Photonic Pathways FOCUS
Biopsies are the main source of diagnostic tissue. When CT or MRI imaging identifies a suspicious lesion, clinicians extract small tissue samples for analysis. However, because it can be difficult to obtain representative material, several samples( often 2 – 12) must be taken to ensure that diagnostic material is included. If all are non-representative, the biopsy must be repeated. In pulmonology and gastroenterology, rapid on-site evaluation( ROSE) partly mitigates this by allowing cytopathologists to inspect fine-needle aspiration smears within minutes. Yet ROSE assesses only single cells, not histological structure, and reaches predictive values of about 70 %( 1).
In the operating room, similar challenges arise. Surgeons must decide which tissue to remove or preserve, decisions that can determine the outcome of lung, brain, or endocrine surgery. To aid them, intraoperative frozen section( FS) analysis provides rapid histopathology: tissue is frozen, sectioned, and stained for microscopic examination. FS offers high diagnostic accuracy( 95 – 98 %)( 2), but the process is slow, technically demanding, and interrupts the operation for at least 25 – 45 minutes. In many hospitals, especially those without on-site pathology facilities, it is logistically challenging and sometimes even requires transport of the tissue to a larger hospital by a taxi. What surgeons and pathologists need is a fast, reliable, and label-free method to assess tissue morphology and cellularity in real time, ideally within 10 – 15 minutes, with minimal handling and submicron resolution. nonlinear interactions occur only at the focal volume, enabling intrinsic optical slicing and eliminating the need for physical sectioning.
Several nonlinear contrasts can reveal complementary aspects of tissue architecture. Raman-based methods, such as coherent anti-Stokes Raman scattering( CARS) and stimulated Raman scattering( SRS), visualize molecular vibrations and have been translated into stimulated Raman histology( SRH), a computationally rendered, label-free analog of H & E staining pioneered by academic( 3) and industrial groups, including LightCore and Invenio. Multiphoton fluorescence microscopy exploits intrinsic chromophores such as NAD( P) H and flavins to visualize cellular metabolism. Second harmonic generation( SHG) highlights ordered, non-centrosymmetric structures like collagen fibers, while third harmonic generation( THG) maps optical interfaces, visualizing cells, nuclei, and extracellular structures, without dyes or labels. Firms like Flash Pathology and Eleuthera Photonics use SHG, THG, and multiphoton autofluorescence to visualize unprocessed tissue in real time. A linear approach towards real-time histology is confocal microscopy combined with rapid fluorescent staining, implemented by Vivascope and SamanTree Medical, providing another route to fast histological imaging. Together, these advances outline a clear translational pathway toward rapid, photonics-based intraoperative pathology.
HIGHER HARMONIC GENERATION MICROSCOPY( HHGM) Our laboratory focuses on the translation of higher harmonic generation microscopy( HHGM), which integrates SHG, THG,
Figure 1. Schematic of the transportable HHG microscope developed by Flash Pathology BV. A 1070 nm is focused by a high NA objective, resulting in a resolution of 0.4 × 0.4 × 2.7 µ m 3. Generated signals are detected in epi-direction and divided into separate channels through dichroic mirrors. Photomultiplier tubes are used to detect the four channels simultaneously. Galvo mirrors( GM) are used to perform a bidirectional raster scan, creating a 2D image with a field of view of around 400 × 400 µ m 2. Larger 2D images are created as a mosaic of smaller images by moving the motorized sample stage in x- and y-direction. 3D images are obtained by moving the stage in vertical( z) direction. Fast overview images are generated at a speed of 7.5 s / mm 2, and high resolution images at 1 minute / mm 2. On average a biopsy is scanned in 5 minutes.
NONLINEAR MICROSCOPY: IMAGING WITHOUT LABELS Nonlinear optical microscopy provides a powerful framework for such real-time histology. By using near-infrared photons(> 900 nm), light penetrates deeper into tissue while limiting photodamage. Crucially,
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