Application Notes

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.

http://adobeassets.evidentscientific.com/content/dam/video/video/library/movie_neurovascular-structures_01(2)_480.mp4

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

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

FV4000

Game-changing dynamic range for imaging from the macro scale to subcellular structures
Multiplex up to six channels simultaneously with TruSpectral technology
Redesigned high-speed, high-resolution scanners for fixed and live cell imaging
Improved depth and photosensitivity with pioneering NIR capabilities and renowned optics
Peace of mind with the reliable, repeatable SilVIR detector
Industry leading ^* ten laser lines with a broader spectral range from 405 nm to 785 nm

_{*As of October 2023.}

UPLXAPO

These extended apochromat objectives offers a high numerical aperture (NA), wide homogenous image flatness, and 400 nm to 1000 nm chromatic aberration compensation. They enable high-resolution, bright image capture for a range of applications, including brightfield, fluorescence, and confocal super resolution microscopy.

High NA, wide homogeneous image flatness, and wide range chromatic aberration compensation from 400 nm to 1000 nm
Highly reliable precision image for wide applications from bright field/fluorescence microscopy to confocal /super resolution microscopy