This methodology combines improvement of clearing protocol with the eye capability of optical sectioning of the Mesos PIM technology allowing us to perform a meso scopic reconstructions at micrometric resolution. It provides the possibility to image massive cardiac tissues or entire mouse hearts entire solution in a single scan without losing the original three-dimensional tissue organization. After the heart has been cannulated by proximal aorta washed in tyro solution and fixed in 4%Paraform aldehyde in 0.01 molar PBS it is ready to start the clearing protocol.
Wash the heart three times in 0.01 molar PBS at four degrees Celsius for 15 minutes. After this step, the heart can be stored in PBS and 0.01%sodium azide at four degrees Celsius for several months. Incubate the heart in 20 milliliters of hydrogel solution in shaking condition at 15 RPM for three days at four degrees Celsius.
De-gas the sample at room temperature using a dryer a vacuum pump, and a tube system that connects the dryer to both the pump band and nitrogen pipeline. Place the sample in the dryer and open the vial keeping the cap on it. Close the dryer and remove the oxygen from the tube by opening the nitrogen pipeline.
Turn on the vacuum pump to remove the oxygen from the dryer for 10 minutes. Turn off the pump and open the knob of the dryer to the nitrogen pipeline. Then open the nitrogen tap.
Once the pressure is equal to the atmospheric pressure carefully open the dryer and quickly close the vial. Keep the heart in the De-gased hydrogel solution at 37 degrees Celsius for about three hours at rest. When the hydrogel is properly polymerized and appears entirely gelatinous, carefully remove the heart from it and place it in a clearing solution in the vial with clearing solution.
Change the clearing solution in the vial once every three days to speed up the clarification procedure. Once the heart appears completely clarified remove it from the vial and wash it in 50 milliliters of warmed up PBS for 24 hours in shaking condition. Wash again in 50 milliliters of one X PBS T for 24 hours in shaking condition.
Incubate the sample in 0.01 milligrams per milliliters wheat germ aglutinin Alexa floor 633 in three milliliters of one X PBS T in shaking condition at 50 RPM at room temperature for seven days. After the seven day incubation, wash the sample in 50 milliliters of one X PBS T at room temperature in shaking for 24 hours. Incubate the sample in 4%PFA for 15 minutes and then wash it three times in 0.01 molar PBS for five minutes each.
Incubate the heart successively in 20 and 47%TDE in 0.01 molar PBS for eight hours each. And then finally at 68%TDE in 0.01 molar PBS to provide the required refractive index of 1.46. Gently fill about 80%of the external quartz quvet with the refractive index medium.
Fill the internal quartz quvet with the same refractive index medium. Immerse the sample inside the internal quvet. Gently move the sample to the bottom of the quvet using thin tweezers and arrange the heart with its longitudinal axis parallel to the quvet's main axis to minimize the excitation light path across the tissue during the scanning.
Carefully fix the tailored plug above the internal quvet with two screws. Mount the sample to the microscope stage using magnets. Translate the vertical sample stage manually to immerse the internal quvet into the external one.
Turn on the excitation light source with a wavelength of 638 nanometers and set it at low power in the order of three milliwatts. Move the sample using the motorized translator to illuminate an inner plate of the tissue. Turn on the HC image live camera software and set the camera trigger on external edge trigger light sheet mode to drive the acquisition trigger of the camera by the custom software controlling the entire setup.
Enable auto save in the camera software and set the output folder where the images need to be saved. Manually adjust the sample position in the X Y plane with the linear translators to move the sample to the center of the field of view of the camera sensor. Move the sample along the Z axis using the linear motorized translator to identify heart borders for tomographic reconstruction.
Increase the laser power to approximately 20 milliwatts before beginning for the imaging session. Start the tomographic acquisition by clicking the start button in the capture panel of the imaging software, and at the same time start moving the sample along the Z-axis at the constant velocity of six micrometers per second using the motorized translator. The combination of the clarity methodology with TDE as refractive index medium does not significantly change the sample's final volume nor leads to an isotropic deformation of the specimen.
The excitation optics produced a light sheet with a minimum waste of about six micrometers that diverged up to 175 micrometers at the edge of the field of view. The synchronization of the camera rolling shutter with the axial scan of the light sheet waste ensured to collect the emission signal only in the sample portion excited with the waste of the light sheet resulting in an average F W H M of about 6.7 micrometers along the entire field of view. The Z point spread function of the microscope was also estimated by a tomographic reconstruction of the fluorescent nanosphere and an F W H M of 6.4 micrometers was estimated by the fit.
The potentiality of the system to resolve single cellular membranes in three dimensions with a sufficient signal to noise ratio in the entire organ was also confirmed. The de-gassing procedure is the most critical step of the protocol. If it is not properly performed the tissue could encounter damages indicating during the incubation in a cleaning solution.
The presented protocol can be combined with a multi staining protocol to achieve a whole organ reconstruction integrating different biological structures and can be applied to study pathological models.
