ausblenden:
Schlagwörter:
axial resolution; CLSM; confocal; DMD; micromirror device; ML-deconvolution; optical sectioning; scanning
Zusammenfassung:
A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane, where it is used to define a pattern of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non-conjugate image (Inc) 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 microscopes (CLSM) and greater utilization of the available emission light. It thus 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 behavior of the novel Inc image characterized. As in systems in which only Ic 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 Ic 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 Inc image (non-conjugate subtraction approach) or, alternatively a scaled widefield image (widefield subtraction approach) from the Ic image. Based on the comparative noise levels of the two methods, the non-conjugate subtraction was significantly superior. A point spread function for Ic and Inc was simulated and properties of the optical transfer functions (OTF) are compared. Simulations of the OTF in non-conjugate imaging did not suffer from the missing cone problem, which enabled a high quality deconvolution of the non-conjugate side on its own. We also investigated the properties of images obtained by subjecting the Ic and Inc data to a combined maximum likelihood (ML) deconvolution.
