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The implementation of digital photomicrography has expanded the capabilities of microanalysis for quality control. But if used incorrectly, the technique can hurt more than help.
Contaminants, such as hairs and fibers, in a pharmaceutical solution are sometimes visible during postfilling inspection. If undetected, these undesirable materials can potentially threaten the manufacturer’s reputation and the patient’s well being. When a product fails inspection, manufacturing companies need to identify the cause of the contamination before production can continue.
Many companies rely on various microanalytical techniques, along with digital photomicrography, to aid in contaminant identification. During microscopic analysis, personnel can attach a digital camera to the light microscope to capture digital images of the sample.
The implementation of digital photomicrography has expanded the capabilities of microanalysis. But if used incorrectly, the technique can hurt more than help. Digitally altering images after they have been captured to make the contaminant more pronounced, or to highlight certain elements within the sample, reduces the validity of the scientific data.
Without full confidence in the microanalytical data, pharmaceutical manufacturers risk missing important information that could help them identify the source of the contamination and ensure product safety. The strength of digital photomicrography is that it supports or validates the microscopists’ findings, which may be difficult to convey in writing. As a result, it is important to capture images properly without using image-editing or -manipulating software.
The type and condition of the microscopes and equipment are crucial in capturing high-quality images. All microscopes must be cleaned and properly aligned. High magnification and high numerical aperture objectives, as well as high-resolution cameras, are critical when trying to capture images of microscopic contamination. Every lens within the microscope and camera must be particle-free. The cleanliness of the slides, coverglasses, instruments, and mounting media are all critical to image quality as well. Dust or foreign debris in the field of view, or air bubbles introduced during sample preparation, can resemble contamination to the untrained eye, thus causing confusion and leading to incorrect conclusions.
Once the equipment has been checked and samples have been prepared, the final step in producing high-quality images is applying proper photomicrographic techniques. Samples are observed in a cleanroom to avoid cross-contamination. Initial examination of an isolated sample is usually performed with a low-magnification stereomicroscope or a high-magnification compound polarized-light microscope. Once the microscope has been turned on, the rheostat should be adjusted to the optimum color temperature and intensity using neutral density and color-correction filters. The compound microscope should be configured to Köhler-type illumination, which maximizes the amount of even illumination from inhomogeneous light sources. The last and most critical step is the proper adjustment of the aperture diaphragm, which controls the resolving power, depth of field, and contrast.
Once the scientist has optimized the visual image while looking through the microscope’s eyepiece, he or she must adjust the camera settings and electronic controls to duplicate the eyepiece view on the monitor. Most digital cameras used with microscopes are operated by software that controls the camera settings and functionality. Typically, camera software settings may need adjustment before the image may be captured. First, white balance allows the image sensor to adjust to the color temperature of the microscope’s light. Second, the exposure, or camera aperture setting, is adjusted to control the amount of light reaching the image sensor. Third, the saturation setting helps control the image’s color intensity. Finally, contrast adjusts the tones in the image, and can be used to match the adjustments made with the aperture diaphragm. These settings should be adjusted in an effort to match what is seen through the microscope eyepiece with the preview or “live” view in the software just before capturing the image.
The main goal of capturing images through the microscope is to achieve an image that accurately represents the view seen through the eyepieces. Image-editing and -manipulating software should never be used to compensate for poor microscopy. Instead, personnel should use proper microscopy techniques to ensure quality images. Accurate images of contamination, along with analytical data to support the images, provide a solid understanding of the source of the problem, and ultimately help the manufacturer take appropriate steps toward corrective action.
Kristen Wiley is a senior research microscopist at McCrone Associates, the analytical division of the McCrone Group, 850 Pasquinelli Dr., Westmont, IL 60559, tel. 630.887.7100, [email protected]. Wiley is also an instructor of sample preparation, isolation, and manipulation at Hooke College of Applied Sciences, the education division of the McCrone Group.