Hanley Q S, Verveer P J, Gemkow M J, Arndt-Jovin D, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen, Germany.
J Microsc. 1999 Dec;196(Pt 3):317-31. doi: 10.1046/j.1365-2818.1999.00602.x.
The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x-z sections and less intensity drop-off with scanning depth.
可编程阵列显微镜（PAM）的显著特征是在图像平面中存在空间光调制器。单独使用或作为照明和检测的匹配对使用的空间光调制器可用于生成光学切片。在大多数情况下，光学切片PAM的基本光学特性与旋转尼普科夫盘的光学特性相似。然而，图案生成方法在根本上是不同的，它允许在可编程控制下生成任意照明图案，并根据特定实验条件快速改变切片策略。我们报告了一种包含数字微镜器件的PAM的特性，包括对荧光薄膜的轴向切片响应和生物样本的成像。比较了三种轴向切片策略：线扫描、点阵扫描和伪随机序列扫描。当用于实际生物样本时，这三种策略在光通量、切片强度和稳健性方面有很大差异。对薄荧光膜的轴向响应表明，随着定义图案的单位单元变小，半高宽（FWHM）持续减小，同时偏移增加。实验轴向响应曲线代表给定照明点的响应与相邻点的串扰之和。在最佳聚焦平面中，串扰最小，并且与单点响应一起测量时，会导致FWHM减小。在具有恒定通量的图案中，FWHM和偏移大小之间似乎存在权衡。使用生物样本将PAM与共聚焦激光扫描显微镜进行了比较。尽管采集时间较短，但PAM显示出更高的信号水平和动态范围。它还在x-z截面中揭示了更多结构，并且随着扫描深度的增加，强度下降更少。
