folder where the images need to be saved.
12. Manually adjust the sample position in the XY plane with the linear translators to move the sample to the center of the FoV of the camera sensor.
13. Move the sample along the Z-axis using the linear motorized translator to identify heart borders for tomographic reconstruction.
14. Increase the laser power to ~20 mW, ready for the imaging session.
15. Start the tomographic acquisition, click the Start button in the Sequence Panel of the imaging software, and at the same time move the sample along the Z-axis at the constant velocity of 6 µm/s using the motorized translator.

Results

The developed passive clearing setup allows to obtain a cleared adult mouse heart (with a dimension of the order 10 mm x 6 mm x 6 mm) in about 3 months. All the components of the setup are mounted as shown in Figure 1. The negligible temperature gradient between each clearing chamber (of the order of 3°C) allows maintaining the temperature in a proper range across all chambers.

Discussion

In this work, a successful approach to clear, stain, and image a whole mouse heart at high resolution was introduced. First, a tissue transformation protocol (CLARITY) was optimized and performed, slightly modified for its application on the cardiac tissue. Indeed, to obtain an efficient reconstruction in 3D of a whole heart, it is essential to prevent the phenomenon of light scattering. The CLARITY methodology allows us to obtain a highly transparent intact heart, but it requires long incubation times when performed pas...

Materials

Name	Company	Catalog Number	Comments
2-2’ Thiodiethanol	Sigma-Aldrich	166782
Acrylamide	Bio-Rad	61-0140
AV-044 Initiator	Wako Chemicals	AVP5874
Bis-Acrylamide	Bio-Rad	161-042
Boric Acid	Sigma-Aldrich	B7901
Camera	Hamamatsu	Orca flash 4.0 v3
Camera software	Hamamatsu	HC Image
Collimating lens	Thorlabs	AC254-050-A-ML
Detection arm	Integrated optics	0638L-15A-NI-PT-NF
Excitation lens	Nikon	91863
Exteraìnal quartz cuvette	Portmann Instruments	UQ-753
Fold mirrors	Thorlabs	BBE1-E02
Galvanometric mirror	Thorlabs	GVS211/M
Glucose	Sigma-Aldrich	G8270
HCImage Live	Hamamatsu	4.6.1.19
HEPES	Sigma-Aldrich	H3375
Internal quartz cuvette	Portmann Instruments	UQ-204
KCl	Sigma-Aldrich	P4504
Laser source	Integrated Optics	0638L-15A-NI-PT-NF
Long-pass filter	Thorlabs	FELH0650
Magnetic base	Thorlabs	KB25/M
MgCl2	Chem-Lab	CI-1316-0250
Motorized traslator	Physisk Instrument	M-122.2DD
NaCl	Sigma-Aldrich	59888
Objective	Thorlabs	TL2X-SAP
Paraformaldehyde	Agar Scientific	R1018
Phosphate Buffer Solution	Sigma-Aldrich	P4417
Polycap AS	Whatman	2606T
Relay lens	Qioptiq	G063200000
Sodium Dodecyl Sulfate	Sigma-Aldrich	L3771
Tube lens	Thorlabs	ACT508-200-A-ML
Tunable lens	Optotune	EL-16-40-TC-VIS-5D-1-C
Vacuum pump	KNF Neuberger Inc	N86KT.18
Water bath	Memmert	WTB