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Education in Microscopy and Digital Imaging 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Understanding Digital Imaging Contact Us | Carl Zeiss Education in Microscopy and Digital Imaging ZEISS Home ¦ Products ¦ Solutions ¦ Support ¦ Online Shop ¦ ZEISS International ZEISS Campus Home Interactive Tutorials Basic Microscopy Spectral Imaging Spinning Disk Microscopy Optical Sectioning Superresolution Introduction Article Quick Links Introduction Live-Cell Imaging For the most of the twentieth century, a photosensitive chemical emulsion Acquisition Fluorescent Proteins spread on film was used to reproduce images from the optical microscope. It Resolution has only been in the past decade that improvements in electronic camera Microscope Light Sources Transfer Function and computer technology have made digital imaging faster, cheaper, and Digital Image Galleries far more accurate to use than conventional photography. A wide range of Bit Depth Applications Library new and exciting techniques have subsequently been developed that Histograms enable researchers to probe deeper into tissues, observe extremely rapid Digital Cameras Reference Library biological processes in living cells, and obtain quantitative information Performance about spatial and temporal events on a level approaching the single molecule. The imaging device is one of the most critical components in 3D Imaging Image Display Search optical microscopy because it determines at what level fine specimen detail may be detected, the relevant structures resolved, and/or the dynamics of a Print Version process visualized and recorded. The range of light detection methods and the wide variety of imaging devices currently available to the microscopist make the equipment selection process difficult and often confusing. This discussion is intended to aid in understanding the basics of light detection, the fundamental properties of digital images, and the criteria relevant to selecting a suitable detector for specific applications. Introduction Image Formation Microscope Resolution Point-Spread Function Microscope Optical Train Köhler Illumination Optical Systems Microscope Objectives Enhancing Contrast Fluorescence Microscopy Reflected Light Microscopy Reflected Light Contrast Recording images with the microscope dates back to the earliest days of microscopy. The first zeiss-campus.magnet.fsu.edu/articles/basics/digitalimaging.html 1/16 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Understanding Digital Imaging Digital Imaging Basics single lens instruments, developed by Dutch scientists Antoni van Leeuwenhoek and Jan Microscope Practical Use Swammerdam in the late 1600s, were used by these pioneering investigators to produce highly detailed drawings of blood, microorganisms, and other minute specimens. British scientist Microscope Ergonomics Robert Hooke engineered one of the first compound microscopes and used it to write Microscope Care Micrographia, his hallmark volume on microscopy and imaging published in 1665. The History of the Microscope microscopes developed during this period were incapable of projecting images, and observation was limited to close visualization of specimens through the eyepiece. True photographic images were first obtained with the microscope in 1835 when William Henry Fox Talbot applied a chemical emulsion process to capture photomicrographs at low magnification. Between 1830 Microscope Lightpaths and 1840 there was an explosive growth in the application of photographic emulsions to recording microscopic images. For the next 150 years, the art and science of capturing images Objective Specifications through the microscope with photographic emulsions co-evolved with advancements in film Optical Pathways technology. During the late 1800s and early 1900s, Carl Zeiss and Ernst Abbe perfected the Microscope Alignment manufacture of specialized optical glass and applied the new technology to many optical instruments, including compound microscopes. Concept of Magnification Conjugate Planes The dynamic imaging of biological activity was introduced in 1909 by French doctorial student Fixed Tube Microscope Jean Comandon, who presented one of the earliest time lapse videos of syphilis producing spirochaetes. Comandon's technique enabled movie production of the microscopic world. Infinity Corrected Optics Between 1970 and 1980 researchers coupled tube based video cameras with microscopes to Infinity Optical System produce time lapse image sequences and real-time videos. In the 1990s the tube camera gave Field Iris Diaphragm way to solid state technology and the area array charge coupled device (CCD), heralding a new era in photomicrography. Current terminology referring to the capture of electronic images with Numerical Aperture the microscope is digital or electronic imaging. Airy Disk Formation Spatial Frequency Digital Image Acquisition: Analog to Digital Conversion back to top ^ Conoscopic Images Image Resolution Regardless of whether light focused on a specimen ultimately impacts on the human retina, a film emulsion, a phosphorescent screen or the photodiode array of a CCD, an analog image is Airy Disk Basics produced. These images can contain a wide spectrum of intensities and colors. Images of this Oil Immersion type are referred to as continuous tone because the various tonal shades and hues blend Substage Condenser together without disruption, to generate a diffraction limited reproduction of the original specimen. Continuous tone images accurately record image data by using a sequence of electrical signal Condenser Aperture fluctuations that vary continuously throughout the image. Condenser Light Cones Coverslip Thickness As we view them, images are generally square or rectangular in dimension thus each pixel is represented by a coordinate pair with specific x and y values, arranged in a typical Cartesian Focus Depth coordinate system (Figure 1c). The x coordinate specifies the horizontal position or column Reflected Light Pathways location of the pixel, while the y coordinate indicates the row number or vertical position. Thus, a digital image is composed of a rectangular or square pixel array representing a series of intensity values that is ordered by an (x, y) coordinate system. In reality, the image exists only as a large serial array of data values that can be interpreted by a computer to produce a digital Basic Principles representation of the original scene. Optical Systems Specimen Contrast The horizontal to vertical dimension ratio of a digital image is known as the aspect ratio and can be calculated by dividing the image width by the height. The aspect ratio defines the geometry of Phase Contrast the image. By adhering to a standard aspect ratio for display of digital images, gross distortion of DIC Microscopy the image is avoided when the images are displayed on remote platforms. When a continuous Fluorescence Microscopy tone image is sampled and quantized, the pixel dimensions of the resulting digital image acquire the aspect ratio of the original analog image. It is important that each pixel has a 1:1 aspect ratio Polarized Light (square pixels) to ensure compatibility with common digital image processing algorithms and to Microscope Ergonomics minimize distortion. Spatial Resolution in Digital Images back to top ^ The quality of a digital image, or image resolution, is determined by the total number of pixels and the range of brightness values available for each pixel. Image resolution is a measure of the degree to which the digital image represents the fine details of the analog image recorded by the microscope. The term spatial resolution is reserved to describe the number of pixels utilized in constructing and rendering a digital image. This quantity is dependent upon how finely the image is sampled during digitization, with higher spatial resolution images having a greater number of pixels within the same physical image dimensions. Thus, as the number of pixels acquired during sampling and quantization of a digital image increases, the spatial resolution of the image also increases. zeiss-campus.magnet.fsu.edu/articles/basics/digitalimaging.html 2/16 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Understanding Digital Imaging The optimum sampling frequency, or number of pixels utilized to construct a digital image, is determined by matching the resolution of the imaging device and the computer system used to visualize the image. A sufficient number of pixels should be generated by sampling and quantization to dependably represent the original image. When analog images are inadequately sampled, a significant amount of detail can be lost or obscured, as illustrated by the diagrams in Figure 2. The analog signal presented in Figure 2(a) shows the continuous intensity distribution displayed by the original image, before sampling and digitization, when plotted as a function of sample position. When 32 digital samples are acquired (Figure 2(b)), the resulting image retains a majority of the characteristic intensities and spatial frequencies present in the original analog image. When the sampling frequency is reduced as in Figure 2(c) and (d), frequencies present in the original image are missed during analog-to-digital (A/D) conversion and a phenomenon known as aliasing develops. Figure 2(d) illustrates the digital image with the lowest number of samples, where aliasing has produced a loss of high spatial frequency data while simultaneously introducing spurious lower frequency data that don't actually exist. The spatial resolution
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