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Programmable LED ring enables label-free 3D tomography for conventional microscopes

The cell is the structural and functional unit of life. Live cells are highly dynamic in all three dimensions. The visualization of cell dynamics in 3D, such as membrane fluctuations, mass transport, growth, and motility, has been a long-standing pursuit in optical microscopy. However, the cytoplasm and most organelles of biological cells have very weak absorption, so they produce very little contrast under normal illumination in a traditional brightfield microscope. To overcome this difficulty, conventional approaches have relied heavily on exogenous labeling techniques, such as fluorescence confocal microscopy, that require fluorescent dyes and proteins as biomarkers, and are thus ill-suited for samples that are non- fluorescent or cannot be easily fluorescently tagged. Besides, the photobleaching and phototoxicity of the fluorescent agents prevent live cells imaging over extended periods of time.

Although most biological cells are transparent and do not change the amplitude of the light passing through them, they introduce phase delays due to different structural regions of nonuniform optical density. In 1942, Zernike invented the Zernike phase contrast (ZPC) microscopy to visualize phase optically. The method uses a phase mask to shift only the unperturbed incident field component by a quarter of a wavelength, such that it interferes with scattered field with higher spatial frequencies, rendering the maximum contrast on the interference image. This provides a simple, efficient method for converting phase difference into intensity contrast, thus greatly improves the contrast of the transparent phase object under an optical microscope. This led to a revolution in biological imaging, as the significant contrast gain enabled the observation of unstained biological cells and tissues that were nearly invisible before, and the invention earned Zernike the 1953 Nobel prize in physics.

Over the half century, phase contrast microscope has gone on to become a ubiquitous tool in cell biology laboratories, and Zernike’s idea has continually inspiring the invention of novel label-free microscopic techniques, among which quantitative phase microscopy (QPM)1 is probably one of the most promising developments. QPM differs from phase-contrast microscopy in that it quantifies the phase shifts induced by the sample, instead of simply exploiting them for contrast enhancement. By combining the QPM with Computerized Tomography (CT) technique (sample rotation or beam scanning), the optical diffraction tomography (ODT) allows for depth-resolved true 3D imaging of the thick sample, just like confocal microscopy, but in a label-free manner. However, conventional ODT techniques require laser illumination and interferometric measurement, producing coherent imaging artifacts such as speckle. Moreover, most of them require complicated optical systems which are not typically available to most biologists, prohibiting their widespread use in biological and medical science.

Figure 1. Commonly used labeling and label-free microscopic techniques in biological and medical science

Published in Advanced Photonics, Vol. 1, Issue 6, 2019(Jiaji Li, Alex Matlock, Yunzhe Li, Qian Chen, Chao Zuo, Lei Tian. High-speed in vitro intensity diffraction tomography[J]. Advanced Photonics, 2019, 1(6): 066004), researchers from the Nanjing University of Science and Technology and Boston University report a new form of ODT, which they name annular illumination intensity diffraction tomography (aIDT).

Interestingly, in their method, the illuminator of a conventional microscope is replaced by a programmable LED ring (costs only one US dollar) for label-free noninterferometic tomographic imaging, which promises to greatly facilitate its adoption and implementation in biological laboratory. The LED ring is optimally fit with the edge of the circular pupil of the objective lens3, and only 8 intensity images under different illumination angles are acquired for the RI reconstruction based on the 4D deconvolution algorithm4, as the Figure 2 shows. By making use of the full NA range of imaging optics, the matched annular illumination provides a broadband 3D frequency coverage and enhanced response in both low- and high-frequencies, which are crucial for both raw data reduction and high-quality RI reconstruction at incoherent diffraction-limited resolution (both laterally and axially). With a 40×, 0.65 NA microscope objective, aIDT achieves a resolution of 350nm transversely and 890 nm axially across the object volume of 350 μm× 100μm× 20 μm at an imaging speed up to 10 Hz. It recovers high-quality, dynamic, volumetric morphological details through a Caenorhabditis elegans (C. elegans) worm. In Figure 3(b), an example raw captured intensity measurement shows that the asymmetric illumination creates strong intensity contrast for phase features. And Figures 3(b) and 3(c) give the recovered RI slice located at central depth position and depth color coding of 3D RI distribution within the whole field of view.

Figure 2. Illustration of aIDT imaging system. (a) A photo of aIDT system consisting of a standard microscope equipped with an LED ring. (b) An LED ring illumination unit is placed underneath the sample. The distance between LED and sample is tuned such that the illumination angle is matched with the objective NA. (c) Each IDT image measures the interference between the scattered and the unperturbed fields. (d) The absorption and phase transfer functions at various illumination angles and sample depths.

Compared with the conventional ODT techniques, aIDT has the following three advantages:

1) Non-laser light source: only a low-cost programmable LED ring is used.

2) No mechanic scanning: compatible with traditional microscopes.

3) High speed: 3D RI tomographic imaging of living, dynamic biological samples at 10 Hz.

The programmable LED ring endows conventional microscopes completely new features: aIDT enables label-free tomography of dynamic biological samples at sub-micron resolution without exogenous contrast agents such as fluorescent dyes. It provides a label-free, motion-free, simple, low-cost, high-speed imaging modality for life science and biomedicine research. Just like the relationship between phase contrast microscopy and fluorescence microscopy, aIDT is expected to become a new partner of fluorescence confocal microscopy, providing the complementary 3D ‘context’ of the whole cell body within which labelled structures can be identified by fluorescence microscopy. The next step researchers undertake is to further improve the imaging resolution of aIDT by utilizing high NA illumination and imaging optics, and explore its new applications in cell physiology, immuno-oncology, and disease diagnosis.

Figure 3. aIDT Imaging results of C. elegans. (a) A sample raw intensity image under annular illumination. (b) Reconstructed RI cross-sections demonstrate the sectioning capability enabled by the aIDT. (c) Depth color coding of 3-D RI measurements of sample in the whole field of view.

References:

1. Park, Y., Depeursinge, C., and Popescu, G., Quantitative phase imaging in biomedicine. Nature Photonics 12, 578 (2018).

2. Li, J., Matlock, A., Li, Y., Chen, Q., Zuo, C., and Tian, L., High-speed in vitro intensity diffraction tomography. Adv. Photon. 1, 1 (2019).

3. Zuo, C., Sun, J., Li, J., Zhang, J., Asundi, A., and Chen, Q., High-resolution transport-of-intensity quantitative phase microscopy with annular illumination. Sci Rep 7, 7654 (2017).

4. Ling, R., Tahir, W., Lin, H.-Y., Lee, H., and Tian, L., High-throughput intensity diffraction tomography with a computational microscope. Biomed. Opt. Express 9, 2130 (2018).