The overall goal of this protocol is to chemically clear the heart of a mouse, with an induced cardiac arrhythmia for a whole organ visualization of the leukocytic cardiac infiltrate by Fluorescence Light Sheet Microscopy. This method can help answer key questions in the field of whole organ research about prosthesis and structures at the cellular resolution level. The main advantage of this technique is that it allows both the quantification and the localisation of inflammation in whole organs.
Giving detailed insides into neurological prosthesis. We first had the idea for this method when we were in urgent need of a visualization tool to identify cellular explanations for cardiac arrhythmia in the depletion module. After exposition of the heart from an eight to 10 week old mouse, use a 21 gauge catheter and penetrate the right ventricle.
Incise the aorta to allow blood drainage and then perfuse with five milliliters of PBS plus EDTA, with a slow constant pressure. Follow the PBS EDTA with five milliliters of 4%paraformaldehyde. At the end of the perfusion, use serrated forceps to gently grasp the fixed heart, and pull the organ slightly upwards to cut all of the tissue connections.
Including the incoming and outgoing arteries and veins. Then, store the heart for four hours in fresh 4%PFA for further fixation. To dehydrate the tissue, incubate the heart in an ascending ethanol series, in black five milliliter polypropylene reaction tubes in the dark, with constant slow rotation on a tube rotator, to prevent air bubble formation.
Then, clear the tissue with a 98%dibenzyl ether incubation with constant rotation overnight. To conduct Light Sheet Fluorescence Microscopy, first place dehydrated agarose blocks into the standard sample holder, to hold the sample in place. Then, transfer the sample onto the sample holder.
Next, transfer the sample holder into an appropriately sized dibenzyl ether filled cuvette. Use the appropriate excitation and emission filter settings, to detect the fluorescence signals of interest. So correct positioning and fixation of the sample is fundamental to minimize shadowing effects during imaging.
Further, a loose specimen can cause moving artifacts, which are hard to illuminate during post-processing. After requiring all of the images, open the appropriate 3D and 4D image processing analysis software, and select surpass. Choose file and open, and select the folder containing the data from the first acquired channel to open the image stacks.
Select edit and add channels, to add the appropriate acquired fluorescent channel data. Next, click edit and select image properties, to correct the x, y and z voxel dimensions. Adjusting the parameters in the newly opened window.
To adjust the fluorescent signals, first open the edit menu and select, show display adjustment. A new window very display all of the open channels. To change the autofluorescence channel to greyscale, select the autofluorescence channel, choose the white display style and click okay.
Then, adjust the view by removing the grid. Then, zoom in and deselect one channel. Then, adjust the black level value to exclude any unwanted background signals that do not represent the sample structures, the signal intensity and the contrast.
Adjust the antibody standing channel in relation to the revised autofluorescence channel signal. Setting the channel mode, fire to set the visualization of the antibody signal to a heat map color lookup table. Then, select the blend mode, to visualize the tissue structures in detail.
To virtually cut open the organ, before the 3D scene export, select the clipping plane icon in the object list. A yellow frame and a white spindle manipulator will appear in the image view. Use the mouse to rotate the spindle at the thinner end, to change the orientation of the clipping plane, and move the thicker middle part of the spindle to select the desired depth of clipping.
Then, uncheck the respective check boxes to hide the frame and manipulator and create the desired snapshots. The acquisition of an entire image stack, composed of two fluorescent channel signals, elicits an artificial 3D rendering, in which the leukocyte distribution is displayed in a heat map view. The strongly inflamed areas of hearts from a Diphtheria toxin treated animal can be perceived by their reddish, whitish appearances.
Particularly in the regions of the cardiac conduction system, such as the atrial ventricular bundle and the Purkinje fibers. In contrast, the hearts of controlled PBS treated animals, do not demonstrate this pattern of inflammation. Once mastered, this technique allows the digital ready analysis of a target organ, from its harvest to its computer assisted rendification in four to five days.
After its development, this technique paved the way for researchers in the field of emmenology, to explore cellular distribution patterns in whole mouse organs. This may age in the analysis and the treatment of a variety of pathophysiological disease prosthesis. After watching this video, you should have a good understanding of how to prepare whole mouse organs for Fluorescence Light Sheet Microscopy and how to generate and process 3D data stacks.
