【Newsachieve.com】A new approach for high-throughput quantitative phase microscopy

The hybrid bright/dark field intensity transfer (HBDTI) approach for high-throughput quantitative phase microscopy greatly expands the spatial bandwidth product of a conventional microscope, extending the available spatial sample frequencies in Fourier space well beyond the traditional limit of coherent diffraction. Credit: Lingpeng Lu, NJUST.

Cell organelles are involved in various cellular life processes. Their dysfunction is closely related to the development and metastasis of cancer. The study of subcellular structures and their abnormal states facilitates understanding of the mechanisms of pathologies, which may allow early diagnosis for more effective treatment.

The optical microscope, invented over 400 years ago, has become an indispensable and ubiquitous tool for studying micro-objects in many fields of science and technology. In particular, fluorescence microscopy has made several leaps, from 2D wide-field to 3D confocal, and then to super-resolution fluorescence microscopy, which has significantly contributed to the development of modern life sciences.

Using conventional microscopes, researchers are currently trying to create sufficient internal contrast for unstained cells due to their weak absorption or weak scattering. Special dyes or fluorescent labels can aid in imaging, but long-term observation of living cells is still difficult to achieve.

Recently, Quantitative Phase Imaging (QPI) has shown promising results due to its unique ability to quantify the phase delay of unlabeled samples in a non-destructive manner. However, the bandwidth of the imaging platform is mainly limited by the spatial bandwidth product of the optical system (SBP), and the magnification ofthe SBP of a microscope is fundamentally mixed with the scale-dependent geometric aberrations of its optical elements. This results in a trade-off between the achievable image resolution and the field of view (FOV).

Lead author Lingpeng Lu, a PhD student at SCILab, provides a colorful hand-drawn animation as a helpful summary of the report. Credit: Lu et al., doi10.1117/1.AP.4.5.056002.

A label-free microscopic imaging approach with high resolution and a large field of view is essential to enable accurate detection and quantification of subcellular features and events. To this end, researchers from the Nanjing University of Science and Technology (NJUST) and the University of Hong Kong have recently developed a label-free high-performance microscopy method based on hybrid bright/dark field illumination.

As reported in advanced photonics The Hybrid Bright-Dark-Field Intensity Transfer (HBDTI) approach for high-throughput quantitative phase microscopy greatly expands the available spatial sample frequencies in Fourier space, increasing the maximum achievable resolution by about five times compared to coherent imaging diffraction limit.

Based on the principle of multiplexing illumination and synthetic aperture, they create a model for direct visualization of the nonlinear transfer of light and darkfield intensity. This model gives HBDTI the ability to provide performance beyond the limits of coherent diffraction.

QPI results of unlabeled HeLa cells. (a) Approximately 4,000 HeLa cells per ~7.19 mm² field of view. (b1) and (c1) Low-resolution bright-field (BF) images in focus with intensities of regions 1 and 2 in (a), respectively. (b2) and (c2) Intense darkfield (DF) images with low resolution in focus (b1) and (c1), respectively.(b3) and (c3) Recovery phase results of (b1) and (c1) using the conventional intensity transfer equation (TIE) phase recovery method based on the FFT, respectively. (b4) and (c4) Results of the search phase (b1) and (c1)   using the new HBDTI method, respectively. Credit: Lu et al., doi 10.1117/1.AP.4.5.056002.

Using a commercial microscope with 4x magnification and a numerical aperture of 0.16,the team demonstrated high-performance HBDTI imaging, achieving image resolution at a FWHM of 488 nm within a field of view of approximately 7.19mm², which leads to a 25-fold increase in SBP compared to the case of coherent illumination.

Non-invasive high-throughput imaging enables the delineation of subcellular structures in large-scale cell studies. According to corresponding author Chao Zuo, Principal Investigator at the Intelligent Computational Imaging Laboratory (SCILab) at NJUST, “HBDTI offers a simple, high-performance, low-cost, and versatile imaging tool for quantitative analysis in life sciences and biomedical research.” Its high-throughput QPI capability 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 advance the high-speed uptake of HBDTI in large-scale living cell analysis.

Additional Information:

Linpeng Lu et al, A hybrid bright-field and dark-field intensity transfer approach for high-throughput quantitative phase microscopy, advanced photonics (2022). DOI:10.1117/1.AP.4.5.056002

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

This document is protected by copyright. Except in any fair dealing for the purpose of private study or research, no part may be reproduced without written permission. Content is provided for informational purposes only.