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Introduction

Digitization of a video or electronic image captured through an optical microscope results in a dramatic increase in the ability to enhance features, extract information, or modify the image. When compared to the traditional mechanism of image capture, photomicrography on film, digital imaging and post-acquisition processing enables a reversible, essentially noise-free modification of the image as an ordered matrix of integers rather than a series of analog variations in color and intensity. This section addresses a variety of current topics in image acquisition and processing for optical microscopy.

Introduction to Digital Imaging in Microscopy

Part I: Basic Imaging Concepts - This article describes the fundamentals of digital image acquisition, spatial resolution, contrast, brightness, bit depth, dynamic range, CCD fundamentals, and performance measures as well as image display and storage issues. Starting with a historical perspective, the conversion of images from analog to digital format is reviewed followed by discussions of the contrast transfer function, histograms, quantum efficiency, noise, cooling, binning, and linearity.

Part II: Basic Microscopy Concepts - The primary considerations in imaging living and fixed cells in the microscope with a digital camera are detector sensitivity (signal-to-noise), the required speed of image acquisition, and specimen viability. The relatively high light intensities and long exposure times that are typically employed in recording images of fixed cells and tissues (where photobleaching is the major consideration) must be strictly avoided when working with living cells. In virtually all cases, live-cell microscopy represents a compromise between achieving the best possible image quality and preserving the health of the cells. Rather than unnecessarily oversampling time points and exposing the cells to excessive levels of illumination, the spatial and temporal resolutions set by the experiment should be limited to match the goals of the investigation.

Digital Imaging New Opportunities for Microscopy - Digital imaging is increasingly applied to image capture for microscopy - an area that demands high resolution, color fidelity and careful management of, often, limited light conditions. The latest digital cameras combined with powerful computer software now offer image quality that is comparable with traditional silver halide film photography. Moreover, digital cameras are also easier to use and offer greater flexibility for image manipulation and storage.

Concepts in Digital Imaging Technology - Explore the basic concepts in digital imaging with our illustrated discussions and interactive Java tutorials. Topics covered include CCD operation, binning, blooming, image capture, dynamic range, electronic shutters, CCD clocking schemes, quantum efficiency, photodiodes, photomultipliers, digital manipulation of images and a wide spectrum of other issues in this emerging field.

Basic Properties of Digital Images - Continuous-tone images are produced by analog optical and electronic devices, which accurately record image data by several methods, such as a sequence of electrical signal fluctuations or changes in the chemical nature of a film emulsion that vary continuously over all dimensions of the image. In order for a continuous-tone or analog image to be processed or displayed by a computer, it must first be converted into a computer-readable form or digital format. This process applies to all images, regardless the origin and complexity, and whether they exist as black and white (grayscale) or full color. A digital image is composed of a rectangular (or square) pixel array representing a series of intensity values and ordered through an organized (x,y) coordinate system.

Electronic Imaging Detectors - The range of light detection methods and the wide variety of imaging devices currently available to the microscopist make the selection process difficult and often confusing. This discussion is intended to aid in understanding the basics of light detection and to provide a guide for selecting a suitable electronic detector (CCD or video camera system) for specific applications in optical microscopy.

Introduction to Charge-Coupled Devices (CCDs) - In modern scientific grade CCD camera systems, the light sensitivity, dynamic range, linearity, spatial resolution, and readout speed combine to provide unexcelled performance. Compared to a film emulsion of similar signal-to-noise ratio, the light gathering efficiency of a CCD would produce an ISO rating of approximately 100,000. Their linear performance enables CCD cameras to function as imaging spectrophotometers for quantitative scientific analysis.

Fundamentals of Video Imaging - Optical images produced in the microscope can be captured using either traditional film techniques, digitally with electronic detectors such as a charge-coupled device (CCD), or with a tube-type video camera. When a dynamic event must be recorded in real time, a video camera is often the most suitable resource for the task.

Introduction to CMOS Image Sensors - CMOS image sensors are designed with the ability to integrate a number of processing and control functions, which lie beyond the primary task of photon collection, directly onto the sensor integrated circuit. These features generally include timing logic, exposure control, analog-to-digital conversion, shuttering, white balance, gain adjustment, and initial image processing algorithms. Inexpensive CMOS image sensors are entering the field of optical microscopy in educational instruments that combine acceptable optical quality with user-friendly control and imaging software packages.

Basic Concepts in Digital Image Processing - Digital image processing enables the reversible, virtually noise-free modification of an image in the form of a matrix of integers instead of the classical darkroom manipulations or filtration of time-dependent voltages necessary for analog images and video signals. Even though many image processing algorithms are extremely powerful, the average user often applies operations to digital images without concern for the underlying principles behind these manipulations. The images that result from careless manipulation are often severely degraded or otherwise compromised with respect to those that could be produced if the power and versatility of the digital processing software were correctly utilized.

Introduction to Image Processing and Analysis - John Russ has taught hands-on courses and extended workshops in image processing and analysis to more than 3000 students, worldwide, over the course of his career. His one-day tutorials and lectures, sponsored by various professional societies and other organizations, have reached several thousand more. But the need to have a basic understanding of these topics is far wider than he can ever reach in person. Potentially everyone working with images, and certainly that includes every microscopist, needs to be aware of the possibilities (and limitations) of computer-based image processing and measurement. The descriptive reviews and interactive tutorials in this section cover most of the topics that the author discusses in typical one-day tutorials.

Recommended Strategy for Processing Digital Images - Depending upon the illumination conditions, specimen integrity, and preparation methods, digital images captured in the optical microscope may require a considerable amount of rehabilitation to achieve a balance between scientific accuracy, cosmetic equilibrium, and aesthetic composition. When first acquired by a charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) image sensor, digital images from the microscope often suffer from poor signal-to-noise characteristics, uneven illumination, focused or defocused dirt and debris, glare, color shifts, and a host of other ailments that degrade overall image quality.

Deconvolution in Optical Microscopy - Deconvolution is a computationally intensive image processing technique that is being increasingly utilized for improving the contrast and resolution of digital images captured in the microscope. The foundations are based upon a suite of methods that are designed to remove or reverse the blurring present in microscope images induced by the limited aperture of the objective. Practically any image acquired on a digital fluorescence microscope can be deconvolved, and several new applications are being developed that apply deconvolution techniques to transmitted light images collected under a variety of contrast enhancing strategies. One of the most suitable subjects for improvement by deconvolution are three-dimensional montages constructed from a series of optical sections.

Color Balance in Digital Imaging - The acquisition of accurately color balanced images in the optical microscope can be a challenge even to experienced microscopists, regardless of whether they are employing traditional photographic film emulsions or newer solid-state digital camera systems. Utilization of electronic image capture technology relies upon the same familiar properties of light as does conventional film-based photomicrography, but the capability of performing white balance adjustment for color balancing is a unique function of electronic image sensors that is not at all intuitive to investigators seeking to capture digital images from the microscope.

Digital Image Processing Interactive Java Tutorials - Explore the basic concepts of digital image processing applied to specimens captured in the microscope. Techniques reviewed include contrast, color balance, spatial resolution, image sampling frequency, geometric transformation, averaging, measurements, histogram manipulation, convolution kernels, filtering digital images, compression, noise reduction, and binary digital images.

Background Subtraction Toolkit - Because of the wide spectrum of illumination modes available with the optical microscope, images can suffer from brightness variations that are manifested by gradients appearing in the background. These fluctuations often lead to contrast and brightness deficiencies in the specimen region and can seriously affect the quality of an otherwise acceptable digital image. This section discusses important details concerning the Molecular Expressions Background Subtraction Toolkit, which is designed to assist image processing applications by providing uniform backgrounds for specimens captured digitally with an optical microscope.

Background Subtraction Toolkit Download - The Molecular Expressions digital microscope image Background Subtraction Toolkit is a stand-alone Java application program designed for the Windows operating system, which can be utilized to produce uniform backgrounds for digital images captured with this unique inverted optical microscope. Use this link to visit the download area for additional information and to download the software to client computers.

Digital Camera Resolution Requirements for Optical Microscopy - The ultimate resolution of a charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) image sensor is a function of the number of photodiodes and their size relative to the image projected onto the surface of the imaging array by the microscope optical system. When attempting to match microscope optical resolution to a specific digital camera and video coupler combination, use this calculator for determining the minimum pixel density necessary to adequately capture all of the optical data from the microscope.

Determining the Signal-to-Noise Ratio in Digital Cameras - For any electronic measuring system, the signal-to-noise ratio (SNR) characterizes the quality of a measurement and determines the ultimate performance of the system. With a CCD (charge-coupled device) image sensor, the SNR value specifically represents the ratio of the measured light signal to the combined noise, which consists of undesirable signal components arising in the electronic system, and inherent natural variation of the incident photon flux. Because a CCD sensor collects charge over an array of discrete physical locations, the signal-to-noise ratio may be thought of as the relative signal magnitude, compared to the measurement uncertainty, on a per-pixel basis. The three primary sources of noise in a CCD imaging system are photon noise, dark noise, and read noise, all of which must be considered in the SNR calculation.

MicroscopyU Digital Imaging Center - For the past fifty years, the primary medium for photomicrography has been film, which has served the scientific community well by faithfully reproducing countless images from the optical microscope. It has only been in the past decade that improvements in electronic camera and computer technology have made digital imaging cheaper and easier to use than conventional photography. This section explores new concepts in digital imaging technology and reviews both fundamental concepts and advanced techniques involved in digital imaging. In addition several of the current camera systems designed for optical microscopy are explored.

Digital Cameras for Optical Microscopy

Olympus DP70 Digital Camera System - The latest generation of digital cameras designed for wide-ranging applications in optical microscopy combine excellent resolution, high sensitivity, and rapid data transfer to a host computer. The Olympus DP70 is a 12.5 million-pixel cooled digital color camera system that incorporates the latest innovations in imaging technology to enable the capture of superb images in the most demanding current microscopy applications, including differential interference contrast (DIC), darkfield, phase contrast, polarized light, and most widefield fluorescence techniques.

Digital Sight Camera System - The DS-5M-L1 Digital Sight Camera System is Nikon's innovative digital imaging system for microscopy that emphasizes the ease and efficiency of an all-in-one concept, incorporating a built-in LCD monitor in a stand-alone control unit. The system optimizes the capture of high-resolution images up to 5 megapixels through straightforward menus and pre-programmed imaging modes for different observation methods. The stand-alone design offers the advantage of independent operation including image storage to a CompactFlash Card housed in the control/monitor unit, but has the versatility of full network capabilities if desired. Connection is possible to PCs through a USB interface, and to local area networks or the Internet via Ethernet port. Web browser support is available for live image viewing and remote camera control, and the camera control unit supports HTTP, Telnet, FTP server/client, and is DHCP compatible.

Digital Sight ACT-1 for L-1 Software - The automatic camera tamer software (referred to as the ACT-1 for L-1 Version) is an application program designed to allow operation of the Nikon Digital Sight DS-L1 camera control unit from a networked high-performance PC. Multiple Digital Sight camera systems, connected to the network, can be controlled from the PC, with the selection of the desired camera being accomplished by simple menu selection. The ACT-1 program allows basic digital imaging operations such as image capture, saving, printing, and deleting to be performed, and in addition provides access to more advanced image processing, display manipulation, and image analysis functions.

Nikon Digital Eclipse DXM 1200 - The Digital Eclipse DXM 1200 is Nikon's new high-resolution color digital camera designed exclusively for photography through the microscope. This system provides real photo-quality digital imaging at a resolution of up to 12 million pixels with low noise, superb color rendition, and high sensitivity.

Nikon DN100 Digital Network Camera - The DN100 Digital Network Camera is Nikon's new platform-independent, Internet-capable digital camera system that can be utilized to deliver live or captured images to a local computer in the laboratory or to a remote computer anywhere in the world.

Olympus DP-10 Digital Camera - Olympus has designed a revolutionary new digital camera specifically designed for critical color photomicrography.

DXM 1200 Digital Eclipse Screen Saver - Digital imaging through the microscope has reached a new level of performance with the Nikon DXM 1200 electronic camera system. Enjoy full-color images captured through advanced electronic photomicrography with Nikon MicroscopyU screen savers. The DXM 1200 screen saver has been developed for computers utilizing the Windows (95, 98, NT, and 2000) operating system and is available as a free download to Molecular Expressions visitors.

Contributing Authors

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

John C. Russ - Materials Science and Engineering Department, North Carolina State University, Raleigh, North Carolina, 27695.

Renato Turchetta - Microelectronics Group, Instrumentation Department, Rutherford Appleton Laboratory, Chilton, Didcot, OX11 0QX, United Kingdom.

Matthew J. Parry-Hill, John C. Long, Thomas J. Fellers, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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