Numerous cellular life processes include the organelles of cells. Their malfunction and the growth and spread of cancer are strongly connected. Understanding the mechanisms underlying diseases is made easier through the exploration of subcellular structures and their aberrant states, which could help with early diagnosis and more successful treatment.
Since its creation more than 400 years ago, the optical microscope has been used in a wide range of fields of science and technology to examine objects at the microscopic scale.
Fluorescence microscopy, in particular, has advanced significantly, moving from 2D wide-field to 3D confocal and finally to super-resolution fluorescence microscopy. This has considerably aided the advancement of contemporary life sciences.
Due to unstained cells’ poor absorption or weak scattering qualities, researchers now fail to produce enough intrinsic contrast for them using standard microscopes. Visualization can be aided by specific dyes or fluorescent markers, but it is still challenging to observe live cells for an extended period of time.
Quantitative phase imaging (QPI), which provides the unique capacity to non-destructively measure the phase delay of unlabeled specimens, has recently demonstrated promise.
However, the space-bandwidth product (SBP) of an imaging platform’s optical system essentially determines its throughput, and the scale-dependent geometric aberrations of a microscope’s optical elements fundamentally complicate efforts to raise SBP. As a result, the possible image resolution and field of view (FOV) are compromised.
To enable accurate detection and quantitative analysis of subcellular features and events, a method for attaining label-free, high-resolution, and large FOV microscopic imaging is required.
To do this, scientists from the University of Hong Kong and Nanjing University of Science and Technology (NJUST) recently created a label-free high-throughput microscopy technique based on hybrid bright/darkfield illuminations.
According to a study published in Advanced Photonics, the “hybrid brightfield-darkfield transport of intensity” (HBDTI) method for high-throughput quantitative phase microscopy significantly increases the sample spatial frequencies that can be accessed in the Fourier space, thereby increasing the maximum achievable resolution by about five times over the coherent imaging diffraction limit.
They establish a forward imaging model for nonlinear brightfield and darkfield intensity transport based on the illumination multiplexing and synthetic aperture principles. The coherent diffraction limit can now be exceeded by HBDTI with this model.
Using a commercial microscope with a 4×, 0.16 NA objective lens, the team performed HBDTI high-throughput imaging, achieving 488-nm half-width imaging resolution within a FOV of roughly 7.19 mm2, resulting in a 25× increase in SBP over the case of coherent illumination.
In large-scale cell research, noninvasive high-throughput imaging enables subcellular structure delineation.
HBDTI offers a simple, high-performance, low-cost, and universal imaging tool for quantitative analysis in life sciences and biomedical research. 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.”
Chao Zuo, Study Corresponding Author and Principal Investigator, Smart Computational Imaging Laboratory, Nanjing University of Science and Technology
Zuo points out that more work is required to encourage the rapid application of HBDTI in large-group live cell analysis.
Lu, L., et al. (2022). Hybrid brightfield and darkfield transport of intensity approach for high-throughput quantitative phase microscopy. Advanced Photonics. doi.org/10.1117/1.AP.4.5.056002