The speed of data acquisition in angle scan optical diffraction tomography (ODT) and synthetic aperture tomography is ultimately limited by the mechanical scanning of galvo mirror or sample motion across the light sheet. An alternative three-dimensional cell imaging technique that completely obviates the need for scanning optical elements of a microscope has also been developed at the LBRC. In this method, the depth information is acquired by scanning the wavelength of illumination, which was first proposed in the ultrasound regime [1].
Figure 1 shows the interferometric setup, adapted from diffraction phase microscopy, to record the complex amplitude (both amplitude and phase) of the scattered field in the visible wavelengths [2]. A broadband source (Fianium SC-400) is tuned to a specific wavelength through a high-resolution acousto-optic tunable filter (AOTF) operating in the range from 400 to 700 nm. The output of the AOTF is coupled to a single-mode fiber, collimated and used as the input beam to the setup. A Ronchi grating (40 lines/mm, Edmund Optics Inc.) is placed at a plane (IP0) conjugate to the sample plane (SP) in order to generate three beams incident on the sample via a condenser lens (Olympus 60 X, 0.8 NA) at different angles. The three transmitted beams pass through the microscope objective lens (Nikon 60 X, NA = 1.2) and arrive at a second Ronchi grating (140 lines/mm, Edmund Optics Inc.) placed at the imaging plane (IP2) with the grating vector orthogonal to that of the first grating. As shown in Fig. 1, there are nine diffracted orders at the Fourier plane, as opposed to three in the conventional DPM. Three columns correspond to three angles of incidence on the sample while three rows are due to diffraction by the grating in the detection arm. We apply a low-pass filter on the strongest diffracted beam at the center to use it as a reference for the interference. The recorded interferogram provides the information of the sample carried by the three beams. While the three interference patterns (corresponding to incident beams) may not be visibily distinguishable in the raw interferogram, one can isolate the information for each incident angle in the spatial frequency domain, i.e., by taking the Fourier transform of the interference pattern.
By scanning the color of three beams in our setup provides spatial-frequency coverage similar to scanning the angle of illumination along one direction in angle-scan tomography. After completing the mapping, one can obtain the refractive index map through the three-dimensional inverse Fourier transform. This approach also, similar to angle-scan tomography suffers from the missing cone problem, indicating that data in the cone-shaped region near the origin of frequency coordinates is not collected. However, using a priori information of the object such as non-negativity and piecewise-smoothness constraint , we can fill the missing region and improve the quality of refractive index reconstruction. To demonstrate 3-D label-free imaging of hematopoietic stem cells (HSCs), we image the morphology and structure of HSCs using SCOT. Figures 2(a-c) show horizontal cross-sections of the specimen at 2 µm intervals after tomographic reconstruction, clearly demonstrating 3-D imaging capability of the proposed method. These internal structures are not visible in the cumulative phase map shown in the Fig. 2(d). Moreover, Figs. 2(e) and 2(f) show 3-D rendering of the refractive index map at two different angles.