GRadient INdex (GRIN) System
Challenges in imaging Ca2+ in large numbers of endothelial cells in physiologically-relevant pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca2+ signalling.
We have developed a miniature wide-field, gradient index (GRIN) optical probe designed to fit inside an intact, pressurized artery. Using this setup, we are able to image Ca2+ signals with subcellular resolution in a large number (~200) of naturally connected endothelial cells at various pressures.
This cartoon shows the GRIN microprobe assembly inside a pressurized artery and endothelium.
The GRIN microprobe consists of a single pitch GRIN relay lens (SRL-050; Nippon Sheet Glass, USA) with dimensions ø = 0.5 mm and length = 30.2 mm.
A 0.5 mm x 0.5 mm x 0.5 mm aluminium-coated micro-prism (66-771; Edmund Optics, USA) is attached to the distal surface using UV-curing optical epoxy (NAO 68; Norland Products, USA). The distal end of the GRIN rod with the prism attached is shown in the high resolution microscope image below.
The lens was sheated in a surgical stainless steel tube (0.71 mm outder diameter) for mechanical protection.
In this single lens configuration, the GRIN rod acts to reconjugate the image plane of detection optics through the length of the cylinder.
Wilson, C., Saunter, C.D., Girkin, J.M. and McCarron, J.G., 2015. Pressure‐dependent regulation of Ca2+ signalling in the vascular endothelium. The Journal of physiology, 593(24), pp.5231-5253. DOI: 10.1113/JP271157
The Setup
The Setup
Imaging using the GRIN system
A Time series Ca2+ images of endothelial cells showing the progression of the Ca2+ response evoked by ACh (100 μm; bath applied; 1 μm estimated at the vessel lumen) recorded at 60 mmHg. Images are composed of instantaneous Ca2+ activity (green) overlaid on standard deviation images (greyscale) indicative of total Ca2+ activity. Scale bar: 100 μm. B ACh‐evoked Ca2+ signals from the same cells shown in A. In the left panel (unaligned data) the range of times for Ca2+ to increase for each of the ∼200 cells is approximately 4 s. The spread of responses resulted in mean data representing the data poorly. The position of the first peak of derivative signals was used to align Ca2+ signals, synchronizing the Ca2+ rises occurring in each due to the action of ACh (middle panel ‘aligned’) and illustrating total Ca2+ activity (red line). On successive application of ACh (20 min reequilibration following wash), the response to ACh was reproducible. C Select examples of unaligned (left) and aligned (right) cellular Ca2+ responses from ROIs shown in A. The grey box illustrates the time point of measurements from aligned signals. D Summary data illustrating the reproducibility of Ca2+ responses upon repeat application (wash + 20 min reequilibration) with ACh.
Wilson, C., Saunter, C.D., Girkin, J.M. and McCarron, J.G., 2015. Pressure‐dependent regulation of Ca2+ signalling in the vascular endothelium. The Journal of physiology, 593(24), pp.5231-5253. DOI: 10.1113/JP271157
Curved image plane correction in the GRIN probe
In uniform optics, field curvature arises from the use of curved optics producing images on a curved plane. This is a problem when imaging as the majority of detectors are flat.
For GRIN optics, which may be very narrow and long, far-field waves travel further than near field ones due to the refractive index changes in the material , causing off-axis rays to be focussed at a different focal plane