Stimulated emission depletion microscopy

摘要

Aberrations in stimulated emission depletion microscopy are corrected using an adaptive optics approach using a metric which combines both image sharpness and brightness. Light modulators (22,32) are used to perform aberration correction in one or more of the depletion path (10), the excitation path (12), or the emission path from sample to detector.

主权项

1. A method of stimulated emission depletion microscopy using adaptive optics comprising:
(a) capturing a fluorescence image from a stimulated emission depletion microscope having an excitation light path and a first light modulator in a depletion light path defining a pattern on the first light modulator, wherein the first light modulator is a spatial light modulator or a deformable mirror; (b) calculating a measure of image sharpness of the fluorescence image, calculating a measure of image brightness of the fluorescence image, and calculating a metric combining the measure of image brightness and the measure of image sharpness; wherein the metric M is defined by
M=S+σβBf(S)  (2)wherein M is the metric, S an image sharpness measure, B an image brightness measure, σ is constant that has a value −1 or 1 depending on whether the aberration mode being corrected produces a maximum or minimum brightness, β is a constant and f(S) is a function of S that has a larger absolute value for a value of S above a threshold value ST and a lower absolute value for a value of S below the threshold value ST:
(c) adjusting the pattern on the first light modulator; and repeating (a), (b) and (c) to maximise or minimise the metric combining the measure of image brightness and the measure of image sharpness.