Intrinsic dark-field Fourier ptychographic diffraction tomography under non-matched illumination
image: A comparison of imaging results on a 15-micrometer polystyrene microsphere. a–c: Lateral and axial slice reconstruction results of microsphere simulations using FPDT and DF-FPDT methods under 40×/0.65NA, 40×/0.95NA, and 100×/1.4NA objectives respectively. d–f: Experimental lateral and axial slice reconstructions of the microspheres using the FPDT and DF-FPDT methods under 40×/0.65NA, 40×/0.95NA, and 100×/1.4NA objectives respectively. Scale bars: (a, b, d, e) 10 μm; (c, f) 5 μm.
Credit: Habib Ullah et al.
Observing living cells in their natural, three-dimensional state within standard laboratory dishes is crucial for biomedical research. However, it poses a challenge for high-resolution microscopes. The geometry of common multi-well plates restricts the lighting angles, leading to blurry and low-contrast images.
Now, researchers from Nanjing University of Science and Technology have turned this problem into a solution. Their new method, called dark-field Fourier ptychographic diffraction tomography (DF-FPDT), cleverly uses the same restricted lighting to selectively enhance the fine details of a cell while suppressing the blurry background.
“Instead of fighting the physical constraints of the culture dish, our method embraces them,” says Professor Chao Zuo, corresponding author of the study. “We use an algorithm to tell the reconstruction process to ignore the missing 'fuzzy' information and only piece together the sharp, high-frequency details that are naturally encoded in the images.”
The result is a 3D image with much clearer contrast, similar to that from a more complex dark-field microscope.
The team validated DF-FPDT by imaging live cells and other samples. “The results showed dramatically improved visibility of intracellular structures like mitochondria, which are often hard to see with standard label-free methods under these conditions,” adds Zuo.
The technique also successfully captured dynamic processes, such as mitochondria fusing and dividing over time.
“This innovation provides biologists with a powerful, label-free tool for long-term 3D observation of cells in their most common and practical growing environment,” says Zuo. “It requires no special sample preparation, fluorescent dyes, or hardware modifications to standard microscopes, making it readily accessible for labs.”
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Contact the author: Chao Zuo, Professor, Nanjing University of Science and Technology
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