Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO

Abstract

Scanning laser ophthalmoscopy (SLO) employs the eye’s optics as a microscope objective for retinal imaging in vivo. The mouse retina has become an increasingly important object for investigation of ocular disease and physiology with optogenetic probes. SLO imaging of the mouse eye, in principle, can achieve submicron lateral resolution thanks to a numerical aperture (NA) of ~0.5, about 2.5 times larger than that of the human eye. In the absence of adaptive optics, however, natural ocular aberrations limit the available optical resolution. The use of a contact lens, in principle, can correct many aberrations, permitting the use of a wider scanning beam and, thus, achieving greater resolution then would otherwise be possible. In this Letter, using an SLO equipped with a rigid contact lens, we report the effect of scanning beam size on the lateral resolution of mouse retinal imaging. Theory predicts that the maximum beam size full width at half-maximum (FWHM) that can be used without any deteriorating effects of aberrations is ~0.6 mm. However, increasing the beam size up to the diameter of the dilated pupil is predicted to improve lateral resolution, though not to the diffraction limit. To test these predictions, the dendrites of a retinal ganglion cell expressing YFP were imaged, and transverse scans were analyzed to quantify the SLO system resolution. The results confirmed that lateral resolution increases with the beam size as predicted. With a 1.3 mm scanning beam and no high-order aberration correction, the lateral resolution is ~1.15 μm, superior to that achievable by most human AO-SLO systems. Advantages of this approach include stabilization of the mouse eye and simplified optical design.

Temporal speckle-averaging of optical coherence tomography volumes for in-vivo cellular resolution neuronal and vascular retinal imaging

“…However, the ability to visualize the highly transparent somas of the neurons in the mouse retina without the use of AO makes the TSA very attractive. As an alternative to AO, it is possible to use a somewhat larger imaging beam size on the mouse pupil to increase the system NA, 29 or switch to shorter wavelength (visible light OCT 61 , 62 ) imaging. Both strategies will predictably improve the system resolution and yield a better contrast of cell somas.…”

“… 13 The beam at the mouse pupil was 0.5 mm, which corresponds to a NA of 0.12 for the imaging system. 29 The light power is for SLO (488 nm laser, Coherent) and for OCT (center wavelength 860 nm with a bandwidth of 132 nm, SLD, Superlum Ltd.). A custom spectrometer using Balser line scan camera with 2048 pixels was used to acquire the OCT spectra.…”

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

“…Mouse rod outer segments (OS) are in effect genetically manipulable, femtoliter test tubes whose protein constituents (15,16), as well as their biochemical reactions (17)(18)(19) and light-stimulated translocations (20)(21)(22), are well known and have been measured primarily with ex vivo methods. Advances in imaging technology have now made it possible to achieve subcellular-resolution imaging of the mouse retina in vivo (23)(24)(25)(26). We recently developed a multimodal ocular imaging system for mice that combines confocal laser scanning ophthalmoscopy (SLO) and OCT (27) and used this system to quantify photoactivation (bleaching) of rhodopsin in vivo (28).…”

Aperture phase modulation with adaptive optics: a novel approach for speckle reduction and structure extraction in optical coherence tomography

“…The imaging beam at the mouse pupil has a diameter of 0.93 mm, a size for which ocular aberration is non-negligible [41,42]. The mouse eye's aberrations were first corrected using wavefront sensorless (WFSL) aberration correction software [36,43,44] with image intensitybased optimization metric.…”