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Digital camera systems, incorporating a variety of charge-coupled device (CCD) detector configurations, are by far the most common image capture technology employed in modern optical microscopy. Until recently, specialized conventional film cameras were generally used to record images observed in the microscope. This traditional method, relying on the photon-sensitivity of silver-based photographic film, involves temporary storage of a latent image in the form of photochemical reaction sites in the exposed film, which only becomes visible in the film emulsion layers after chemical processing (development).
Digital cameras replace the sensitized film with a CCD photon detector, a thin silicon wafer divided into a geometrically regular array of thousands or millions of light-sensitive regions that capture and store image information in the form of localized electrical charge that varies with incident light intensity. The variable electronic signal associated with each picture element (pixel) of the detector is read out very rapidly as an intensity value for the corresponding image location, and following digitization of the values, the image can be reconstructed and displayed on a computer monitor virtually instantaneously.
Several digital camera systems designed specifically for optical microscopy are illustrated in Figure 1. The Nikon Digital Eclipse DXM1200 provides high quality photo-realistic digital images at resolutions ranging up to 12 million pixels with low noise, superb color rendition, and high sensitivity. The camera is controlled by software that allows the microscopist a great deal of latitude in collecting, organizing, and correcting digital images. Live color monitoring on the supporting computer screen at 12 frames per second enables easy focusing of images, which can be saved with a choice of three formats: JPG, TIF, and BMP for greater versatility.
The DS-5M-L1 Digital Sight camera system (Figure 1) 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. The camera systems illustrated in Figure 1 represent the advanced technology currently available for digital imaging with the optical microscope.
Perhaps the single most significant advantage of digital image capture in optical microscopy, as exemplified by CCD camera systems, is the possibility for the microscopist to immediately determine whether a desired image has been successfully recorded. This capability is especially valuable considering the experimental complexities of many imaging situations and the transient nature of processes that are commonly investigated. Although the charge-coupled device detector functions in an equivalent role to that of film, it has a number of superior attributes for imaging in many applications. Scientific-grade CCD cameras exhibit extraordinary dynamic range, spatial resolution, spectral bandwidth, and acquisition speed. Considering the high light sensitivity and light collection efficiency of some CCD systems, a film speed rating of approximately ISO 100,000 would be required to produce images of comparable signal-to-noise ratio (SNR). The spatial resolution of current CCDs is similar to that of film, while their resolution of light intensity is one or two orders of magnitude better than that achieved by film or video cameras. Traditional photographic films exhibit no sensitivity at wavelengths exceeding 650 nanometers in contrast to high-performance CCD sensors, which often have significant quantum efficiency into the near infrared spectral region. The linear response of CCD cameras over a wide range of light intensities contributes to the superior performance, and gives such systems quantitative capabilities as imaging spectrophotometers.
A CCD imager consists of a large number of light-sensing elements arranged in a two-dimensional array on a thin silicon substrate. The semiconductor properties of silicon allow the CCD chip to trap and hold photon-induced charge carriers under appropriate electrical bias conditions. Individual picture elements, or pixels, are defined in the silicon matrix by an orthogonal grid of narrow transparent current-carrying electrode strips, or gates, deposited on the chip. The fundamental light-sensing unit of the CCD is a metal oxide semiconductor (MOS) capacitor operated as a photodiode and storage device. A single MOS device of this type is illustrated in Figure 2, with reverse bias operation causing negatively charged electrons to migrate to an area underneath the positively charged gate electrode. Electrons liberated by photon interaction are stored in the depletion region up to the full well reservoir capacity. When multiple detector structures are assembled into a complete CCD, individual sensing elements in the array are segregated in one dimension by voltages applied to the surface electrodes and are electrically isolated from their neighbors in the other direction by insulating barriers, or channel stops, within the silicon substrate.
The light-sensing photodiode elements of the CCD respond to incident photons by absorbing much of their energy, resulting in liberation of electrons, and the formation of corresponding electron-deficient sites (holes) within the silicon crystal lattice. One electron-hole pair is generated from each absorbed photon, and the resulting charge that accumulates in each pixel is linearly proportional to the number of incident photons. External voltages applied to each pixel’s electrodes control the storage and movement of charges accumulated during a specified time interval. Initially, each pixel in the sensor array functions as a potential well to store the charge during collection, and although either negatively charged electrons or positively charged holes can be accumulated (depending on the CCD design), the charge entities generated by incident light are usually referred to as photoelectrons. This discussion considers electrons to be the charge carriers. These photoelectrons can be accumulated and stored for long periods of time before being read from the chip by the camera electronics as one stage of the imaging process.
Image generation with a CCD camera can be divided into four primary stages or functions: charge generation through photon interaction with the device’s photosensitive region, collection and storage of the liberated charge, charge transfer, and charge measurement. During the first stage, electrons and holes are generated in response to incident photons in the depletion region of the MOS capacitor structure, and liberated electrons migrate into a potential well formed beneath an adjacent positively-biased gate electrode. The system of aluminum or polysilicon surface gate electrodes overlie, but are separated from, charge carrying channels that are buried within a layer of insulating silicon dioxide placed between the gate structure and the silicon substrate. Utilization of polysilicon as an electrode material provides transparency to incident wavelengths longer than approximately 400 nanometers and increases the proportion of surface area of the device that is available for light collection. Electrons generated in the depletion region are initially collected into electrically positive potential wells associated with each pixel. During readout, the collected charge is subsequently shifted along the transfer channels under the influence of voltages applied to the gate structure. Figure 3 illustrates the electrode structure defining an individual CCD sense element.
In general, the stored charge is linearly proportional to the light flux incident on a sensor pixel up to the capacity of the well; consequently this full-well capacity (FWC) determines the maximum signal that can be sensed in the pixel, and is a primary factor affecting the CCD’s dynamic range. The charge capacity of a CCD potential well is largely a function of the physical size of the individual pixel. Since first introduced commercially, CCDs have typically been configured with square pixels assembled into rectangular area arrays, with an aspect ratio of 4:3 being most common. Figure 4 presents typical dimensions of several of the most common sensor formats in current use, with their size designations in inches according to a historical convention that relates CCD sizes to vidicon tube diameters.CCD Formats
The rectangular geometry and common dimensions of CCDs result from their early competition with vidicon tube cameras, which required the solid-state sensors to produce an electronic signal output that conformed to the prevailing video standards at the time. Note that the ’inch’ designations do not correspond directly to any of the CCD dimensions, but represent the size of the rectangular area scanned in the corresponding round vidicon tube. A designated ’1-inch’ CCD has a diagonal of 16 millimeters and sensor dimensions of 9.6 x 12.8 millimeters, derived from the scanned area of a 1-inch vidicon tube with a 25.4-millimeter outside diameter and an input window approximately 18 millimeters in diameter. Unfortunately, this confusing nomenclature has persisted, often used in reference to CCD ’type’ rather than size, and even includes sensors classified by a combination of fractional and decimal terms, such as the widely used 1/1.8-inch CCD that is intermediate in size between 1/2-inch and 2/3-inch devices.
Although consumer cameras continue to primarily employ rectangular sensors built to one of the ’standardized’ size formats, it is becoming increasingly common for scientific-grade cameras to incorporate square sensor arrays, which better match the circular image field projected in the microscope. A large range of sensor array sizes are produced, and individual pixel dimensions vary widely in designs optimized for different performance parameters. CCDs in the common 2/3-inch format typically have arrays of 768 x 480 or more diodes and dimensions of 8.8 x 6.6 millimeters (11-millimeter diagonal). The maximum dimension represented by the diagonal of many sensor arrays is considerably smaller than the typical microscope field of view, and results in a highly magnified view of only a portion of the full field. The increased magnification can be beneficial in some applications, but if the reduced field of view is an impediment to imaging, demagnifying intermediate optical components are required. The alternative is use of a larger CCD that better matches the image field diameter, which ranges from 18 to 26 millimeters in typical microscope configurations.
An approximation of CCD potential-well storage capacity may be obtained by multiplying the diode (pixel) area by 1000. A number of consumer-grade 2/3-inch CCDs, with pixel sizes ranging from 7 to 13 micrometers in size, are capable of storing from 50,000 to 100,000 electrons. Using this approximation strategy, a diode with 10 x 10 micrometer dimensions will have a full-well capacity of approximately 100,000 electrons. For a given CCD size, the design choice regarding total number of pixels in the array, and consequently their dimensions, requires a compromise between spatial resolution and pixel charge capacity. A trend in current consumer devices toward maximizing pixel count and resolution has resulted in very small diode sizes, with some of the newer 2/3-inch sensors utilizing pixels less than 3 micrometers in size.
CCDs designed for scientific imaging have traditionally employed larger photodiodes than those intended for consumer (especially video-rate) and industrial applications. Because full-well capacity and dynamic range are direct functions of diode size, scientific-grade CCDs used in slow-scan imaging applications have typically employed diodes as large as 25 x 25 micrometers in order to maximize dynamic range, sensitivity, and signal-to-noise ratio. Many current high-performance scientific-grade cameras incorporate design improvements that have enabled use of large arrays having smaller pixels, which are capable of maintaining the optical resolution of the microscope at high frame rates. Large arrays of several million pixels in these improved designs can provide high-resolution images of the entire field of view, and by utilizing pixel binning (discussed below) and variable readout rate, deliver the higher sensitivity of larger pixels when necessary.Readout of CCD Array Photoelectrons
Before stored charge from each sense element in a CCD can be measured to determine photon flux on that pixel, the charge must first be transferred to a readout node while maintaining the integrity of the charge packet. A fast and efficient charge-transfer process, as well as a rapid readout mechanism, are crucial to the function of CCDs as imaging devices. When a large number of MOS capacitors are placed close together to form a sensor array, charge is moved across the device by manipulating voltages on the capacitor gates in a pattern that causes charge to spill from one capacitor to the next, or from one row of capacitors to the next. The translation of charge within the silicon is effectively coupled to clocked voltage patterns applied to the overlying electrode structure, the basis of the term ’charge-coupled’ device. The CCD was initially conceived as a memory array, and intended to function as an electronic version of the magnetic bubble device. The charge transfer process scheme satisfies the critical requirement for memory devices of establishing a physical quantity that represents an information bit, and maintaining its integrity until readout. In a CCD used for imaging, an information bit is represented by a packet of charges derived from photon interaction. Because the CCD is a serial device, the charge packets are read out one at a time.
The stored charge accumulated within each CCD photodiode during a specified time interval, referred to as the integration time or exposure time, must be measured to determine the photon flux on that diode. Quantification of stored charge is accomplished by a combination of parallel and serial transfers that deliver each sensor element’s charge packet, in sequence, to a single measuring node. The electrode network, or gate structure, built onto the CCD in a layer adjoining the sensor elements, constitutes the shift register for charge transfer. The basic charge transfer concept that enables serial readout from a two-dimensional diode array initially requires the entire array of individual charge packets from the imager surface, constituting the parallel register, to be simultaneously transferred by a single-row incremental shift. The charge-coupled shift of the entire parallel register moves the row of pixel charges nearest the register edge into a specialized single row of pixels along one edge of the chip referred to as the serial register. It is from this row that the charge packets are moved in sequence to an on-chip amplifier for measurement. After the serial register is emptied, it is refilled by another row-shift of the parallel register, and the cycle of parallel and serial shifts is repeated until the entire parallel register is emptied. Some CCD manufacturers utilize the terms vertical and horizontal in referring to the parallel and serial registers, respectively, although the latter terms are more readily associated with the function accomplished by each.
A widely used analogy to aid in visualizing the concept of serial readout of a CCD is the bucket brigade for rainfall measurement, in which rain intensity falling on an array of buckets may vary from place to place in similarity to incident photons on an imaging sensor (see Figure 5 (a)). The parallel register is represented by an array of buckets, which have collected various amounts of signal (water) during an integration period. The buckets are transported on a conveyor belt in stepwise fashion toward a row of empty buckets that represent the serial register, and which move on a second conveyor oriented perpendicularly to the first. In Figure 5(b), an entire row of buckets is being shifted in parallel into the reservoirs of the serial register. The serial shift and readout operations are illustrated in Figure 5(c), which depicts the accumulated rainwater in each bucket being transferred sequentially into a calibrated measuring container, analogous to the CCD output amplifier. When the contents of all containers on the serial conveyor have been measured in sequence, another parallel shift transfers contents of the next row of collecting buckets into the serial register containers, and the process repeats until the contents of every bucket (pixel) have been measured.
There are many designs in which MOS capacitors can be configured, and their gate voltages driven, to form a CCD imaging array. As described previously, gate electrodes are arranged in strips covering the entire imaging surface of the CCD face. The simplest and most common charge transfer configuration is the three-phase CCD design, in which each photodiode (pixel) is divided into thirds with three parallel potential wells defined by gate electrodes. In this design, every third gate is connected to the same clock driver circuit. The basic sense element in the CCD, corresponding to one pixel, consists of three gates connected to three separate clock drivers, termed phase-1, phase-2, and phase-3 clocks. Each sequence of three parallel gates makes up a single pixel’s register, and the thousands of pixels covering the CCD’s

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