Abstract
Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light. In thick unlabeled specimens, multiple scattering randomizes the phase of the field and absorption attenuates light that travels long optical paths. These obstacles limit the performance of transmission imaging. To mitigate these challenges, we developed an epi-illumination gradient light interference microscope (epi-GLIM) as a label-free phase imaging modality applicable to bulk or opaque samples. Epi-GLIM enables studying turbid structures that are hundreds of microns thick and otherwise opaque to transmitted light. We demonstrate this approach with a variety of man-made and biological samples that are incompatible with imaging in a transmission geometry: semiconductors wafers, specimens on opaque and birefringent substrates, cells in microplates, and bulk tissues. We demonstrate that the epi-GLIM data can be used to solve the inverse scattering problem and reconstruct the tomography of single cells and model organisms.
Highlights
Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light
Optical coherence tomography (OCT) is an established label-free imaging technique, which provides depth-sectioning via low-coherence interferometry[9]
Exploiting the phase information provided by epi-Gradient light interference microscopy (GLIM), we extracted the nanoscale topography of the wafer across the entire field of view
Summary
As the shear in GLIM renders the transfer function nonradially symmetric, this approach is informative, but cannot be used to perform 3D reconstructions We apply epi-GLIM to larger, more turbid structures that highlight the system’s ability to suppress multiple scattering This suppression is acomplished by combining phase-shifting with white light interferometry. When illuminated under a low numerical aperture, the maximum contrast is typically obtain at the sharp discontinuity between cellular material and surrounding media In this sample, we note that the largest spheroids are relatively flat, meaning, that their transverse size is larger than their thickness. Because we used a full-field imaging system we were able to acquire the whole tomographic series, consisting of 469 slices, before the animal swam away
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