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应用资料

Discovering Fine Neurovascular Structures in Tibial Epiphysis Using the FLUOVIEW FV3000 Microscope


Imaging of Fine and Complex Tissue Structures while Reducing Bleaching

Imaging blood vessels and sensory nerves in the epiphysis of a knee joint is difficult because the nerves and vessels form a complex structure within a narrow area. Owing to its high transmission efficiency, the FLUOVIEW FV3000 confocal laser scanning microscope enables bright, high-resolution imaging of fine structures while using low laser power, which helps reduce photobleaching in the sample. Using this capability of the FV3000 microscope, we were able to successfully image a complex 3D structure of sensory nerves and their surrounding vasculature penetrating a foramen in the tibial epiphysis.

Figure 1: Sensory nerves and surrounding vasculature penetrate a foramen in the tibial epiphysis (3D image) Sensory nerves (EYFP, cyan), blood vessels (Alexa Fluor 594, magenta), nuclei (DAPI, orange)

Figure 1: Sensory nerves and surrounding vasculature penetrate a foramen in the tibial epiphysis (3D image)
Sensory nerves (EYFP, cyan), blood vessels (Alexa Fluor 594, magenta), nuclei (DAPI, orange)


Imaging equipment
Microscope: FLUOVIEW FV3000 System
Objective: 100X oil immersion objective (UPLSAPO100XO)


Discovery of Neurovascular Structures in Tibial Epiphysis

Understanding the vascular and neural projections to the knee joint is important for relieving pain in knee arthropathy. However, until now, researchers have been unable to fully observe the fine structures formed by sensory nerves and blood vessels throughout the knee joint. With the FV3000 microscope, these structures are clearly visible for the first time. We observed that sensory nerves in the knee joint exist not only in the meniscus, but also in the tibial epiphysis. These sensory nerves are entwined with surrounding blood vessels, and together the neurovascular structure penetrates a foramen in the tibial epiphysis.

Reference: Koichi Matsuo, et al. “Innervation of the tibial epiphysis through the intercondylar foramen.” Bone, 120 (2019) 297–304

Figure 2:Neural and vascular foramen
Figure 2:Neural and vascular foramen


How the FV3000 Confocal Microscope Facilitated Our Experiment

Highly Sensitive TruSpectral Detector Performs Imaging with Low Phototoxicity

The TruSpectral detector is equipped with a transmission-type diffraction grating that can transmit fluorescent signals with more than 40% higher efficiency compared to conventional reflection-type diffraction gratings. Because of the enhanced tranmission, less laser power is needed to acquire images, thus reducing phototoxicity.

Acquire High Signal-to-Noise Ratio Images under Low Excitation Light

The GaAsP photomultiplier tube (PMT) incorporates up to 4 channels with a maximum quantum efficiency of 45%, enabling users to view samples that were too dim to view with conventional equipment. Peltier cooling reduces background noise by 20% for high S/N ratio images under exceptionally low excitation light.


Comment by Dr. Katsuhiro Kawaai

Dr. Katsuhiro Kawai

To image the fine and complex 3D structure of neurites in a narrow area, a 100X oil immersion objective with a high numerical aperture (NA) was required; however, bleaching caused by concentrated laser power was a concern. Fortunately, owing to the high sensitivity of the FV3000 microscope, we were able to keep laser power low while obtaining high-resolution images from 50 Z planes at 0.45μm intervals without photobleaching our sample.


Acknowledgments
This application note was prepared with the help of the following researchers:
Dr. Katsuhiro Kawaai and Dr. Koichi Matsuo, Laboratory of Cell and Tissue Technology, Keio University School of Medicine

Products used for this application

激光扫描共聚焦显微镜

FV3000

  • 可提供配置常规扫描的FV3000或配置常规/共振混合扫描的FV3000RS
  • 所有通道均可进行全新高效且精确的全真光谱TruSpectral检测
  • 针对活细胞成像的高灵敏度和低光毒性优化
超平场超级复消色物镜

UPLXAPO

  • 高数值孔径,高平场性,从400nm到1000nm宽波段色差校准
  • 极其精准的成像效果使之具有广泛的应用,从明场/荧光显微镜到共聚焦/超高分辨率显微镜都可使用

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