Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

Abstract

, "Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue," J. Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Review of methods for intraoperative margin detection for breast conserving surgery

“…This shows the correlation between the false-color images created using nonlinear microscopy and H&E images. 106 The study concluded that confocal florescence microscopy offered similar information to nonlinear microscopy, but at much lower cost. When magnification was increased significantly, a blurring effect did occur with confocal fluorescence microscopy (CFM); however, nonlinear microscopy offers molecular information with very strong sensitivity and specificity.…”

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

“…However, the ability to image through surgical debris, blood and tissue marking ink is an important advantage of NLM that enables imaging of tissue aspects that have surface contamination typical of breast surgical specimens. In a previous manuscript, we evaluated CFM and found its ability to image subsurface features to be significantly more limited than NLM [28], while MUSE can only image a single plane on the tissue surface and so cannot perform subsurface assessment [32]. OCT provides excellent subsurface imaging but has limited nuclear contrast.…”

“…Because physical sectioning accounts for the vast majority of processing delay for both FFPE and FSA histology, optical sectioning can dramatically reduce processing times and enable intraoperative histological examination in scenarios where FFPE and FSA histology are too time consuming. Various methods have been proposed for imaging breast, prostate and other surgical margins without physical sectioning, including optical coherence tomography (OCT) [18][19][20][21][22], reflectance confocal microscopy (RCM) [23,24], confocal fluorescence microscopy (CFM) [25][26][27][28][29], structured illumination microscopy (SIM) [30], light sheet microscopy [31], microscopy with ultraviolet surface excitation (MUSE) [32,33], and nonlinear microscopy (NLM) [34][35][36][37][38]. Stimulated Raman scattering (SRS) has also been demonstrated for surgical histology [39], but typically has appreciably lower imaging speed or signal to noise ratio when performed without physical sectioning in reflectance mode.…”

Label-free optical imaging technologies for rapid translation and use during intraoperative surgical and tumor margin assessment

“…This is an attractive strategy that can seamlessly integrate the technical advantages of multiple techniques while minimizing the practical limitations of each. Giacomelli et al 20 and Yoshitake et al 21 demonstrated the use of confocal and multiphoton microscopy for virtual H&E imaging of excised sliced breast specimens, enabling determination of various breast pathologies. Glaser et al 22 recently demonstrated volumetric imaging of fresh slices of breast and prostate tissue immersed in silicone oil using light sheet microscopy.…”

“…21,41,42 Journal of Biomedical Optics 021104-5 February 2018 • Vol. 23 (2) Recently, Tu et al 43 and Tu and Boppart 44 developed a platform system based on a pumped photonic crystal fiber source that produced widely coherent supercontinuum suitable for pulse compression and shaping to efficiently drive label-free nonlinear processes of two-and three-photon autofluorescence, secondand third-harmonic generation, and broadband hyperspectral coherent anti-Stokes Raman scattering.…”