Lyubovitsky Julia G, Krasieva Tatiana B, Xu Xiaoman, Andersen Bogi, Tromberg Bruce J
University of California, Beckman Laser Institute, Laser Microbeam and Medical Program, Irvine, California 92612, USA.
J Biomed Opt. 2007 Jul-Aug;12(4):044003. doi: 10.1117/1.2764462.
We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations.
我们报告了对小鼠毛囊进行多光子原位光学切片以及对转基因小鼠模型中病理性毛囊的初步研究。利用这种成像技术，我们快速获得了单个毛囊的详细三维（3-D）重建图像。由于多光子显微镜可将细胞的双光子激发荧光（TPF）与细胞外基质（ECM）的二次谐波产生（SHG）信号进行共定位，因此无需染色或机械切片。这些信号非常适合用于估计分子编码的毛囊三维几何结构，包括毛囊口的大小及其相对于皮肤表面的角度。在正常毛囊中，毛囊ECM产生的SHG信号与固有细胞荧光的SHG信号在光谱上分离，揭示了细胞成分与周围结缔组织之间复杂的空间相互作用。在病理性毛囊中，这些相互作用明显改变。特别是在转基因小鼠中，我们观察到细胞荧光缺失，且毛囊口相对于皮肤表面的角度明显变浅。TPF与SHG的结合对正常毛囊生理和特定基因改变引起的细胞和细胞外基质结构变化敏感。
