Multiphoton microscopy of fixed tissue is normally limited to shallow penetration depths of just a few hundred microns. We recently demonstrated the first use of multiphoton microscopy as deep as several millimeters into fixed mouse organs using optical clearing with the solution of benzo alcohol and benzoyl benzoate. Our initial demonstration, the technique imaged intrinsic tissue fluorescence.
However, there is increasing interest in using optical clearing methods with tissue expressing fluorescent proteins such as GFP. Here we present a protocol for multiphoton microscopy of cleared brain tissue with the same benzo alcohol solution that preserves the fluorescence of yellow fluorescent protein expressed by the thigh. One promoter in mouse brain High resolution whole mouse organ imaging using multiphoton microscopy is made possible by optically clearing the organ before imaging without clearing.Multiphoton.
Imaging of the brain is limited to 300 microns beneath the tissue surface due to highlights scattering effects created by the differences in refractive indices of water and the proteins that make up the brain tissue. To overcome this limitation, the organ is dehydrated and the water is replaced with a fluid that has a similar refractive index as the proteins that make up the tissue. This process of optical clearing helps to greatly reduce light scattering to offer better imaging farther beneath the tissue surface.
Images from Batta et al demonstrate how this technique can be applied to view the histology of different mouse organs using intrinsic fluorescence and second harmonic generation. This first image is of the mouse testicle, taken 1.4 millimeters beneath the tissue surface. The high resolution property of multiphoton microscopy allows us to zoom in and get a clear look at the individual sperm cells forming in the seminiferous tubulars.
As seen on the far right here, we show an image of the mouse lung, which was also taken 1.4 millimeters beneath the tissue surface. This image is a demonstration of two channel multiphoton imaging in which the elastin components are labeled in red while the collagen is labeled in green, zooming in individual oli are easily distinguishable. Finally, we show high resolution images of the inner regions of the mouse brain taking 850 microns beneath the tissue surface By zooming in on the neocortex and hippocampus of the brain, individual astrocytes and neuron cell bodies can clearly be seen.
However, when optically cleared organs that can contain fluorescent proteins such as YFP, the optical clearing protocol of badra at all does not preserve the fluorescent signal of these fluorescent proteins. The protocol presented here is a novel way in which to perform whole organ optical clearing and imaging of mouse sprain while preserving the fluorescent signal of YFP and neurons dehydrating the brain using an ethanol graded series and clearing with a one to two solution of benzo alcohol benzoate, otherwise known as BAB has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal. For multiphoton imaging, YFP mice are first weighed and then anesthetized with an intraperitoneal injection of ketamine xylazine.
A surgical plate of anesthesia must be confirmed before proceeding to surgery. The animal should be checked every five minutes to see if it reacts to a firm toe or tail pinch. If the animal reacts a S supplemental dose of ketamine xylazine is required.
Once anesthetized, the mouses are staying by adhering each limb to a surgical bed using lab tape so that the mouse is in a supine position exposing his chest for surgery To begin, an incision is made below the xiphoid process in order to make a cut along the base of the rib cage using scissors and tweezers to pull back the skin as the cut is being made, two cuts are then made up either side of the mouse sternum to create a flap of tissue that is held away from the chest cavity using a hemostat to leave the heart exposed. Next, a 23 gauge needle is inserted into the left ventricle of the heart, and a small incision is made in the muscle wall of the right atrium to allow blood to escape. Immediately after the right atrium is cut, a perfusion with four degrees Celsius phosphate buffered saline is begun until no more blood is draining from the right atrium.
During the perfusion, a epistolic pump is used and set to a pumping power that ejects fluid 1.5 to two inches away from the tip of the needle. Once all the blood has been drained, the perfusion medium is switched to a 4%paraform aldehyde solution at four degrees Celsius until the mouse's body becomes noticeably rigid and stiff. After perfusion, the mouse is removed from the surgical bed and decapitated to begin the excision of the brain using forceps and iris scissors.
The skull is removed in small sections starting from the back of the skull and moving forward. Small cuts are made every two to four millimeters with the scissors across the skull while the forceps are used to carefully pull the bone away from the brain in small sections, this is done until the entire top surface of the brain is exposed. The brain is then excised from the skull using a surgical spatula and placed to take glass vial of 4%paraform aldehyde to fix for six hours.
While we are primarily focusing on the imaging of mouse brain expressing YFP for this demonstration, we will also be optically clearing a mouse, hind leg and section a small intestine to best show the clearing capabilities of this procedure. After post fixation, the brain and other tissues are washed twice in PBS. The tissue samples are then dehydrated at room temperature by a series of ethanol incubations at concentrations of 50%70%90%and 100%ethanol.
Each incubation lasts two hours and then a second. 100%ethanol incubation of 12 hours is conducted to efficiently extract the water from the fixed tissue. After dehydration, the last 100%ethanol solution is poured out and the tissue samples are submerged in a one-to-one solution of ethanol to BAB for two hours before being immersed in 100%BAB clearing solution.
Once in bab, the brain and other tissue samples will become noticeably transparent within four to five hours. Here we show a time-lapse video of the first six hours of the optical clearing process. The skip marks the time when the one-to-one solution of ethanol to BAB was replaced with 100%BAB solution.
At the end of six hours, all organs show sign of transparency, for example, the bone in the hind leg of the mouse is now clearly visible for best clearing results. The brain should be left to clear for six days at room temperature while shielded from bright light. Once the brain is cleared and ready for imaging, it is affixed to the bottom of your Petri dish using Sano acrylic or super glue.
After the glue dries, the brain is emerged in BAB and the Petri dish is placed under the objective for imaging. For imaging, we use a multiphoton microscope that incorporates a MI tie titanium sapphire laser adjustable between a 710 to 990 nanometer excitation wavelength. The excitation wavelength we use to generate YFP signals is 886 nanometers.
The reflective fluorescent signal is then captured using a NIK icon five x objective that allows for large field of view imaging. The reflective fluorescent signal is filtered using a 5 35 50 band pass filter and collected using a high quantum efficiency folder. Multiplier two from OMA Matsu images are processed using scan image software at a resolution of 2048 by 2048 pixels using a scan rate of two milliseconds per line to generate high resolution YFP images.
Once imaging is complete, the brain is removed from the P two dish and stored in BAB and shielded from light for future imaging. The representative images and videos shown here demonstrate the high resolution multi photon imaging capability made possible by optical clearing. Whole brain imaging allows for YFP label neurons in the different layers of the hippocampus and neocortex to be clearly visible as deep as two millimeters beneath the tissue surface.
Here we show a video of a 1.2 millimeter image stack of whole mouth sprain, taken 0.8 to two millimeters into the brain to reveal the different anatomical layers of the hippocampus. The following is a representative image of a coronal section 1.94 millimeters called to Bergman taken from the two millimeter deep image stuck in which the different layers of the hippocampus have been labeled. By zooming in on the neocortex, the individual axons and neuron cell bodies of layer five peral neurons of the neocortex are clearly distinguishable up to 1.02 millimeters beneath the tissue surface.
Here we show an image stuck of layer five neurons taken between 700 to 1020 microns beneath the tissue surface. The following is a representative image from that stack taken 774 microns into the brain. Using this same image stack, A 3D reconstruction of the neuron region was made using Image J software.
The high resolution capability of multi bot microscopy also allows us to present images of whole individual neurons. Here we show a reconstruction of a layer five neuron of the neocortex in which D genetic processes are clearly visible. This concludes our presentation of optical clearing, whole mouse sprain expressing YFP.
Performing this technique is relatively simple and as we have shown, can be used to view and image many different regions and structures of the mouse sprain. Thank you for watching and good luck.
