Recently, the research group led by Prof. Qian Chen and Prof. Chao Zuo at the School of Electronic and Optical Engineering, Nanjing University of Science and Technology (NJUST) has published a research paper entitled “Quasi-Isotropic High-Resolution Fourier Ptychographic Diffraction Tomography with Opposite Illuminations” in ACS Photonics (IF = 7) and it was selected as the journal’s cover feature. PhD student Ning Zhou is the first author of this paper, and Prof. Qian Chen and Prof. Chao Zuo are the co-corresponding authors of this paper. NJUST is the first completing and corresponding institution.
Article Link: https://doi.org/10.1021/acsphotonics.3c00227
Optical diffraction tomography (ODT) is a new three-dimensional label-free microscopic imaging technology that can perform 3D visualization or quantitative characterization of the internal characteristics of transparent biological samples. Compared with traditional fluorescence imaging technology, ODT effectively avoids the phototoxicity and photobleaching of fluorescent dyes in biological samples and enables noninvasive, label-free 3D volume imaging. Therefore, it has been widely used in biophysics, cell biology, hematology, and microbiology. It is expected to provide more accurate and effective analysis methods for biomedical research and clinical treatment. However, due to the limited projection angle imposed by the numerical aperture of the single objective lens in traditional ODT microscopes, the three-dimensional optical transfer function can only cover a saucer-shaped region in the Ewald limit sphere (Figure 1). As a result, the highest axial resolution can only reach one-third of the lateral resolution, about 600 nm.
Figure 1. Schematic diagram of three-dimensional optical transfer function and imaging system resolution
In recent years, to record characteristic frequencies beyond the aperture limit of the single objective lens so as to achieve isotropic ODT imaging, methods such as dual objective lens opposite detection and transmission combined with reflection illumination have received increasing attention. However, for more than half a century, quantitative phase measurements have been based on the principle of interferometry. To realize diffraction tomography, it is necessary to obtain the phase distribution of the sample at different angles using interference holography and then perform spectrum synthesis. The extra back interference light path makes the structure of the imaging system more complicated, and, therefore, it is difficult for it to be compatible with existing microscopes. At the same time, the ODT technology using time-coherent illumination inevitably suffers from speckle noise and parasitic interference. The backscattered field is a dark field distribution for weakly scattering transparent biological cells. After the coherent light source’s speckle noise is superimposed, it will become a speckle field. Therefore, it is difficult to measure the total field complex amplitude using interference methods accurately. This poses a severe challenge to motion-free isotropic high-resolution ODT based on backscattering detection.
To address the above issues, the research group of Prof. Qian Chen and Prof. Chao Zuo from NJUST proposed a quasi-isotropic high-resolution Fourier Ptychographic diffraction tomography technology based on opposite illuminations. The uniquely iterative Ptychographic method is used to solve the nonlinear inverse problem from dark-field images to complex refractive index, making it possible to obtain backscattering dark-field information. This method effectively expands the spectral coverage support domain of the object function by integrating transmission angle scanning and reflection wavelength scanning illumination schemes (Figure 2), achieving 3D near-isotropic high-resolution 3D diffraction tomography and alleviating the distortion problem of refractive index values.
Figure 2. Schematic diagram of angle scanning (transmission) and wavelength scanning (reflection) opposite illuminations and reconstruction algorithms for quasi-isotropic high-resolution Fourier Ptychographic diffraction tomography technology
The research team has established an associated experimental platform for opposite illumination microscopy (Figure 3). The system uses a surface-mounted programmable LED array illumination for forward angle scanning illumination and a supercontinuum laser source combined with an acoustic-optical tunable filter for reflection wavelength scanning illumination. The LED array and AOTF in the system are triggered and monitored synchronously with the camera by the FPGA controller via two coaxial cables.
Figure 3. Quasi-isotropic high-resolution three-dimensional microscopic imaging system with opposite illuminations
Figure 4. Experimental results of tailor-made fiberglass sample
The quasi-isotropic high-resolution 3D diffraction tomography imaging ability of the above system has been verified in imaging experiments on tailor-made fiberglass samples, as shown in Figure 4. These results show that the two types of fiberglass reconstructed by the transmission-only method (FPDT) are aliased together at the intersection due to the missing cone problem. By introducing backscattering information, the opposed illumination method (OI-FPDT) significantly expands the spectral support domain of the object function in a non-interferometric and sample motion-free manner, and finer structures in the axial direction can be distinguished. The results show that this technology improves the axial resolution of the instrument three times through coupling transmission angle scanning and reflection wavelength scanning illumination and alleviates the distortion of 3D RI reconstruction.
Figure 5 Experimental results of onion epidermal cells
Figure 5 shows the 3D RI reconstruction results of an onion epidermal cell. Here, we dissected the onion from the surface, separated them into peels with only one cell wall thickness (∼7 μm), and then cleaned and fixed them on the resinous slide. Two intensity sets are acquired by transmissive angle-scanning and reflective wavelength-scanning illumination schemes. Experimental results confirm that the resolution and signal-to-noise ratio of the axial RI reconstruction of complex biological samples are significantly reduced due to the limitation of the illumination angle imposed by the single objective lens in the conventional transmission-only method. The opposite-illumination method can significantly improve the resolution along the Z-axis and obtain more explicit edge information, which is expected to provide important imaging support for life sciences and basic medical research.
复审 | 左超
终审 | 徐峰