Tumor progression in breast cancer is significantly influenced by its interaction with the surrounding stromal tissue. Specifically, the composition, orientation, and alignment of collagen fibers in tumor-adjacent stroma affect tumor growth and metastasis. Most of the work done on measuring this prognostic marker has involved imaging of collagen fibers using second-harmonic generation microscopy (SHGM), which provides label-free specificity. Here, we show that spatial light interference microscopy (SLIM), a label-free quantitative phase imaging technique, is able to provide information on collagen-fiber orientation that is comparable to that provided by SHGM. Due to its wide-field geometry, the throughput of the SLIM system is much higher than that of SHGM and, because of the linear imaging, the equipment is simpler and significantly less expensive. Our results indicate that SLIM images can be used to extract important prognostic information from collagen fibers in breast tissue, potentially providing a convenient high throughput clinical tool for assessing patient prognosis.
Optical microscopy is a powerful tool for understanding the fundamentals of the microscopic world. However, for centuries its resolving ability remained limited by the optical diffraction limit. Super‐resolution fluorescence microscopy (SRFM) has been introduced to break the diffraction limit and significantly expand the fields in which optical microscopy can be applied. Unfortunately, SRFM contributes little towards axial resolution enhancement, rendering observation of the axial and three‐dimensional structures of biological tissues difficult; this may yield a misunderstanding of intracellular interactions. Based on the existing literature, the development of axial SRFM is still behind that of lateral SRFM. In light of this, this Review presents a comprehensive summary of the principles, development, characteristics, and applications of existing techniques for improving the axial resolution. This Review will provide a guide to researchers and promote further development of related technology.
Studying the coherence of an optical field is typically compartmentalized with respect to its different optical degrees of freedom (DoFs) -spatial, temporal, and polarization. Although this traditional approach succeeds when the DoFs are uncoupled, it fails at capturing key features of the field's coherence if the DOFs are indeed correlated -a situation that arises often. By viewing coherence as a 'resource' that can be shared among the DoFs, it becomes possible to convert the entropy associated with the fluctuations in one DoF to another DoF that is initially fluctuation-free. Here, we verify experimentally that coherence can indeed be reversibly exchanged -without loss of energy -between polarization and the spatial DoF of a partially coherent field. Starting from a linearly polarized spatially incoherent field -one that produces no spatial interference fringes -we obtain a spatially coherent field that is unpolarized. By reallocating the entropy to polarization, the field becomes invariant with regards to the action of a polarization scrambler, thus suggesting a strategy for avoiding the deleterious effects of a randomizing system on a DoF of the optical field.
We introduce the second-harmonic patterned polarization-analyzed reflection confocal (SPPARC) microscope-a multimodal imaging platform that integrates Mueller matrix polarimetry with reflection confocal and second-harmonic generation (SHG) microscopy. SPPARC microscopy provides label-free three-dimensional (3-D), SHG-patterned confocal images that lend themselves to spatially dependent, linear polarimetric analysis for extraction of rich polarization information based on the Mueller calculus. To demonstrate its capabilities, we use SPPARC microscopy to analyze both porcine tendon and ligament samples and find differences in both circular degree-of-polarization and depolarization parameters. Moreover, using the collagen-generated SHG signal as an endogenous counterstain, we show that the technique can be used to provide 3-D polarimetric information of the surrounding extrafibrillar matrix plus cells or EFMC region. The unique characteristics of SPPARC microscopy holds strong potential for it to more accurately and quantitatively describe microstructural changes in collagen-rich samples in three spatial dimensions.
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