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

A programmable array microscope (PAM) incorporates a. spatial light modulat or (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describ e the characteristics of a special type of PAM collecting two images simult aneously. The conjugate image (I-c) is formed by light originating from the object plane and returning along the optical path of the illumination ligh t. The nonconjugate 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 scanni ng microscope (CLSM) and greater utilization of the available emission ligh t. It has superior noise characteristics in comparison to single-sided inst ruments. The axial responses of the system under a variety of conditions we re measured and the behaviour of the novel I-c image characterized. As in s ystems in which only I-c images are collected (Nipkow-disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent fil ms showed a sharpening of the axial response as the unit cell of the repeti tive patterns decreased in size. The dual-signal PAM can be adapted to a wide range of data analysis and col lection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse pattern s lead to an I-c image formed by the superposition of the many parallel bea ms, 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, wer e 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, alternativel y, a scaled widefield image from the I-c, image. Based on the comparative n oise levels of the two methods, the non-conjugate subtraction was significa ntly superior. A point spread function for I-c, and I-nc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulati ons of the OTF in non-conjugate imaging did not suffer from the missing con e 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.