Back-reflection focusing cameras

The most precise measurement of lattice parameter is made in the back-reflection region, as discussed in greater detail in Chap. 11. The most suitable camera for such measurements is the symmetrical back-reflection focusing camera illustrated in Fig. 6-8. 

Fig. 6-8 Symmetrical back-reflection focusing camera. Only one hkl reflection is shown.

Fig. 6-9 Powder photograph of tungsten made in a symmetrical back-reflection focusing camera, 4.00 in. (10.16 cm) in diameter. Unfiltered copper radiation.

Fig.1 Schematic of the LAPAP optical setup. Two mirrors, M1 and M2, are used to adjust the beam direction, before it reaches the sample. The back-reflection from the coverslip is directed to a camera using a wedge window, W1. The image is focused by alens, L2,to monitor the position of the laser focus on top of the sample surface (color figure online)...

Briefly, the substrate is incubated in BSA to minimize nonspecific binding. The sample is placed on the holder, the laser is turned on, and the sample is moved up and down to adjust the focus until the back-reflected spot on a camera is as small as possible. The laser is turned off and a drop of B4F is placed on the sample. The program that controls the laser and motors is started. After laser illumination, the sample can usually be observed in a fluorescence microscope it looks dim because most of the dye has been bleached, but it is usually still visible. The rest of the reagents are incubated in order. It is recommended to begin with fast and simple patterns of a few hundred microns long. Grids can easily be found visually and they... 

In this configuration, when the sample is positioned at the focus of the objective, the size of the back-reflection spot on the CCD is minimum. Thus, the sample-objective distance can be adjusted by slowly moving the z-axis until the laser spot on the camera becomes the smallest. When using coverslips and high NA objectives, while the distance from the sample to the objective is changed, one can notice two positions at which the image of the back-reflection is the smallest. These two positions correspond to the reflections originating at the two interfaces of the coverslip, and one needs to identify the one that corresponds to the upper surface, where the biotin solution will be placed. 

Figure 17 Principle of ellipsometric microscopy. Full arrows symbolise the light path of illumination, broken arrows stand for the observation, respectively. A parallel beam of polarised light is focussed into an ofT-axis spot in the back focal plane of the lens. In the front focal plane of the lens, where the object is located, this results In a parallel polarised beam of light hitting the object under a shallow angle of incidence. Light reflected by the object is collected by the lens, passes a motorised polarization analyzer and is focused onto a digital CCD camera. For clarity, several optical elements are omitted, (redrawn from [36])...

One of the most critical aspects of observation using any optical microscope is the specimen illumination. Two illumination systems are commonly used in optical microscopy transmitted light and reflected light (Fig. 2). Transmitted, also called diascopic illumination, requires the specimen to be transparent. It is used primarily to examine thin sections of biological or material samples. Reflected light, or episcopic illumination (epi-illumination), is most commonly used for fluorescence microscopy, where fluorphores inside the specimen are excited to produce fluorescent light. The fluorescence is then emitted, or reflected back to the objective and collected by the detector (eyes, or camera). The reflected light is also used to study the surfaces of opaque specimens, which is the focus of this session. 

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