Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope

Abstract

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to artifacts on the tissue surface from electrocautery, surgical ink or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin fixed, paraffin embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in-situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins

“…While evaluation of multiple depth serial sections per bread loaf is not typically performed for breast margin assessment, the ability to image through surgical debris is likely to be important. Combined with our recent demonstration that inexpensive and compact ytterbium fiber lasers are effective for NLM imaging of surgical specimens [43], the ability to operate at long wavelengths may be important for clinical translation of compact, affordable real-time surgical equipment that can image through surgical debris and tissue marking ink.…”

“…Tissue specimens were inked to simulate surgical margins and then grossed into 'bread loaf' specimens. Following grossing, specimens were fluorescently labeled using a protocol described previously [43] that consists of 2 minutes immersion in 40 μg/ml acridine orange (AO) combined with 40 μg/ml sulforhodamine 101 (SR101) dissolved in 50% ethanol/water solution followed by 20 seconds rinse in buffered saline.…”

“…The system lateral and axial resolution was degraded because of a substantially thicker imaging glass (400 μm) than the objectives' design thickness (180 μm), although the effect is modest at 0.45 NA and insignificant at 0.25 NA. In a previous study, we evaluated attenuation at both wavelengths in normal human breast tissue and obtained an e −1 depth of ~34 μm at 780 nm and ~52 μm at 1030 nm [43]. NLM resolution was further assessed by imaging discarded human breast tissue taken from the superficial aspect of a mastectomy specimen using both 5x/0.25 and 10x/0.45 objectives.…”

“…By reproducing classic histological features, these techniques enable direct histological evaluation of tissue, but without the delay that makes paraffin embedding or cryosectioning prohibitively time-consuming in many surgeries. Furthermore, several groups have demonstrated extraction of exogenous labels following fluorescent imaging, greatly reducing the possible risk of interference with postoperative immunohistochemistry or DNA assays, while retaining the high contrast enabled by molecularly specific labels [32,43]. Of these techniques, NLM is particularly attractive because of its very high imaging speed and ability to image below the tissue surface through blood and surgical debris.…”

Non-contact reflection-mode optical absorption spectroscopy using photoacoustic remote sensing

“…These methods have yet to widely adopted because they do not fully address the requirements described above. Techniques such as fluorescence microscopy [6][7][8][9] structured light microscopy 10,11 , light-sheet microscopy (LSM) 12 and microscopy with ultraviolet surface excitation (MUSE) 13,14 have demonstrated promising results in providing H&E-like contrast on tissue mounted on microscope slides or freshly excised tissue. However, these methods cannot image unstained tissue and require the application of fluorescence dyes to the sample, adding time, expense, and the potential for occupational exposure to these chemicals.…”