Shaping light for volumetric microscope imaging in the heart

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Recent advances in light sheet fluorescence microscopy have allowed biomedical researchers to watch and study living animals such as the zebrafish as they grow from a single cell to a fully functioning organism. However obtaining continuous 3D images presents a particular challenge in the heart (since it is constantly beating) and images become clouded and blurred as the animal grows larger and it is necessary to image through increasing amounts of overlying tissue to see the organ of interest.We will acquire microscope images using specific new illumination and imaging techniques we will develop, to allow us to obtain higher quality images than previously possible inside living tissue. In their raw form these images will not resemble conventional images, but with the help of the powerful imaging processing capabilities of modern computers, we will be able to analyze and combine the raw images to recover better images than would have otherwise been possible with conventional microscope imaging.Specifically, we will research and implement three techniques:1. Speckle light sheet imaging. Here instead of illuminating our sample with uniform light we will illuminate it with a random speckle field. Our raw images will therefore appear "dappled" and unclear, but following computer image processing the resultant images will be much sharper and less affected both by shadowing effects and by the overlying tissue that the light has passed through.2. Wavefront coding for focus-invariant synchronized heart imaging. When we take 3D video images of the heart, we have to cope with the fact that the heart is beating faster than we can normally obtain a complete 3D image of it. We overcome this by using image analysis and computer control to synchronize our image acquisition with the heartbeat. However this is particularly difficult because we would usually move the sample around in order to take the 3D image, and this spoils the synchronization. Wavefront coding lets us acquire images that no longer look like a clear image of the heart, but which remain the same as we move the sample, thus allowing us to build a much simpler and cheaper synchronized imaging system.3. Wavefront coding for snapshot volume imaging. Our synchronized imaging technique assumes that the heart is beating regularly, and by definition that will not be the case in many diseased hearts - which biologists are particularly interested in studying. We will overcome this problem by developing a method for extremely fast volume imaging. Normally the imaging speed is limited by how fast we can change the focus of our microscope, but wavefront coding will allow us to do the refocusing on a computer afterwards, thus allowing us to obtain 3D images much faster.These techniques together will offer new and improved methods for microscope imaging to look inside living animals, to help biologists better understand how the heart develops and functions - with the ultimate aim of improving medical treatments for human heart diseases.