Image formation in image scanning microscopy, including the case of two-photon excitation

Abstract

The effect of combining the image scanning microscopy (ISM) technique with two-photon fluorescence microscopy is analyzed. The effective spatial frequency cutoff can be doubled, as compared with conventional two-photon fluorescence microscopy, and the magnitude of the optical transfer function near the cutoff of conventional two-photon microscopy is increased by orders of magnitude. For the two-photon case, it is found that the optimum pixel reassignment factor in ISM is not equal to one half, as is often assumed in single-photon fluoresence image scanning microscopy, because the excitation and detection point spread functions are different. The optimum reassignment factor depends on the noise level, and in general the useful cutoff spatial frequency is about 1.8 times that for conventional two-photon microscopy. The effect of altering the reassignment factor in single-photon fluorescence ISM with a Stokes shift is also investigated. Illumination using pupil filters, such as by a Bessel beam, is considered. Using a ring detector array is found to result in good imaging behavior, exhibiting a sharpening of the point spread function by a factor of 1.7 compared with conventional fluorescence. Image formation in ISM can be considered in a four-dimensional spatial frequency space, giving new insight into the imaging properties. This approach is related to phase space representations such as the Wigner distribution function and the ambiguity function. A noniterative algorithm for image restoration is proposed.

Super resolution measurement of collagen fibers in biological samples: Validation of a commercial solution for multiphoton microscopy

“…ISM is a detection-based technique where the point-spread function (PSF) of the diffracted light from sub-resolution objects are collected, and this additional information is used to improve the resolution of the resulting image [16][17][18][19][20][21]. The effect of combining ISM with two-photon fluorescence microscopy has been evaluated in detail by Sheppard et al [22], and SR-MP has been implemented by Gregor et al [17], who demonstrated an all-optical ISM design that allows straightforward implementation into existing microscopes. However, all SR-MP systems currently require custom-built or heavily modified systems, which are difficult to implement for biology focused laboratories.…”

Image scanning microscopy (ISM) with a single photon avalanche diode (SPAD) array detector

“…If a scanning illumination spot is combined with a detector array [8], we have a 4 dimensional signal, and unlike in the case of a confocal microscope with a small pinhole (single-pixel detector) we detect almost all the light from the object, which is particularly important for fluorescence microscopy when the signal is weak. The image signal is basically a cross-correlation, and is highly redundant [9,10]. It has more than sufficient information to reconstruct the image.…”

“…The effective imaging point is midway strictly only for the case when the point spread functions for illumination and detection are identical. Otherwise, the optimum reassignment factor takes a slightly different value [10]. Ref.…”

“…Hence, it is possible to investigate improvements in terms of resolution and signal-to-noise ratio of the proposed approach. Experimental results, in The imaging performance of ISM has been explored in a few papers [8,10,12,28,49]. For a confocal fluorescence microscope with a small pinhole, the intensity point spread function (PSF) is given by the product of the illumination and detection PSFs: H 1 (x)H 2 (x) .…”

“…The effect of Stokes shift was explored in Ref. [10]. In fact, any value of reassignment factor can be used, a reassignment factor of zero (back to the scan position) giving a scanning image with an effective pinhole size equal to the array size, and a reassignment factor of unity (i.e.…”