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Researchers Improve Microscopic Imaging of Unstained Biological Tissues

A new study published in Nature Communications by the research group of Professor Wonshik Choi at the Department of Physics at Korea University demonstrates a lensless, Fourier holographic imaging fiber endoscope that performs label-free imaging of unstained biological tissues. This ultrathin fiber endoscope performs faster and provides a more accurate diagnosis than previous methods.

Study: Flexible-type ultrathin holographic endoscope for microscopic imaging of unstained biological tissues. Image Credit: Photobank gallery/Shutterstock.com

Limitations of Optical Microscopes

Endoscopes were developed to extend the capabilities of optical microscopic imaging of biological material for the early diagnosis and prevention of diseases. While some of the limitations of optical microscopes were overcome by fiber endoscopes, probe size and calibration and dependence on fluorescence measurements have remained a challenge.

Optical microscopes have long aided medical diagnosis, providing early detection of tissue alterations due to harmful agents.

The image resolution afforded by microscopic imaging and its non-invasive application was transformational in the medical industry. However, imaging samples located inside curved passages within physiological materials was challenging.

Optical microscopy relies on fluorescence activity specific to certain molecular interactions and cannot provide label-free imaging. In most cases, the sample region studied has to be stained with a particular material to enhance fluorescence. Due to light scattering, target regions beneath the optically active region are also not detected efficiently by microscopic imaging. The chemicals staining the samples can also negatively affect the imaging process.

Fiber Endoscopes

Endoscopes were developed to image hard-to-reach areas using an invasive method. Advancements in endoscopic probe designs, optical delivery, and detection methods have had a vast positive impact on the early diagnosis of diseases. 

Microscopic imaging with endoscopes typically uses thin, flexible fibers. The fibers deliver light to the target region in the sample. The size of the fibers and the methods implemented on the fiber tips to scan the target region can vary. Since fiber endoscopy is an invasive imaging method, a balance in size between the delivery channel and scanning mechanism has to be maintained to minimize discomfort for the patient. The different fiber endoscopic designs developed so far have had some drawbacks in providing high resolution and label-free imaging while maintaining low levels of complications. They have also not been able to utilize stain-free tissue samples.

New Ultrathin, Lensless Fiber Endoscopes

The Choi group, also associated with the Center for Molecular Spectroscopy and Dynamics, devised a novel experimental scheme to implement fiber endoscopy. Their design scheme used bare fiber bundles and the tips of the fibers were devoid of any scanners and was lens-free.

In this study, the fiber cores were not imaged pixels like the ones employed in traditional fiber endoscopy. Fiber bundles with 200 μm or 350 μm diameter was chosen for this experiment. This ensured that the invasive probe size was ultrathin, limited to the fiber bundle size. The target region to be imaged was set at least 400 μm from the fiber tip.

Light travels through each fiber core before being delivered to the target. The reflected light waves from the target region spread back to the fibers. Detecting the weak, reflected a holographic field measurement technique carries out light. This particular arrangement of light delivery and detection uniquely filters any noise from the back reflection from the incident light.

Holographic field measurements are performed using Fresnel diffraction methods based on Fourier transform. The research group used the phrase ‘Fourier holographic endoscopy’ to describe their experimental technique. A novel algorithm had to be devised to reconstruct the image of the target.

When light travels through bends and twists along the fiber, the various phases of light concerning the different fiber core positions change. The reconstruction algorithm had to account for these delays. The mathematical description developed by the research group could reconstruct pixilation-free and diffraction-limited images. Using this scheme, a fiber endoscope with arbitrary length variation should be able to image a stain-free tissue sample successfully.

Tests on Live Tissues

The proof of this experimental concept was verified by imaging unstained intestine tissues of a rat. This target sample is invisible to other microscopic imaging methods used in traditional endoscopy.

As elaborated in the Nature Communications publication, the results of the tests exhibited spatial resolution and contrast that were far better than traditional methods. This new ultrathin, stain-free, fiber endoscopic technology is positioned to speed-up diagnostic analysis with minimal discomfort for patients.

References

Choi, W., Kang, M., Hong, J.H. et al. Flexible-type ultrathin holographic endoscope for microscopic imaging of unstained biological tissues. Nat Commun 13, 4469 (2022). https://www.nature.com/articles/s41467-022-32114-5

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Written by

Ilamaran Sivarajah

Ilamaran Sivarajah is an experimental atomic/molecular/optical physicist by training who works at the interface of quantum technology and business development.

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