Spatial Light Interference Tomography (SLIT)

Posted on March 5, 2012 by admin

Z. Wang, D. L. Marks, P. S. Carney, L. J. Millet, M. U. Gillett, A. Mihi, P. V. Braun, Z. Shen, S. G. Prasanth and G. Popescu, “Spatial Light Interference Tomography (SLIT)“, Opt. Exp., 19(21), 2011.

Due to the combination of white light illumination, high numerical aperture, and phase-resolved detection, Spatial Light Interference Microscopy (SLIM) has the ability to provide optical sectioning. Thus, in SLIM the out-of-focus blur is suppressed by the micron-range coherence length. In addition to suppressing the speckle effects that generally degrade laser light imaging, SLIM can also provide depth-resolved imaging.  Thus Spatial light interference tomography (SLIT), is a labelfree method for 3D imaging of transparent structures such as live cells. SLIT uses the principle of interferometric imaging with broadband fields and combines the optical gating due to the micron-scale coherence length with that of the high numerical aperture objective lens. Measuring the phase shift map associated with the object as it is translated through focus provides full information about the 3D distribution associated with the refractive index. Using a reconstruction algorithm based on the Born approximation, we show that the sample structure may be recovered via a 3D, complex field deconvolution. We illustrate the method with reconstructed tomographic refractive index distributions of microspheres, photonic crystals, and unstained living cells.

In order to obtain a tomographic image of a sample, we perform axial scanning by translating the sample through focus in step sizes of less than half the Rayleigh range, with an accuracy of 20 nm. At each axial position, we record a quantitative phase image using the principle of spatial light interference microscopy (SLIM). In SLIM, the image is considered an interferogram between the scattered and unscattered fields. Shifting the relative phase between these two fields in 4 successive steps of π/2 and recording the 4 corresponding images, we can quantitatively extract the pathlength map associated with the specimen with sub-nanometer sensitivity. In order to obtain a tomographic image of a sample, we translate the sample through focus in step sizes of less than half the depth of field with an accuracy of 20 nm. Figures 1b-d illustrate this approach with quantitative phase images obtained on a live neuron. While there is certain elongation in the z-axis, as indicated especially by the shape ofthe cell body in Fig. 1b, it is evident that SLIM provides optical sectioning without further processing. Specifically, at the substrate plane (z = 2 μm) the neuronal processes are clearly in focus and the nucleolus is absent. However, 6 μm above this plane, the cell body and nucleolus are clearly in focus, while the contributions from the processes are subdominant. Starting with these quantitative phase images, we solve the inverse scattering problem.

Perhaps one of the most appealing applications of SLIT is the 3D imaging of live, unstainedcells. We performed SLIM experiments on live neuron cultures. Results obtained from asingle neuron are shown in Fig. 2. Thus, Figs. 2a-b show two sections separated by 5.6 μm.Notably, for some regions of the cytoplasm, the refractive index distribution is below 1.39,which is compatible with previous average refractive index measurements on other cell types.

Our results demonstrate that rich quantitative information can be captured from both fixed structures and cells using SLIT. In essence, SLIT combines microscopy and interferometry to solve the inverse scattering problem. Because of its implementation with existing phase contrast microscopes, SLIT has the potential to make a broad impact and elevate phase-based imaging from observing to quantifying over a broad range of spatiotemporal scales. We anticipate that the studies allowed by SLIT will further our understanding of basic phenomena related to biological applications as well as material science research.