Correction-free remotely scanned two-photon in vivo mouse retinal imaging

Abstract

Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications. The in vivo two-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue. However, to date, this has only been possible using relatively complex adaptive-optics systems. Here, the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality, optically sectioned fundus images. By remotely scanning the focus using an electronically tunable lens, high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired, thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging. Moreover, the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator, GCaMP6s. These results and the simplicity of the new add-on optics are an important step toward several structural, functional, and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.

Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases

“…In the case of RPE cells affixed on a scaffold, the cells may not migrate and the entire transplant could be extracted and would be an invasive procedure, whereas locating and selecting suspended single cells from RPE replacement or any preservation therapy would be nearly impossible. In vivo imaging systems, such as two-photon imaging, are able to detect differences in retinal thickness, light stimulation, and single cells in animal models, but detecting and then extracting these cells would not be feasible in humans, and the patient would be subjected to any lingering adverse effects of the cells (Bar-Noam et al, 2016; Maeda et al, 2014; Sharma et al, 2016). …”

In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina

“…In addition, several retinal imaging tools for the murine eye have been developed. [2][3][4][5][6][7][8] A commercially available retinal imaging microscope was generated from a prototype system with a 4 3 48 field of view and further developed with a wider image view and better image contrast. 9 However, the ophthalmic features of rodents, including size and optical features, compared with human eyes limit the acquisition of high-quality images in these small animals.…”

Simultaneous in vivo confocal reflectance and two-photon retinal ganglion cell imaging based on a hollow core fiber platform

“…4). Even at low-power levels, (close to the human use safety threshold), 9 individual RGCs were clearly visible. The quantity of fluorescence photons detected was too low to calculate an additional lifetime map.…”

“…Reporter molecules in cell bodies can be excited with IR light to allow differential activation of rod and cone photoreceptors by wavelengths in the visually sensitive range to evoke responses in the retina. [6][7][8][9] An additional advantage of TPE is reduced phototoxicity. 8 TPE fluorescence imaging enables the study of functional physiological processes, which, in combination with in vivo ophthalmoscopy, represent powerful imaging techniques that are well suited for noninvasive in vivo retinal imaging.…”

“…10,11 Systems used thus far are bulky and complex and require sophisticated adaptive optics (AO) to compensate for the wavefront errors introduced by the lens and cornea, hence achieving a tightly focused spot and increased TPE signal rate. Recently, a non-AO two-photon mouse retinal imaging system was described; 9 however, a water-immersion contact microscope objective lens was used for the focusing optics and the imaging required surgical fixation of the mouse skull.…”