Research On Multiphton Imaging Of Osteocytes And Through Skull Imaging

Multiphoton microscopy(MPM)is anindispensibletechnology for imaging biological tissue dueto its noninvasiveness and deep penetration.MPM excited at the 1700-nm window can reduce the scattering from biological tissue and enable deeper imaging.In vivo imaging in animals,such as mouse skull imaging brain imaging,excitation at theconventional 800-nm window cannot be used to image osteocytes orbrain structurewithout craniotomy,due to the highly scattering skull.The craniotomy for brain imaging may induce brain injury and inflammation.Hence,how toimage osteocytesinmouse skull and avoid theadverse effectincurred by craniotomy in brain imagingremains to be solved.Here we demonstratemultiphoton imaging of osteocytes andthrough the skull brain MPM excited at 1700-nm window.Using this technique,we can image osteocytes and brain structures without craniotomy,with a maximumpenetration depth of 750 ?m below the surface of the brain.Our research work specificallyincludes the following:1.Self-referenced axial chromatic dispersion measurement inmultiphoton microscopyand its applicationto simultaneous imaging of two focal planesHere we demonstrate 2-color THG imaging to measure axial chromatic dispersion inmultiphoton microscopy.The self-referenced technology can eliminate measurement error due to mechanical movement of the stage.Harnessing this axial chromatic dispersion,we further demonstrate2-color,2 focal plane THG imaging of osteocytes in an excised mouse skull.This technique canreduce the imaging timeby half.2.Improving mouse operationCatering to the needs of MPM,we demonstrate mouse head-fixeddevice and improvedmouse operation.These operation procedurescan minimize operation time,and boostsuccess rate of head fixing.3.Label-free THG imaging of osteocytes in mouse skull in vivoWe demonstrate 1700-nm third-harmonic-generation(THG)imaging and three-photon-fluorescence(3PF)imagingof osteocytes in vivo.Combining with selective labeled technology,we can observe the “sandwich” structure of osteocytes in their native environment.This also paves the way for through the skull brain imaging.4.Three-photon brain imaging through the mouse skull in vivoBased on the osteocytes imaging outcome,we demonstratethird-harmonic-generation(THG)imaging and three-photon-fluorescence(3PF)imaging of mouse brain,through the intact skull excited at the 1700-nm window.A maximum imaging depth of 750 ?m below the surface of the brain is reached,which clearly reveals the blood vessels without craniotomy.