Microdensitometer scanning

So far, we have seen that if we measure the Bragg angle of the reflections and successfully index them, then we get information on the size of the unit cell and, if it possesses any translational symmetry elements, also on the symmetry. In addition, we have seen that the intensity of each reflection is different and this too can be measured. In early photographic work, the relative intensities of the spots on the film were assessed by eye with reference to a standard, and later a scanning microdensitometer was used. In modern diffractometers, the beam is intercepted by a detector, either a charge coupled device (CCD) plate or a scintillation counter, and the intensity of each reflection is recorded electronically. 

The accuracy of most current scanning microdensitometers is thought to be in the range of 3%, but enough error sources exist to cast serious doubt upon this estimate. 

The scanning microdensitometer was a modified Jarrell-Ash model 23-500. Figure 2. is a block diagram. 

Werner (21) has made a detailed study of the errors associated with the measurement of intensities from a protein single-crystal X-ray diffraction pattern. He has demonstrated that when a two-dimensional scanning microdensitometer is used, an individual intensity measurement differs by approximately 4% from the mean value of the four symmetry-related reflections. In general, it will not be possible to measure a fibre pattern to this degree of precision because of the various factors which complicate the analysis. If Werner s criteria are applied to a series of intensity measurements made from fibre patterns using AXIS, the precision attained lies between 7% and 10%. While we do not have enough data to draw any firm conclusions at present, this result is sufficiently close to 4% to be encouraging. 

Quantitative measurements are made by means of an automatic scanning microdensitometer. The optical density, D, is obtained in a given small area (raster) of the film by measuring the transmitted, LT, and incident, L0, visible light intensities through the film where... 

In some instances, intensities are still measured by visual estimation, although in general this practice has now been superseded by the use of one-dimensional microdensitometers. Typically, a radial scan is taken through the centre of each spot, the shape of the background is estimated and sketched in, and overlapping reflections are apportioned. The area under each reflection profile is then measured, and an empirical "arcing factor" is applied. In correcting for the Lorentz and... 

Figure 3. Example of a radial scan. The microdensitometer readings Ds are plotted against a radial coordinate. The signal-to-noise ratio is poor because of...

Quantitative Processing. Plates or film with the diffraction patterns were scanned with a Joyce-Loebl microdensitometer. Radial (20) densitometric plots of the crystalline pattern (eventually three successive exposures of the crystalline pattern are analyzed) and of the corresponding amorphous pattern were recorded on the same curve. In this way, the plot of the amorphous pattern was used as a reference standard. The densitometric recording began with the optical density of the non-irradiated emulsion this allowed the evaluation and normalization of the optical density of the diffraction pattern. When the analytical slit passed through the image of the border of the 75 pm objective aperture, the densitometric curve showed a sudden density raise "A d". (Fig. 6) The plots of the amorphous and crystalline patterns were thus normalized to the same reference " A d". Crystallinity was determined on the normalized curves by measuring the areas "C + A" and "A" under the crystalline and amorphous plots respectively. 

The image analysis of UV photomicrographs is carried out by means of a microdensitometer using an X-Y scanning stage or a TV camera. However, the absorbance value obtained may be influenced by the methods of film development or by unevenness of illumination. It is difficult to eliminate the latter completely from the optical system of a UV microscope. 

Figure 3.2 Diffraction pattern of poly(3-hydroxybutyrate), obtainable by a radial microdensitometer scan of the powder diagram such as shown in Figure 3.1. (Courtesy of M. M. Satkowsky.)...

Fig. 6.6.3. Raw microdensitometer scans of the X-ray intensity plotted against diffracting angle (20) for magnetically aligned nematic samples (a) the uniaxial nematic phase of 80CB at 77 °C (b) the biaxial nematic phase of complex A at 168.5 C, (i) meridional scan (parallel to H), (ii) equatorial scan (perpendicular to H). M represents the diffraction peaks from the mylar film which covered the windows of the sample holder and heater assembly. ...

Negatives of the photos (Polaroid 665 for example) of the gels stained with EB or X-ray autoradiographies can be scanned with a microdensitometer. In both cases, avoid an overexposure. 

Figure 5.11 Schematic flowchart showing the stages involved in an interdiffusion experiment (68). The slices are scanned in an IR microdensitometer to obtain the broadened concentration profile, from which D is evaluated.

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