Optimal model-based sensorless adaptive optics for epifluorescence microscopy

Abstract

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Fig 1. Simplified schematic of the proposed experimental setups.

A: Pinhole based confocal microscopes, and B: camera based microscopes. EX—Excitation source, AB/S—Aberration correction (and scanning), OL—Objective lens, S—Sample, TL—Tube lens, PH—Pinhole, DET—detector, CAM—camera. C, D: Example image of fluorescence spots in the presence of an aberration in C, and after correction in D. The metric employed in the optimization is the second moment of the average image of the spots.

Fig 2. Scheme of the physical model of a microscope.

A coherent illumination with an aberration is considered in the pupil plane of the system, and coherently propagated to the object space to obtain the illumination point spread function. Fluorescence emission is calculated as multiplication of the excitation point spread function with the object distribution. Incoherent propagation to camera space is calculated as the convolution of fluorescence emission with the illumination point spread function.

Fig 3. Experimental measurements of the normalized gradient-orthogonal base generated by a 69 actuators DM.

In the red highlight, the three modes used for displacement, and therefore excluded from the aberration correction procedure.

Fig 4. Experimental measurements of parameter P for an optimal, gradient orthogonal base and a Zernike base.

The diagonal values are infinite, and therefore omitted.

Fig 5. Representative results of optimization for small and severe aberrations.

Images reported are: a- Confocal image for non compensated severe aberration. b- Confocal image for severe aberration after two correction iterations with Zernike base. c- Confocal image for severe aberration after two correction iterations with gradient orthogonal base. d- Epifluorescence image for non compensated severe aberration. e- Epifluorescence image for severe aberration after two correction iterations with Zernike base. f- Epifluorescence image for severe aberration after two correction iterations with gradient orthogonal base.