Optical photon reassignment microscopy (OPRA)

Abstract

To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used (Sheppard 1988). This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and resolution is obtained. Here we present optical photon reassignment microscopy (OPRA) which realizes this concept in an all-optical way obviating the need for image-processing. With the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light, an image with the advantages of photon reassignment can be acquired. However, just as in computational photon reassignment, a loss in confocal sectioning performance is caused by working with relatively open pinholes. The OPRA system shares properties such as flexibility and ease of use with a confocal laser scanning microscope, and is therefore expected to be of use for future biomedical routine research.

Post-processing strategies in image scanning microscopy

“…This approach offers an improved signal to noise ratio and increased resolution with relatively little modification to the existing hardware of a laser--scanning microscope. There are now several different implementations of ISM, underpinned by the concept of 'pixel reassignment' [1--3], achieved either by optical (ISIM [4,5], OPRA [6]) or computational means (MSIM [7] & spinning disk ISM [8]. This manuscript will explore the potential of computational approaches utilizing methods drawn from single molecule localization microscopy for a posteriori pixel reassignment.…”

“…Assuming that both emission and excitation point spread functions are identical, as would be the case for single photon fluorescence with no Stokes shift, the probability of an excitation and detection event is maximal at the position midway between the excitation and detection maxima. Because the detected light is therefore most likely to have originated from this position, it can be 'reassigned' to a location half the original distance from the excitation focus [1,6,8]. Performing this for each pixel corresponds to scaling the image by a factor of ½ around the excitation focus.…”

“…In general the excitation and emission wavelengths are different, resulting in excitation and detection PSFs of different widths, meaning that the position of the maximum of the resulting multiplication, and therefore the appropriate scaling factor (m), is slightly larger than ½ [6]. Approximating the PSFs as Gaussian, the appropriate theoretical scaling factor is given by equation 1 [6]. In practice, a factor of ½ is used [see 5,6,8].…”

“…The final image is the sum of the images generated at each excitation focus as the laser scans across the specimen. The width of the effective PSF can also be calculated using the standard deviations of the emission and excitation PSFs using equation 2 [6]; this value defines the resolution of the final ISM image and can be used to inform any subsequent deconvolution. The processes involved in ISM are summarised in figure 2.…”

“…Approximating the PSFs as Gaussian, the appropriate theoretical scaling factor is given by equation 1 [6]. In practice, a factor of ½ is used [see 5,6,8].…”