Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

Nathan S Alexander,Maciej Wojtkowski,Grazyna Palczewska,Krzysztof Palczewski,Timothy S Kern,Patrycjusz Stremplewski

doi:10.1364/boe.7.002671

Abstract

Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].

Highlights

Two-photon fluorescence microscopy (TPM) is a powerful tool for imaging and diagnosing the health of the retina [1, 2]
Images of the retina vasculature were collected with TPM after injecting fluorescent dye (Fig. 4)
The present work demonstrates the ability to image retinal pigmented epithelium (RPE) cells with low laser powers and retina capillaries in vivo using two-photon fluorescence microscopy, without reflectance imaging hardware

Summary

Introduction

Two-photon fluorescence microscopy (TPM) is a powerful tool for imaging and diagnosing the health of the retina [1, 2]. In two-photon imaging the fundamental imaging depth limit is at such a distance within a tissue where further increases in the excitation power do not improve imaging contrast This loss of contrast deep within thick absorbing or scattering samples occurs because signaling from fluorophores located within the focal volume is overwhelmed by that from fluorophores located closer to sample surface and excited by scattered light [4]. Taking into account dramatically shorter mean free paths in the RPE and choroid, the theoretical maximum depth is 0.44 mm, still sufficient to image mouse retina and RPE This long penetration depth previously allowed collection of two-photon images of the RPE created by endogenous fluorophores [7]

Methods

Results

Conclusion