New Microscopy Method Enables High-Throughput Quantitative Phase Imaging

Researchers from Nanjing University of Science and Technology (NJUST) and the University of Hong Kong have developed a new approach for high-throughput quantitative phase microscopy that shows potential for delineating subcellular structures in large-scale cell studies. This ability would enable more insight into cell organelles, which are involved in a variety of cellular activities and whose dysfunction is closely related to the development and metastasis of cancer. Exploration of such subcellular structures and their abnormal states could enable early diagnosis for more effective treatment.

Conventional microscopes struggle to generate sufficient intrinsic contrast for unstained cells due to their low absorption or weak scattering properties. Specific dyes or fluorescent labels can help with visualization, but long-term observation of live cells remains difficult to achieve. Recently, quantitative phase imaging (QPI) has shown promise with its unique ability to quantify the phase delay of unlabeled specimens in a nondestructive way. Yet the throughput of an imaging platform is fundamentally limited by its optical system’s space-bandwidth product (SBP), and the SBP increase of a microscope is fundamentally confounded by the scale-dependent geometric aberrations of its optical elements. This results in a tradeoff between achievable image resolution and field of view (FOV).

An approach to achieving label-free, high-resolution, and large FOV microscopic imaging is needed to enable precise detection and quantitative analysis of subcellular features and events. The method developed by NJUST and University of Hong Kong uses a label-free high-throughput microscopy method based on hybrid bright/darkfield illuminations. Dubbed “hybrid brightfield-darkfield transport of intensity” (HBDTI), the high-throughput quantitative phase microscopy approach significantly expands the accessible sample spatial frequencies in the Fourier space, extending the maximum achievable resolution by approximately fivefold over the coherent imaging diffraction limit.

Based on the principle of illumination multiplexing and synthetic aperture, HBDTI establishes a forward imaging model of nonlinear brightfield and darkfield intensity transport. This model endows HBDTI with the ability to provide features beyond the coherent diffraction limit. Using a commercial microscope with a 4x, 0.16NA objective lens, the team demonstrated HBDTI high-throughput imaging, attaining 488-nm half-width imaging resolution within an FOV of approximately 7.19 mm2, yielding a 25× increase in SBP over the case of coherent illumination.

“HBDTI offers a simple, high-performance, low-cost, and universal imaging tool for quantitative analysis in life sciences and biomedical research,” says corresponding author Chao Zuo, principal investigator of the Smart Computational Imaging Laboratory (SCILab) at NJUST. “Given its capability for high-throughput QPI, HBDTI is expected to provide a powerful solution for cross-scale detection and analysis of subcellular structures in a large number of cell clusters.” Zuo notes that further efforts are needed to promote the high-speed implementation of HBDTI in large-group live cell analysis.”

HBDTI is described in more detail in a recent issue of Advanced Photonics.