Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues
Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues
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DOI：
10.1529/biophysj.106.099457
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发表时间：
2007-08-01
影响因子：
3.4
通讯作者：
Gerritsen, Hans C.
中科院分区：
文献类型：
--
作者：
Palero, Jonathan A.;de Bruijn, Henriette S.;Gerritsen, Hans C.
利用多光子显微镜的深部组织穿透性和亚微米空间分辨率，以及光谱仪的高探测效率和纳米光谱分辨率，记录了小鼠皮肤组织本征发射的光谱图像。检测到了来自细胞和细胞外结构的自发荧光，来自胶原的二次谐波信号，以及与胶原的拉曼散射相关的窄带发射。通过波长到RGB彩色图像转换的光谱图像的可视化，使我们能够识别和区分组织结构，如表皮角质形成细胞、富含脂肪的角质形成细胞、细胞间结构、毛囊、胶原、弹性蛋白和真皮细胞。我们的结果还显示了小鼠皮肤切除组织切片、厚切除组织和活体组织样本之间的形态和光谱差异。对不同波长的胶原蛋白激发的结果表明，窄带发射的来源是胶原蛋白的拉曼峰。此外，还实验研究了胶原蛋白二次谐波强度对振荡光谱的依赖关系。总体而言，光谱成像提供了目前传统的多光子成像系统不容易获得的丰富信息。
The deep tissue penetration and submicron spatial resolution of multiphoton microscopy and the high detection efficiency and nanometer spectral resolution of a spectrograph were utilized to record spectral images of the intrinsic emission of mouse skin tissues. Autofluorescence from both cellular and extracellular structures, second-harmonic signal from collagen, and a narrowband emission related to Raman scattering of collagen were detected. Visualization of the spectral images by wavelength-to-RGB color image conversion allowed us to identify and discriminate tissue structures such as epidermal keratinocytes, lipid-rich corneocytes, intercellular structures, hair follicles, collagen, elastin, and dermal cells. Our results also showed morphological and spectral differences between excised tissue section, thick excised tissue, and in vivo tissue samples of mouse skin. Results on collagen excitation at different wavelengths suggested that the origin of the narrowband emission was collagen Raman peaks. Moreover, the oscillating spectral dependency of the collagen second-harmonic intensity was experimentally studied. Overall, spectral imaging provided a wealth of information not easily obtainable with present conventional multiphoton imaging systems.