A new holographic histopathology method for improved and fast label-free diagnosis

Researchers from the Korea Advanced Institute of Science and Technology (KAIST), Department of Physics, Daejeon (Republic of Korea) and KAIST Institute for Health Science and Technology, Daejeon (Republic of Korea) have developed a new optical diffraction tomography method for label-free diagnosis based on the histological evaluation of thick tissue slides. They have also showed that this new optical diffraction tomography method accelerated imaging and improved imaging resolution.

The researchers concluded that this method opens a new avenue for improved label-free diagnostics in histopathology. The study was published in the journal Advanced Photonics on April 29th, 2021.

Limitations of traditional histopathology methods in diagnosis

Histology plays a pivotal role in the diagnosis of cancer, among other diseases. However, traditional histopathology methods involve multiple steps of tissue processing and tissue staining. As these methods are time-consuming, laborious, and tissue destructive, they cannot be used for intraoperative diagnosis or when a rapid, non-destructive diagnosis is needed.

Another critical limitation of traditional histopathology is that it requires the sectioning of tissues into thin specimens (2–10 μM), which further delays diagnosis. Therefore, over the last few years, several efforts have been made to develop novel label-free histopathology methods for fast, accurate, and highly reproducible diagnosis. Emerging label-free microscopy methods include optical coherence tomography, quantitative phase imaging, photoacoustic remote sensing, and optical diffraction tomography.

 Holographic Histopathology Enables Fast, Precise Diagnostics

Volumetric histopathology of unlabeled 100-μm-thick pancreas tissue sample from a patient with intraductal papillary neoplasm of bile duct in the liver. For the purpose of comparison, adjacent tissues were prepared in thin tissue slides with conventional H&E staining method. (the fifth row, 400x magnification). Image credit: Hugonnet et al., doi 10.1117/1.AP.3.2.026004.

Principles of optical diffraction tomography

Optical diffraction tomography is an advanced quantitative phase microscopy method that can be used to uncover the 3D distribution of the refractive index of a tissue sample. This is achieved by combining multiple 2D quantitative phase images acquired from different illumination angles using the Fourier diffraction theorem.1

In optical diffraction tomography, off-axis holography is first used to acquire the scattered light transmitted through the specimen. Multiple scattered fields obtained from different directions are then combined to reconstruct the refractive index of the sample. In contrast to most imaging techniques that require the use of labeling agents or dyes as contrast agents, optical diffraction tomography is based on refractive index, an intrinsic optical parameter, to create imaging contrast.2–4 

Refractive index values vary depending on the number and type of intracellular biomolecules, such as lipids and proteins. Hence, reconstructed optical diffraction tomograms can provide detailed structural and quantitative insight into various unstained biological samples.5

Optical diffraction tomography has been successfully used to acquire high-resolution images of single live cells.6,7 Although this advanced microscopy technique has various potential applications in pathology, the complex distribution of refractive indexes of thick tissues limits its use to thin tissues.8

Adapting optical diffraction tomography to image thicker samples

To extend the use of optical diffraction tomography for thick tissue specimens, Hugonnet et al.5 employed a Mach-Zehnder interferometer, digital refocusing, and automated stitching to establish a 3D label-free quantitative phase imaging technique that provides volumetric imaging information based on optical diffraction.

This new microscopy method enabled them to image 100-μm-thick tissues with a lateral field of view of 2 mm × 1.75 mm and an image resolution of 170 nm × 170 nm × 1400 nm. The high resolution of this method in combination with the wide field of view enabled them to visualize subcellular and mesoscopic structures in unstained thick tissue specimens.

Multiscale label-free volumetric holography for fast and accurate diagnosis

To test the feasibility of using their new microscopy method to image unstained tissue specimens, they evaluated its performance in various tissue samples. To this end, the researchers acquired images of unstained tissue samples from multiple human organs (small intestine, pancreas, and large intestine).

Multiscale label-free volumetric holography enabled the acquisition of millimeter-scale 3D images at a subcellular resolution. The images acquired using this new microscopy method allowed researchers to visualize individual cells, multicellular tissue architectures, and different morphological features in various unstained tissues. Imaging accuracy achieved by multiscale label-free volumetric holography was similar to that of conventional H&E staining.

The researchers also imaged unstained, 100-μm-thick tissues from patients with different cancers, including intraepithelial neoplasia, pancreatic neuroendocrine cancer, and intraductal papillary neoplasms of the bile duct.

Notably, the resolution of images obtained through multiscale label-free volumetric holography was similar to that of images acquired with laborious traditional tissue processing and staining methods. This high subcellular 3D resolution allowed researchers to accurately detect precursor lesions and different types of malignant lesions.

“We developed a method based on optical diffraction tomography to image unstained pathological tissue samples,” said Herve Hugonnet, the lead author of the study. “The resulting images show similar contrast to H&E stained sample and when presented to a pathologist enabled visualization of different morphological features in the various tissues allowing for recognition and diagnosis of precursor lesions and pathologies.”

Future perspectives

The findings of this study suggest that multiscale label-free volumetric holography holds great potential for rapid and high-resolution histopathology of thick tissue sections, bypassing the need for time-consuming tissue processing and chemical staining protocols.

Considering the label-free volumetric imaging ability of this method across a variety of tissue samples, the authors concluded that the technique could be used for near real-time diagnosis of cancer during intraoperative pathology consultations.

However, the setup and sample preparation used in this study can only be used to obtain high-quality images from tissues with thickness up to ∼100 μm regardless of the use of digital refocusing. This limitation is due to multiple light scattering, which results in low-quality tomograms when tissues are thicker than ∼100 μm. Hence, the authors note that further research is required to optimize the protocols for sample preparation, improve reconstruction speed, and minimize artifacts due to multiple scattering.

In addition, more research is warranted to improve the histological interpretation of the refractive index and enhance the adoption of optical diffraction tomography for routine histopathology. The integration of machine learning methods in optical diffraction tomography workflows may accelerate the clinical implementation of optical diffraction tomography.

“We expect our method to speed up diagnosis, especially in intraoperative pathology consultations, due to shortening of the sample preparation time,” said Herve Hugonnet. “Our method could also enable more precise diagnosis since it enables volumetric imaging of the sample. More research on the imaging of fresh tissue and hardware improvement would further demonstrate the applicability of our method.”

To learn more about the use of multiscale label-free volumetric holography in histopathology of thick tissue slides, read the open access article by Herve Hugonnet et al., “Multiscale label-free volumetric holographic histopathology of thick tissue slides with subcellular resolution,” Advanced Photonics 3(2), 026004 (2021).


  1. Kim K, Yaqoob Z, Lee K, et al. Diffraction optical tomography using a quantitative phase imaging unit. Opt Lett. 2014;39(24):6935-6938. doi:10.1364/OL.39.006935
  2. Jin D, Zhou R, Yaqoob Z, So PTC. Tomographic phase microscopy: principles and applications in bioimaging [Invited]. J Opt Soc Am B. 2017;34(5):B64-B77. doi:10.1364/josab.34.000b64
  3. Tahara T, Quan X, Otani R, Takaki Y, Matoba O. Digital holography and its multidimensional imaging applications: a review. Microscopy. 2018;67(2):55-67. doi:10.1093/jmicro/dfy007
  4. Soto JM, Rodrigo JA, Alieva T. Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy \[Invited\]. Appl Opt. 2018;57(1):A205–A214. doi:10.1364/AO.57.00A205
  5. Hugonnet H, Kim YW, Lee M, et al. Multiscale label-free volumetric holographic histopathology of thick-tissue slides with subcellular resolution. Adv Photonics. 2021;3(2):1-8. doi:10.1117/1.AP.3.2.026004
  6. Sung Y, Choi W, Fang-Yen C, Badizadegan K, Dasari RR, Feld MS. Optical diffraction tomography for high resolution live cell imaging. Opt Express. 2009;17(1):266-277. doi:10.1364/oe.17.000266
  7. Lee AJ, Hugonnet H, Park W, Park Y. Three-dimensional label-free imaging and quantification of migrating cells during wound healing. Biomed Opt Express. 2020;11(12):6812-6824. doi:10.1364/BOE.405087
  8. Balasubramani V, Kuś A, Tu HY, et al. Holographic tomography: techniques and biomedical applications [Invited. Appl Opt. 2021;60(10):B65-B80. doi:10.1364/AO.416902

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