测微目镜的校正

------首先，必须有一个经过校正的测微标尺（STAGE MICROMETERS）

利用显微镜可以观察材料的细微组织，同时，配置专用测微目镜的话，还可以测量显微组织的实际物理尺寸。在测量之前，针对整个系统，也就是“测微目镜+某一特定放大倍数物镜”中的，测微目镜中的最小刻度线间距所代表的实际物理尺度，需要进行校正。

测微目镜的校正，必须有一个经过校正的测微标尺（STAGE MICROMETERS）；随后，按照一定的步骤进行校正。

当更换为测微目镜后，通过显微镜可以观察到的图案，除了显微组织外，还可以看到与显微组织重叠在一个画面上的“各种刻线”图案。直线型刻度线是最为简单的，垂直十字刻线也很普通（更多图案的测微目镜，可以参看SPI公司的介绍）。

实际上，即使不经过校正，测微目镜也可以进行有效的测量工作。

第一，如果我们将相邻刻线之间的间距作为一个标准单位的话，显微组织中各种相、组织组成物的尺寸就可以用“多少个间距”来进行对比了。只不过，此时的单位是“间距”而已，而此时得到的“间距”数目，也是以后准确得到显微组织观察中所需要的实际物理尺度所必需的。之后，如果进行过校正，只要进行一下数据计算、单位转换即可。

第二，如果没有测微标尺，当然也就无法进行“校正”；但是，根据仪器、设备的生产要求，在这种情况下，我们可以认为，在100倍观察条件下（通常，测微目镜的放大倍数是10倍），测微目镜中的刻线最小间距所代表的实际物理尺度是0.01mm(10微米)。一般生产、科研条件下，误差绝对可以接受。

那么，如果有一个测微标尺，可以进行校正操作的话，如何进行？最为一般的解释，可以参看ASTM E1951-02(2007)的介绍。

不过，为了尽可能提高校正精度，还是有些技巧、规范的。这可以参考STAGE MICROMETERS一文。实际上就是分成两种情况。一种，视场中可以完整看清楚测微标尺图像；此时，可以通过载物台的移动、目镜的转动，确认完整标尺图像所对应的目镜刻线间距数目。另一种，视场中只有部分标尺图像；此时确认全部目镜测微刻线所对应的标尺刻线间距数目。随后，进行计算，确认目镜刻线最小间距代表的实际物理尺度大小。

校正时，刻线的对齐

下面，提供一些检索到的网上资料，并作简单的描述。

参考资料：

[1]  ASTM E1951-02(2007) Standard Guide for Calibrating Reticles and Light Microscope Magnifications（对十字线和光显微镜放大倍数校准的标准导则）

这一标准中，专门有一项内容“5.4 Eyepiece Micrometer Calibration”，就是校正测微目镜中的测量刻线的。

Reticles ：(光学仪器上的)细十字线，标线；分划板。

5.4.1 To calibrate an eyepiece micrometer reticule, view through the eyepiece an image of a stage micrometer using a given objective and intermediate lens combination. Overlay the eyepiece micrometer image on the stage micrometer image, with one end of each coincident upon one another. The measurement should be made consistently from an edge of one division to the corresponding edge of another (Fig. 2). The eyepiece reticule calibration can be determined by dividing the known length of the stage micrometer by the number of overlaid eyepiece micrometer divisions. This calculation yields a length per division value of the micrometer for a given optical setup.

5.4.1校准目测微刻线尺度时，通过目镜看清楚一幅特定物镜以及中间变倍镜头形成的测微标尺（stage micrometer）图像。将测微目镜中的刻线图像与测微标尺合并，即，刻线端部与另一个重合。所谓测量（校正）就是将一方刻线间距的边缘对应到另一个相应的边缘（参看图2，就是下图）。目镜刻线的校正计算公式是：已知测微标尺的长度除以相对应的目镜测微刻线间距数。这一计算得出给定的光学设置下测微刻线最小刻度代表的实际物理长度值。

[2]  http://www.pyser-sgi.com/images/thumbnails/Graticules/Stage Micrometers web.pdf  STAGE MICROMETERS

这一文档，是Structure Probe, Inc.（SPI http://2spi.lookchem.com/About.html ）公司的测微标尺产品介绍，涉及基本概念与实物的对应关系。

the division：相邻刻线间距

eyepiece reticule：目镜测微刻线

[3]  http://www.2spi.com/catalog/ltmic/stage-micrometers-graticules.php  Stage Micrometers from Pyser-SGI

[4]  http://www.tedpella.com/histo_html/2280-10.htm  Standard Series Stage Micrometers for Transmitted and Reflected Light

Line Width and Accuracy of Stage Micrometers

[5]  http://www.mr-damon.com/experiments/1ib_bio/micrometer.htm  Laboratory Investigation：The Micrometer Eyepiece（一个非常容易理解的讲解、示范）

[6]  http://www.microscopyu.com/tutorials/java/reticlecalibration/index.html  Eyepiece Reticle Calibration

这是尼康显微镜的技术资料

Eyepiece Reticle Calibration

Calibration of an eyepiece reticle (determination of the micrometer graduation relationship) for a particular objective is typically conducted by following the recommended procedure described below (also see Figure 4). Note that calibration of an eyepiece reticle holds only for the specific objective/eyepiece combination being tested, and for the specific mechanical tube length of the microscope. To unnecessarily avoid repeating the procedure, the calibration information for each combination should be recorded and stored in a convenient location near the microscope workstation.

·         After ensuring the microscope is aligned and configured for Köhler illumination, insert the proper reticle into the microscope eyepiece and adjust the eye lens so that the engraved scale on the surface of the glass reticle disk appears sharply focused. Carefully check the orientation of the reticle to verify that the numbers positioned above or below the engraved lines are not reversed. This task can be accomplished by holding the eyepiece in front of a bright light source and peering through the eye lens. Finally, adjust the microscope binocular interpupillary spacing and record this value for subsequent measurements. If the microscope is equipped with compensating adjustments on both eyepieces (as is the case with most modern microscopes), the reticle calibration values will be correct for any interpupillary spacing.

·         Place a stage micrometer on the microscope stage and bring the micrometer scale into focus using the microscope coarse and fine focus control knobs. Detecting the scale and translating it into the center of the viewfield is facilitated by the use of a low power objective to first locate the circle surrounding the scale, and then the scale itself. The ring encircling the micrometer scale is visible with the naked eye and should be used to position the stage micrometer in the center of the microscope optical path (stage aperture). In addition, several stage micrometer designs have a line engraved from the ring to the edge of the scale, which is also helpful in locating the scale when using high magnification objectives. Rotate the desired objective into position and ensure that both scales (the stage micrometer and the eyepiece reticle) are visible in the viewfield in simultaneous focus.

·         Translate the stage, using the x-y movement control knobs or handles, and/or rotate the eyepiece (and its reticle) to bring the two scales into parallel alignment (Figure 4(a) and 4(b)). Modern mechanical stages are often provided with a limited degree of rotational movement around the microscope optical axis. In this case, loosen the thumbscrew (usually located at the front of the stage, beneath the specimen platform) and rotate the stage until the micrometer and the eyepiece reticle are parallel.

·         Position the eyepiece reticle directly over the micrometer (with the stage controls) and align the left-hand rule in the reticle with one of the longer, numbered (100 micrometer) division lines on the stage micrometer (Figure 4(b)). Depending upon the objective magnification factor and eyepiece field diameter, a distance ranging between 150 micrometers and 4 millimeters (twice the length of the stage micrometer scale) will be visible in the eyepieces. Over a distance of 100 to 1000 micrometers (10 to 100 rules) on the stage micrometer, determine two points at which the reticle and micrometer scales exactly match (see Figure 4). For the most accurate measurements, utilize the largest possible range of divisions on both scales. Only occasionally do reticle and stage micrometer graduations coincide over the entire length visible in the eyepieces, but this is often the case with reticles manufactured for specific eyepieces. Finally, determine the apparent length of the eyepiece scale in reference to the divisions on the stage micrometer.

·         The micrometer value for the objective in use can be calculated by dividing the known length of the selected region of stage micrometer by the corresponding number of divisions of the eyepiece scale. The result will yield the distance per graduation on the reticle scale for the objective, a quantity often termed the calibration constant. The reticle superimposed on a stage micrometer in Figure 4(b) illustrates alignment of the left-hand rule (marked 0) on the reticle with the stage micrometer division marked 20. Overlap of the two rules is indicated by a red line for clarity. The next area of overlap occurs where the rule labeled 30 on the stage micrometer coincides with the 7.5 mark on the eyepiece reticle. Thus, a 100-micrometer region of the stage micrometer equals 7.5 reticle divisions. Each division of the eyepiece reticle, therefore, corresponds to 13.3 micrometers, for the particular objective/eyepiece combination being calibrated. The number of significant figures appropriate for calculation of the reticle calibration should be carefully scrutinized. Because the minimum resolvable distance in an optical microscope is approximately 0.2 micrometers (under optimal circumstances), a linear measurement below this value cannot be accurately determined.

·         When conducting precise measurements using a stereomicroscope equipped with a zoom optical system, it is necessary to use a stage micrometer for each zoom setting on the microscope. Although many microscope zoom rings and control knobs are graduated with the nominal objective magnification, it is virtually impossible to return the zoom control to exactly the same position, a necessary condition for accurate measurements.

·         After the eyepiece reticle has been calibrated with the stage micrometer, specimen linear dimensions can be measured. For all measurements, the highest magnification objective should be chosen that enables the entire specimen feature of interest to fall within the span of the reticle scale. Orient the reticle scale to coincide with the contour of the specimen region under scrutiny. Next, move the specimen until the left edge coincides with a numbered line on the eyepiece reticle, and count the number of scale divisions spanned by the target region. Carefully estimate any fraction of a division. To increase accuracy, conduct several measurements on large specimens. When circular or oval specimens are being measured (such as blood cells, yeast, bacteria, etc.), record the dimensions of at least 20 candidates from different fields. The specimen being examined in Figure 4(c) is a human scalp hair shaft, which is approximately 93 micrometers in diameter (measured with a calibrated reticle, as discussed above).

The calibration procedure just described must, of course, be repeated for each objective that is to be employed for linear measurements. It should be noted that magnification varies by a few percent for similar objectives (even from the same manufacturer) inscribed with the same magnification factor (for example, 10x), so each objective should be independently measured. If the microscope is regularly used with a number of different objectives, it may be more convenient to plot calibration curves for each objective in graphical form. This provides an easy mechanism to rapidly determine feature sizes while working with the microscope, without having to repeat the arithmetic when applying the micrometer values for all of the objectives used to conduct measurements.

The calibration procedure described above provides a factor that is valid for a specific optical combination, without requiring knowledge of the actual objective magnification, which usually differs from the nominal power that is imprinted on the objective barrel. In utilizing an objective that contains a correction collar to accommodate variations in coverslip thickness, it is important to remember that the magnifying power changes with different settings of the collar. Therefore, a calibration factor determined for such an objective is only valid at the correction collar setting employed for the calibration. Objectives having adjustable collars provide correction for a wide range of coverslip thickness, but also exhibit magnification changes ranging up to 15 percent over the entire adjustment range.

[7]  http://academic.evergreen.edu/curricular/fcb/wk2calibration.doc  Calibration of the Ocular Micrometer on a Microscope

算是一个实验指导书，只是，公式有些费解；似乎“脱裤子放屁”的意思。另外，个人认为：有些题目出的不对，很怪异！