Light field microscopy is a new technique for high-speed volumetric imaging of weakly scattering or fluorescent specimens It employs an array of microlenses to trade off spatial resolution against angular resolution, thereby allowing a 4-D light field to be captured using a single photographic exposure without the need for scanning The recorded light field can then be used to computationally reconstruct a full volume In this paper, we present an optical model for light field microscopy based on wave optics, instead of previously reported ray optics models We also present a 3-D deconvolution method for light field microscopy that is able to reconstruct volumes at higher spatial resolution, and with better optical sectioning, than previously reported To accomplish this, we take advantage of the dense spatio-angular sampling provided by a microlens array at axial positions away from the native object plane This dense sampling permits us to decode aliasing present in the light field to reconstruct high-frequency information We formulate our method as an inverse problem for reconstructing the 3-D volume, which we solve using a GPU-accelerated iterative algorithm Theoretical limits on the depth-dependent lateral resolution of the reconstructed volumes are derived We show that these limits are in good agreement with experimental results on a standard USAF 1951 resolution target Finally, we present 3-D reconstructions of pollen grains that demonstrate the improvements in fidelity made possible by our method

Wave optics theory and 3-D deconvolution for the light field microscope

We demonstrate that a deep neural network can significantly improve optical microscopy, enhancing its spatial resolution over a large field-of-view and depth-of-field. After its training, the only input to this network is an image acquired using a regular optical microscope, without any changes to its design. We blindly tested this deep learning approach using various tissue samples that are imaged with low-resolution and wide-field systems, where the network rapidly outputs an image with remarkably better resolution, matching the performance of higher numerical aperture lenses, also significantly surpassing their limited field-of-view and depth-of-field. These results are transformative for various fields that use microscopy tools, including e.g., life sciences, where optical microscopy is considered as one of the most widely used and deployed techniques. Beyond such applications, our presented approach is broadly applicable to other imaging modalities, also spanning different parts of the electromagnetic spectrum, and can be used to design computational imagers that get better and better as they continue to image specimen and establish new transformations among different modes of imaging.

Deep Learning Microscopy

Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings.

Wide-field computational imaging of pathology slides using lens-free on-chip microscopy

Lenses with tunable focal length play important roles in nature by helping species avoid predators and capture prey. Many practical devices mimic lens concept for imaging, sensing, and detection. This review covers fundamental optics of lenses and its extension to lenses made of liquid crystals (LCs). Three main types of LC lenses are described, namely, lenses with curved surfaces, flat gradient-index lenses and composite lenses. The review discusses advantages of LC lenses over their isotropic counterparts, challenges in their fabrication and control, as well as a variety of potential applications. We also discuss the current challenges associated with nematic LC lenses and their solutions. LC lenses are already having significant impacts on optics and optometry, and these impacts will grow with discovering new LC materials and new lens designs.

Liquid crystal lenses with tunable focal length

Full-color, three-dimensional images of objects under incoherent illumination are obtained by a digital holography technique. Based on self-interference of two beam-split copies of the object’s optical field with differential curvatures, the apparatus consists of a beam-splitter, a few mirrors and lenses, a piezo-actuator, and a color camera. No lasers or other special illuminations are used for recording or reconstruction. Color holographic images of daylight-illuminated outdoor scenes and a halogen lamp-illuminated toy figure are obtained. From a recorded hologram, images can be calculated, or numerically focused, at any distances for viewing.

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Full color natural light holographic camera

Fresnel Incoherent Correlation Holography (FINCH) enables holograms to be recorded from incoherent light with just a digital camera and spatial light modulator. We previously described its application to general three dimensional incoherent imaging and specifically to fluorescence microscopy, wherein one complex hologram contains the three dimensional information in the field of view, obviating the need for scanning or serial sectioning. We have now further analyzed FINCH in view of linear system theory and in comparison to conventional coherent and incoherent two dimensional imaging systems. We demonstrate, theoretically and experimentally, improved resolution by FINCH, when compared to conventional imaging.

Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging.

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

We report a new optical arrangement that creates high-efficiency, high-quality Fresnel incoherent correlation holography (FINCH) holograms using polarization sensitive transmission liquid crystal gradient index (TLCGRIN) diffractive lenses. In contrast, current universal practice in the field employs a reflective spatial light modulator (SLM) to separate sample and reference beams. Polarization sensitive TLCGRIN lenses enable a straight optical path, have >90% transmission efficiency, are not pixilated, and are free of many limitations of reflective SLM devices. For each sample point, two spherical beams created by a glass lens in combination with a polarization sensitive TLCGRIN lens interfere and create a hologram and resultant super resolution image.

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In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens

The use of birefringent crystals improves super-resolution Fresnel incoherent correlation holography (FINCH) microscopy.

High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers.

Fresnel Incoherent Correlation Holography (FINCH) can faithfully reproduce objects above and below the optical plane of focus. However, as in optical imaging, the transverse magnification and optimal reconstruction depth changes based on the longitudinal distance of objects from the focal plane of the input lens with the exception that objects above and below the focal plane are in focus with FINCH and out of focus by standard optical imaging. We have analyzed these effects both theoretically and experimentally for two configurations of a FINCH fluorescence microscopy system. This information has been used to reconstruct a test planar object placed above or below the optical plane of focus with high dimensional and image fidelity. Because FINCH is inherently a super-resolving system, this advance makes it possible to create super-resolved 3D images from FINCH holograms.

Nisan Siegel

Papers

Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens

High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers.

Reconstruction of objects above and below the objective focal plane with dimensional fidelity by FINCH fluorescence microscopy