In vivo Fluorescence Imaging with Lensless Microscopes
Adams, Jesse Kenneth
Robinson, Jacob T; Veeraraghavan, Ashok
Doctor of Philosophy
Fluorescence microscopy is an essential tool for studying the brain. Not only can it provide sub-cellular information about brain structure, but it can also capture dynamic electrical and chemical activity from calcium- and voltage-sensitive indicators. An ideal fluorescence microscope would simultaneously image all the neurons in an animal with the temporal resolution to identify individual action potentials and would not restrict animal behavior. Unfortunately, lenses in traditional microscopes enforce a trade-off between size and weight, resolution, and field-of-view. It is not currently possible to simultaneously achieve cellular resolution, high frame rate, and large fields of view, with a small and lightweight microscope . Recent developments in computational imaging have made it possible to reconstruct images without the use of lenses , thus overcoming many constraints of traditional microscopy. Here we show the first demonstration of a lensless microscope that can perform structural and functional imaging of biological samples in vivo. Specifically, by replacing lenses with an optimized phase mask and computational image reconstruction algorithms we achieve cellular-resolution fluorescence imaging of fixed biological samples . We also demonstrate 2D and 3D in vivo imaging of neurons and muscle cells in millimeter-sized Hydra vulgaris, including measurement of dynamic calcium activity . Finally, we reconstruct stimulus-evoked calcium activity from neurons in mouse cortex . Further miniaturization of this lensless microscope by reducing the size of the electronic packaging will enable flat, and potentially fully implantable devices that can study neural activity over large areas of the brain as animals behave freely. We also anticipate that this new imaging capability can be used in other areas including endoscopy and point-of-care diagnostics, where the small form factor, large field of view, and high temporal resolution will provide advantages compared to current lens-based microscopes.