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

被引:51
作者
Heintzmann, R [1 ]
Hanley, QS [1 ]
Arndt-Jovin, D [1 ]
Jovin, TM [1 ]
机构
[1] Max Planck Inst Biophys Chem, Dept Mol Biol, D-37070 Gottingen, Germany
来源
JOURNAL OF MICROSCOPY-OXFORD | 2001年 / 204卷 / 02期
关键词
axial resolution; CLSM; confocal; DMD; micromirror device; ML-deconvolution; optical sectioning; scanning;
D O I
10.1046/j.1365-2818.2001.00945.x
中图分类号
TH742 [显微镜];
学科分类号
摘要
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 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 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-c 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.
引用
收藏
页码:119 / 135
页数:17
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