Measurement of the three‐dimensional microscope point spread function using a Shack–Hartmann wavefront sensor

Abstract

SummaryWe present a technique to measure the wavefront in the exit pupil of a microscope to determine the microscope's threedimensional point spread function (PSF) experimentally. The wavefront yields the microscope PSF through a Fourier transform that models propagation of light from the exit pupil to the image plane. A Shack-Hartmann wavefront sensor is used to measure the wavefront shape by recording lateral displacements of a grid of focused spots created by a lenslet array. The displacement of each spot is related to the local wavefront slope. Thus, with appropriate sampling across the exit pupil, the entire wavefront can be reconstructed. This technique does not require the use of a sub-resolution object to obtain the three-dimensional microscope PSF. Consequently, larger, brighter fluorescent objects may be imaged, thereby reducing the requirements for detector sensitivity and leading to a three-fold increase in the axial range over which the PSF is measured. The Shack-Hartmann technique results in a description of the PSF as a continuous function whose sampling is not dependent on the size of the CCD pixels. The Shack-Hartmann sensor is not limited by the numerical aperture of the objective and can easily be calibrated to measure the PSF at any wavelength.

Adaptive optics for fluorescence wide-field microscopy using spectrally independent guide star and markers

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“…Those "guide stars" or "beacons" allow collection of strong signals, thus providing fast wavefront measurements with high signal-to-noise ratio. 15,16 In Ref. 17, plastic beads focus illumination light inside the sample and generate stars used to improve the precision and strength of optical tweezers.…”

Characterization of spatially varying aberrations for wide field-of-view microscopy

“…Over the past half-century, many unique aberration characterization methods have been reported [5][6][7][8][9][10][11][12][13][14][15][16][17]. Each of these methods attempts to estimate the phase deviations or the frequency response of the optical system under testing.…”

“…Each of these methods attempts to estimate the phase deviations or the frequency response of the optical system under testing. Several relatively simple noninterferometric procedures utilize a Shack-Hartmann wavefront sensor [11][12][13], consisting of an array of microlenses that each focus light onto a detector. The local tilt of an incident wavefront across one microlens can be calculated from the position of its detected focal spot.…”

Phase‐retrieved pupil function and coherent transfer function in confocal microscopy

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