【Dou word】A new approach for quantitative high-throughput phase microscopy

A hybrid bright/dark field intensity transport (HBDTI) approach for high-throughput quantitative phase microscopy significantly extends the space-bandwidth product of a conventional microscope and extends the accessible sample spatial frequencies in Fourier space far beyond the traditional coherent diffraction limit. Photo credit: Linpeng Lu, NJUST.

Cell organelles are involved in a variety of cellular life activities. Their dysfunction is closely related to the development and metastasis of cancer. Research into subcellular structures and their abnormal states facilitates insights into the mechanisms of pathologies, which can allow early diagnos is for more effective treatment.

Invented more than 400 years ago, the optical microscope has become an indispensable and ubiquitous tool for studying micro-scale objects in many fields of science and technology. Fluorescence microscopy in particular has made several leaps – from 2D widefield to 3D confocal to super-resolution fluorescence microscopy, which has greatly advanced the development of modern life sciences.

With conventional microscopes, researchers are currently having difficulties in generating sufficient inherent contrast for unstained cells, as these only have low absorption or weak scattering properties. Specific dyes or fluorescent labels can aid in 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 non-destructively quantify the phase lag of unlabeled samples. However, the throughput of an imaging platform is fundamentally limited by the spatial bandwidth product (SBP) of its optical system, and a microscope’s SBP enhancement is fundamentally confounded by the scale-dependent geometric aberrations of its optical elements. This leads to a trade-off between achievable image resolution and field of view (FOV).

Lead author Linpeng Lu, a graduate student at SCILab, provides a vivid hand-drawn animationas a helpful synopsis of the report. Image credit: Lu et al., doi10.1117/1.AP.4.5.056002.

An approach to achieve label-free, high-resolution, and large FOV microscopic imaging is required to enable precise detection and quantitative analysis of subcellular features and events. To this end, researchers from Nanjing University of Science and Technology (NJUST) and the University of Hong Kong recently developed a label-free high-throughput microscopy method based on hybrid bright/dark field illumination.

As reported in Advanced Photonicsthe “hybrid brightfield-darkfield intensity transport” (HBDTI) approach to high-throughput quantitative phase microscopy significantly extends the accessible spatial frequencies of the sample in Fourier space and extends the maximum achievable resolution by about five times beyond the diffraction limit of coherent imaging.

Based on the principle of illumination multiplexing and the synthetic aperture, they create a forward imaging model of nonlinear brightfield and darkfield intensity transport. This model gives HBDTI the ability to provide features beyond the coherent diffraction limit.

QPI results from unlabeled HeLa cells. (a) Approximately 4000 HeLa cells on a FOV of ∼7.19 mm2. (b1) and (c1) Low-resolution brightfield (BF) in-focus intensity images of regions 1 and 2 in (a), respectively. (b2) and (c2) Low-resolution darkfield (DF) in-focus intensity images of (b1) and (c1), respectively. (b3) and (c3) Retrieval phase results of (b1) and (c1) respectively using the FFT-based traditional Transport of Intensity Equation (TIE) phase retrieval method. (b4) and (c4) Retrieval phase results of (b1) and(c1), respectively, using the novel HBDTI method. Image credit: Lu et al., doi10.1117/1.AP.4.5.056002.

Using a commercial microscope with a 4x 0.16 NA objective, the team demonstrated high-throughput HBDTI imaging, achieving an image resolution of 488 nm at halfwidth within a FOV of approximately 7.19 mm² resulting in a 25-fold increase in SBP over the case of coherent illumination.

Non-invasive high-throughput imaging enables delineation of subcellular structures in large-scale cell studies. According to corresponding author Chao Zuo, principal investigator at the Smart Computational Imaging Laboratory (SCILab) at NJUST,“HBDTI provides a simple, powerful, inexpensive, 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 multi-scale detection and analysis of subcellular structures in large numbers of cell clusters.”

Zuo notes that further efforts are needed to promote high-speed implementation of HBDTI in large-group live cell analysis.

More information:

Linpeng Lu et al, Hybrid brightfield and darkfield transport of the intensity approach for high-throughput quantitative phase microscopy, Advanced Photonics (2022). DOI:10.1117/1.AP.4.5.056002

Citation: A new approach for high-throughput quantitative phase microscopy (2022, November3), retrieved November 3, 2022 from https://phys.org/news/2022-11-approach-high-throughput-quantitative-phase-microscopy.html

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