Rapid histological imaging of bone without microtome sectioning using nonlinear microscopy.

Tissue preparation for histologic evaluation of bone is particularly lengthy, limiting its use in intraoperative or intraprocedural histological evaluation. Nonlinear microscopy (NLM) is an optical sectioning microscopy method that can visualize pathology in freshly excised tissue without requiring physical microtome sectioning. This study describes a rapid protocol for NLM imaging of bone and associated cartilage. NLM imaging was performed on 71 specimens of normal bone as well as arthritic, malignant and inflammatory bone tissue from 40 patients who underwent joint replacement, amputation, bone marrow biopsy or autopsy. Specimens ranged in size from core needle biopsies to transections of entire femoral heads. Specimens were stained with acridine orange and sulforhodamine 101, nuclear and cytoplasmic/stromal fluorescent dyes, for 5 min, then rinsed for 30 s. NLM fluorescent images were displayed using colors analogous to hematoxylin and eosin (H&E) to facilitate interpretation. Pathologists examined NLM images of the specimens in real time by rapidly translating the specimen to areas of interest, similar to a standard transmission light microscope. By adjusting the NLM focus depth, images from a few-μm-thick layer could be obtained down to ~100 μm below the tissue surface, analogous to serial sectioning. Following real-time NLM imaging, the tissue was processed for conventional paraffin histology, and H&E slides were compared to recorded NLM images. Similarities and differences between NLM and paraffin H&E were assessed. NLM enabled visualization of normal bone architecture, including the lamellar matrix and osteocytes of trabecular bone, articular cartilage, as well as pathological bone features such osteoarthritis, osteomyelitis, and malignancy with an appearance resembling the paraffin H&E. Differences such as changes in cell border sharpness, cellular and nucleolar size, and color patterns were noted, suggesting that training is required for accurate evaluation of bone pathology with NLM. Irregular surface contours and debris generated by gross tissue preparation of bone can make some regions difficult to evaluate with NLM, but the ability to perform rapid three-dimensional translation and sub-surface imaging reduced these problems. NLM is a promising technique for rapid evaluation of bone pathology. Further studies assessing diagnostic performance are warranted.