by Christos Evangelou, MSc, PhD – Medical Writer and Editor

In a new study, researchers at Beth Israel Deaconess Medical Center, Harvard Medical School, and Massachusetts Institute of Technology evaluated the use of nonlinear microscopy for rapid examination of resected lung tissues, including lung tumors. The results showed that nonlinear microscopy could replicate the visual information from H&E staining for both lung tumors and non-neoplastic lung structures.1

According to the authors, this study provides evidence that nonlinear microscopy is a promising imaging modality that could enhance and accelerate the pathologic examination of resected lung tissues compared to current techniques such as frozen section analysis.

“We found that nonlinear microscopy reproduces virtually all diagnostically relevant features across a spectrum of neoplastic lung pathologies. Because nonlinear microscopy does not require freezing or physical sectioning, we were able to demonstrate rapid, high-resolution imaging of the lung tissue, even that contained within intact staple lines,”

said Paul A. VanderLaan MD, PhD, who is an associate professor of pathology at Harvard Medical School and the corresponding author of the study.

The report was published in the American Journal of Clinical Pathology.

Bridging the Gap Between Accuracy and Efficiency

Although surgical resection can provide long-term survival benefits for patients with early-stage lung cancer, its success relies on assessment of complete tumor removal with negative margins. Traditionally, the histopathologic evaluation involves formalin fixation, paraffin embedding, and microtome sectioning of resected tissues, followed by H&E staining. However, this process can often take well over 12 hours.1

Frozen section analysis, a quicker alternative, has its own limitations, such as difficulties with fatty and bony tissues, freezing artifacts, and consumption of valuable tissue samples. This study aimed to address these shortcomings by exploring the capabilities of nonlinear microscopy, a fluorescence microscopy technique that bypasses traditional preparation steps, enabling rapid tissue visualization without damage or loss.1

“In neoplastic lung pathology, there are several applications, such as intraoperative tissue diagnosis and surgical resection margin evaluation, where rapid and high-throughput histology applications could make a significant difference in patient care. Towards this end, we conducted a proof-of-concept study using nonlinear microscopy, also known as two-photon microscopy, to subjectively evaluate rapidly acquired images versus conventional paraffin-embedded H&E-stained slides,”

noted Dr. VanderLaan.

Methodology

In this proof-of-principle study, the researchers examined 73 tissue specimens collected from 13 patients who underwent lung resection for pulmonary nodules. Employing nonlinear microscopy, they generated digital images of these tissues, stained with acridine orange and sulforhodamine 101, within minutes.

“Nonlinear microscopy uses a scanned laser to excite fluorescence from a thin plane in the specimen, forming a so-called optical section and obviating the need for a physical, microtomed section. Rapid staining with fluorescent H&E-analogs and a virtual H&E-like display facilitate interpretation by pathologists,”

explained Dr. Yue Sun, who is an assistant professor of pathology at Harvard Medical School and the lead author of the study.

Two pathologists compared the nonlinear microscopy images to the corresponding traditional H&E slides to evaluate their ability to replicate diagnostic features and identify critical patterns of invasion and spread.

Findings

The study showed that nonlinear microscopy accurately recreated the visual information provided by traditional H&E staining and highlighted histological details that are important for diagnosis. Using nonlinear microscopy, the researchers were able to identify various lung tumor types and architectural growth patterns that pinpoint patterns of invasion, such as vascular, pleural, and spread through air spaces.1

Notably, nonlinear microscopy accentuated elastic fibers, a feature that can be challenging to appreciate with conventional H&E staining, aiding in the detection of pleural invasion — a critical factor in staging lung cancers.1

Moreover, the researchers found that nonlinear microscopy could facilitate rapid evaluation of stapled parenchymal margins without the need for staple removal, offering a potential solution to a long-standing challenge in intraoperative margin assessment.1

“In resection specimens containing staple line margins, it is very difficult to perform frozen section analysis of the tissue actually contained within the staple line because the embedded staples interfere with cryotome sectioning and are time-intensive to remove by hand,”

noted Dr. VanderLaan.

Potential Applications and Limitations

Advantages of nonlinear microscopy include rapid imaging within minutes, no tissue damage, the ability to image deeper into the tissue, and compatibility with subsequent histology and ancillary studies. By providing very rapid tissue evaluation, nonlinear microscopy could significantly reduce time-to-diagnosis, enabling quicker treatment initiation and improved patient outcomes.

The authors propose that nonlinear microscopy also has the potential to augment rapid pathologic evaluation in settings such as intraoperative frozen section assessment and examination of small biopsies. In the grossing room, nonlinear microscopy could guide targeted tissue sampling, minimizing the number of cassettes required for traditional histologic processing.

Furthermore, the authors envision a future in which nonlinear microscopy integrates seamlessly with digital pathology and computer-assisted algorithms, enabling real-time diagnostic support, predictive genomic analysis, and enhanced patient care.

However, integrating new technology into established surgical pathology workflows, addressing physical space requirements, specialized training, and process validation are among the challenges that lie ahead.

In addition, the cost of instrumentation, primarily driven by the ultrashort pulse laser, could pose a barrier to the widespread adoption of nonlinear microscopy. Nonetheless, the authors are optimistic about the potential for cost reduction through the use of high-efficiency, fixed-wavelength lasers commonly employed in manufacturing

Looking Ahead

“The study results are promising but are not alone sufficient to immediately change clinical care. Blinded reading studies to establish diagnostic performance in terms of sensitivity and specificity are needed. Non-neoplastic/interstitial lung diseases should also be investigated,”

Dr. VanderLaan acknowledged.

Moreover, prospective studies are needed to evaluate the sensitivity and specificity of nonlinear microscopy image interpretation compared to traditional H&E staining. Researchers also plan to investigate the impact of this novel technique on downstream ancillary studies, such as immunohistochemistry and molecular testing, ensuring that the benefits of nonlinear microscopy do not compromise the accuracy of these crucial analyses.

The study received financial support from the National Cancer Institute of the National Institutes of Health.

References

1. Sun Y, Weber TD, Fujimoto JG, Rosen S, VanderLaan PA. Rapid examination of lung tissues by nonlinear microscopy. Am J Clin Pathol. Published online April 29, 2024. doi:10.1093/ajcp/aqae046