Nov 3 2022Reviewed by Emily Henderson, B.Sc.
Cell organelles perform a range of cellular tasks. Their impairment is closely associated with cancer development and metastasis. Analysis of subcellular structures and their impaired states offers insights into the workings of pathologies, which may allow early diagnosis for more effective treatment.
Invented over 400 years ago, the optical microscope has turned into a vital and universal instrument for the study of microscale objects in numerous areas of technology and science. Specifically, fluorescence microscopy has accomplished more than a few leaps—from two-dimensional (2D) wide-field to three-dimensional (3D) confocal and then to super-resolution fluorescence microscopy, significantly enhancing the development of contemporary life sciences.
At present, researchers using conventional microscopes struggle to create adequate intrinsic contrast for unstained cells because of their weak scattering or low absorption properties. Fluorescent labels or specific dyes can assist with visualization, but long-term observation of live cells is still challenging to accomplish.
In recent times, quantitative phase imaging (QPI) has demonstrated potential with its exclusive ability to measure the phase delay of unlabeled samples in a nondestructive manner.
An imaging system’s throughput is primarily limited by its optical system’s space-bandwidth product (SBP), and the SBP increase of a microscope is essentially stumped by the scale-dependent geometric abnormalities of its optical elements. This results in a compromise between attainable image resolution and field of view (FOV).
An approach is needed to realize label-free, high-resolution, and large FOV microscopic imaging in order to facilitate accurate detection and quantitative examination of subcellular features and events.
Therefore, researchers from Nanjing University of Science and Technology (NJUST) and the University of Hong Kong recently formulated a label-free high-throughput microscopy method established on hybrid bright/darkfield illuminations.
The “hybrid brightfield-darkfield transport of intensity” (HBDTI) method, reported in the journal Advanced Photonics, for high-throughput quantitative phase microscopy considerably expands the accessible sample spatial frequencies in the Fourier space, prolonging the maximum attainable resolution by about fivefold over the coherent imaging diffraction limit.
Established on the basis of illumination multiplexing and synthetic aperture, they set up a forward imaging model of nonlinear brightfield and darkfield intensity transport. This model provides HBDTI with the ability to deliver features outside the coherent diffraction limit.
The researchers, using a commercial microscope with a 4x, 0.16NA objective lens, showed HBDTI high-throughput imaging, achieving 488-nm half-width imaging resolution within a FOV of around 7.19 mm2, yielding a 25× boost in SBP over the case of coherent illumination.
Noninvasive high-throughput imaging allows the delineation of subcellular structures in comprehensive cell studies.
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 observes that additional efforts are required to enhance the high-speed implementation of HBDTI in large-group live cell studies.
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.