A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images
A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images
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DOI：
10.1046/j.1365-2818.2001.00945.x
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发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Jovin, TM
中科院分区：
文献类型：
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作者：
Heintzmann, R;Hanley, QS;Jovin, TM
可编程阵列显微镜 (PAM) 包含：空间光调制器 (SLM) 放置在宽场显微镜的主图像平面中，用于定义照明和/或检测模式。我们描述了同时收集两幅图像的特殊类型 PAM 的特征。共轭图像（I-c）由源自物平面并沿着照明光的光路返回的光形成。非共轭图像 (I-nc) 仅接收来自 SLM 不用于照亮样品的那些区域的光。双信号 PAM 提供比共焦激光扫描显微镜 (CLSM) 更省时的激发，并且可以更好地利用可用的发射光。与单面仪器相比，它具有优异的噪声特性。测量了系统在各种条件下的轴向响应，并对新型 I-c 图像的行为进行了表征。与仅收集 I-c 图像的系统（Nipkow 圆盘显微镜和先前表征的 PAM）一样，随着重复图案的单位单元尺寸减小，对荧光薄膜的轴向响应显示出轴向响应的锐化。双信号 PAM 可适应各种数据分析和收集策略。我们系统地研究了图案和晶胞尺寸对轴向响应的影响。足够稀疏的图案会产生由许多平行光束叠加形成的 I-c 图像，每个平行光束相当于 CLSM 的单个扫描点。系统的切片能力（由其轴向响应给出）对于给定的扫描模式和具有相同晶胞尺寸的处理后的伪随机序列（PRS）扫描来说是相似的。对于 PRS 扫描，光学切片是通过减去 I-nc 图像或从 I-c 图像中减去缩放的宽场图像来实现的。根据两种方法的噪声水平比较，非共轭减法明显优越。模拟了 I-c 和 I-nc 的点扩散函数，并比较了光学传递函数 (OTF) 的特性。非共轭成像中 OTF 的模拟不会遇到缺失锥体问题，从而能够单独对非共轭侧进行高质量反卷积。我们还研究了通过对 I-c 和 I-nc 数据进行组合最大似然反卷积而获得的图像的属性。
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.