A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (I_c) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non‐conjugate image (I_nc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual‐signal PAM provides much more time‐efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single‐sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel I_nc image characterized. As in systems in which only I_c images are collected (Nipkow‐disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual‐signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an I_c image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an I_nc image or, alternatively, a scaled widefield image from the I_c image. Based on the comparative noise levels of the two methods, the non‐conjugate subtraction was significantly superior. A point spread function for I_c and I_nc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non‐conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non‐conjugate side alone. We also investigated the properties of images obtained by subjecting the I_c and I_nc data to a combined maximum likelihood deconvolution.