Abstract
Here, we present a novel microscopic technique for measuring wavelength-dependent three-dimensional (3-D) distributions of the refractive indices (RIs) of microscopic samples in the visible wavelengths. Employing 3-D quantitative phase microscopy techniques with a wavelength-swept source, 3-D RI tomograms were obtained in the range of 450 - 700 nm with a spectral resolution of a few nanometers. The capability of the technique was demonstrated by measuring the hyperspectral 3-D RI tomograms of polystyrene beads, human red blood cells, and hepatocytes. The results demonstrate the potential for label-free molecular specific 3-D tomography of biological samples.
Highlights
Three-dimensional (3-D) molecular imaging has a crucial role in modern biology and medicine
The hyperspectral optical diffraction tomography (HS-ODT) setup consists of two parts: a wavelength-swept source and common-path interferometric microscopy shown in the Fig. 1(a)
The incident angle of the illumination beam is precisely controlled by the dual-axis rotating mirror (GM2, GVS012/M, Thorlabs Inc.) located at the plane conjugated to the sample and projected onto a sample by a condenser lens [PLAPON 60×, air immersion, numerical aperture (NA) = 0.9]
Summary
Three-dimensional (3-D) molecular imaging has a crucial role in modern biology and medicine. Several 3-D molecular imaging techniques exploiting intrinsic contrasts have been introduced, including molecular imaging spectroscopic optical coherence tomography [4] and 3-D coherent anti-stokes Raman scattering microscopy [5] These techniques are limited to specific molecules exhibiting distinct absorption or vibration spectra. Measurements of 3-D RI tomograms of cells at various wavelengths would have much to offer the fields of cell biology and medicine with its non-invasiveness, quantitative imaging capability, as well as molecular information via optical dispersion [29]. We present a novel approach, referred to as hyperspectral optical diffraction tomography (HS-ODT), which measures the 3-D RI tomograms of microscopic samples at multiple wavelengths ranging from 450 to 700 nm with a diffraction-limited spatial resolution. Detailed information on the wavelength sweeping source, common-path interferometric microscopy, and field measurement techniques can be found in the Refss [10, 30,31,32]
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