Application of multiphoton microscopy in dermatological studies: A mini-review

This review summarizes the historical and more recent developments of multiphoton microscopy,as applied to dermatology.Multiphoton microscopy offers several advantages over competingmicroscopy techniques:there is an inherent axial sectioning,penetration depths that competewll with confocal microscopy on account of the use of near-infrared light,and many t wo-pho ton contrast mechanisms,such as second-harmonic generation,have no analogue in one-photonmicroscopy.While the penetration depths of photons into tissue are typically limited on the orderof hundreds of microns,this is of les concern in dermatology,as the skin is thin and readlily.accessible.As a result,multiphoton microscopy in dermatology has generated a great deal ofmterest,much of which is summarized here.The review covers the interaction of light and tissue,as well as the various considerations that must be made when designing an instrument.The stateof multiphoton microscopy in imaging skin cancer and various other diseases is also discussed,along with the investigation of aging and regeneration phenomena,and finally,the use of mul-tiphoton microscopy to analyze the transdermal transport of drugs,cosmetics and other agents issummarized.The review concludes with a look at potential future research directions,especiallythose that are necessary to push these techniques into widespread clinical acceptance.

DEEP-squared:deep learning powered De-scattering with Excitation Patterning

Limited throughput is a key challenge in in vivo deep tissue imaging using nonlinear optical microscopy.Point scanning multiphoton microscopy,the current gold standard,is slow especially compared to the widefield imaging modalities used for optically cleared or thin specimens.We recently introduced“De-scattering with Excitation Patterning”or“DEEP”as a widefield alternative to point-scanning geometries.Using patterned multiphoton excitation,DEEP encodes spatial information inside tissue before scattering.However,to de-scatter at typical depths,hundreds of such patterned excitations were needed.In this work,we present DEEP2,a deep learning-based model that can de-scatter images from just tens of patterned excitations instead of hundreds.Consequently,we improve DEEP’s throughput by almost an order of magnitude.We demonstrate our method in multiple numerical and experimental imaging studies,including in vivo cortical vasculature imaging up to 4 scattering lengths deep in live mice.

Mapping nanoscale topographic features in thick tissues with speckle diffraction tomography

Resolving three-dimensional morphological features in thick specimens remains a significant challenge for label-free imaging.We report a new speckle diffraction tomography(SDT)approach that can image thick biological specimens with~500 nm lateral resolution and~1μm axial resolution in a reflection geometry.In SDT,multiple-scattering background is rejected through spatiotemporal gating provided by dynamic speckle-field interferometry,while depth-resolved refractive index maps are reconstructed by developing a comprehensive inverse-scattering model that also considers specimen-induced aberrations.Benefiting from the high-resolution and full-field quantitative imaging capabilities of SDT,we successfully imaged red blood cells and quantified their membrane fluctuations behind a turbid medium with a thickness of 2.8 scattering mean-free paths.Most importantly,we performed volumetric imaging of cornea inside an ex vivo rat eye and quantified its optical properties,including the mapping of nanoscale topographic features of Dua’s and Descemet’s membranes that had not been previously visualized.

High spatial and temporal resolution synthetic aperture phase microscopy

A new optical microscopy technique,termed high spatial and temporal resolution synthetic aperture phase microscopy(HISTR-SAPM),is proposed to improve the lateral resolution of wide-field coherent imaging.Under plane wave illumination,the resolution is increased by twofold to around 260 nm,while achieving millisecond-level temporal resolution.In HISTR-SAPM,digital micromirror devices are used to actively change the sample illumination beam angle at high speed with high stability.An off-axis interferometer is used to measure the sample scattered complex fields,which are then processed to reconstruct high-resolution phase images.Using HISTR-SAPM,we are able to map the height profiles of subwavelength photonic structures and resolve the period structures that have 198 nm linewidth and 132 nm gap(i.e.,a full pitch of 330 nm).As the reconstruction averages out laser speckle noise while maintaining high temporal resolution,HISTR-SAPM further enables imaging and quantification of nanoscale dynamics of live cells,such as red blood cell membrane fluctuations and subcellular structure dynamics within nucleated cells.We envision that HISTR-SAPM will broadly benefit research in material science and biology.