Slit lamp microscopy on a cellular level using in vivo confocal laser scanning microscopy

Abstract

Purpose : In vivo corneal 3D imaging on a cellular level and cross-section reconstruction is limited by patient’s compliance and mainly by involuntary eye movements, which induce image distortions and in-plane shifts during image acquisition. To overcome this limitation we designed a dedicated contact element with a concave surface fitted onto the corneal spherical surface to constrain eye movements. Furthermore, our concept incorporates a piezo actuator stage adjusting a microscope objective for focal plane positioning.

Methods : For imaging, we used a microscope lens adapted to an HRTII. The customized, modular lens adapter utilizes a piezo actuator for computerized focus control. Image stacks were captured from three human subjects with 30 fps (field of view: 349x349 µm²). To compare the standard planar with the new curved contact cap, we acquired several stacks from each subject. Mean image-to-image in-plane shifts (n=1000 for each subject) were calculated for both designs for quantitative comparison of eye movements. After brightness adjustments and elastic en face image registration [1], 3D and cross-section reconstructions with isotropic resolution were rendered from a stack of images recorded with a constant focus drive speed of 30 µm/s.

Results : Significant (p<0.0001) reduction of image shifts was observed in all three subjects, see Table 1. Successful 3D image reconstruction as well as cross-sectioning with cellular resolution was only possible after elastic registration according to [1]. Figure 1 represents a 3D reconstruction (a) and a cross-section image (b) analogous to a slit lamp image of the anterior cornea, exemplifying the capabilities of this technique to resolve single cells in corneal tissues.

Conclusions : Compared to the conventional approach for in vivo confocal microscopy, the new concept significantly reduces image distortions and offsets during 3D data acquisition. The possibility to get oblique sections in any spatial direction opens the window to slit lamp microscopy on a cellular level. Further effort is necessary to correct axial distortions during image acquisition and to provide real-time capability.

[1] Allgeier et al. Invest Ophthalmol Vis Sci. 2011

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Table 1: Measured mean values of image-to-image in-plane shift for planar (MSP) and curved (MSC) cap.

 

Fig. 1: Volume reconstruction (a) and cross-section (b) of the anterior cornea.