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at the low intensity end of the dynamic range. Ellis and Wright [3] justify this conclusion after a careful examination of the various sources of noise and of the dark current or dark count contribution.

The commercial availability of ready-to-use photon-counting packages is also a reason for the success of this technology. These assemblies are light, compact, and all elements are fully integrated, including the high-voltage source. They only require a low-voltage input and generally all controls are preset to provide the best performance so that no adjustment is required. This allows the construction of sensitive laboratory-assembled luminometers for low light level. Spectroscopic studies may be carried out with some of these instruments [4]. Furthermore, these photon-counting modules are also incorporated in commercial instruments [5]. PMTs are by far the preferred detectors for CL. Recently, however, avalanche photodiodes (AVP) brought the cost benefit of solid-state instrument together with good overall performance to new luminometers. An interesting comparison of PMT and AVP has recently been published by Fullam [6].

A few words should be said about the existence of PMT-based instruments that are developed to solve specific problems in chemior bioluminescence. For instance, marine laboratories have developed and improved over time a range of so-called bathyphotometers for hydrobiophysical measurements (microalgae, zooplankton in the surface waters of the sea) [7].

The high sensitivity and fast counting rate of recent instruments allow a large number of repetitions per sample, which makes possible meaningful statistical analysis. As noted earlier, most luminometers are computer-driven and often benefit from the presence of software. The latter allows choosing procedures, such as setting the parameters for quantification of the light emitted in assays from flashor glow-type reagents (TL, integrated light emission between two time limits, average emission during a selected interval, peak emission), or data handling such as curve fitting for kinetic enzyme studies. In case no software is available, provisions are usually made to transfer results to a spreadsheet.

With today’s instruments the customer may specify an injector or an automatic reagent dispenser, and a choice of sample formats is available: test tube, vial, microplate, Petri dish. The volumes of sample and reagent required to carry out a determination steadily decrease and are typically a few microliters. Sample temperature during the assays is generally controlled.

Flow injection analysis (FIA) is a convenient technique for automatic measurements. It can be easily adapted to measure CL. Typically two or more liquid solutions containing the analyte, reagents, and/or buffers are mixed sequentially in purposely interconnected narrow tubes. A multichannel peristaltic pump insures an even flow. After the last mixing operation the mixture is pushed through a flow cell where the light-emitting reaction occurs. An example of flow cell consists in a quartz coil (typical length 1 m, total volume 50–100 L) positioned in front of the PMT window. FIA was developed before the advent of robotized

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systems to handle microplates. This technique owes much to its versatility. One drawback is its higher consumption of reagent, which means often a higher cost per determination.

Despite all these developments, some problems remain. One of them concerns the comparison of results obtained from different instruments. Solutions involving standards are proposed. But most of them suffer from drawbacks: thermal or photochemical instability, multistep preparation, wavelength inadequacy between standard and actual fluorophore emission. Many solutions have been advocated to calibrate luminometers [8].

2.3 Bibliographic Sources for Commercial Instruments

Scientific and technical literature provide a wealth of information on the detailed historical and technical developments of CL. Early reviews describing commercial apparatus appeared as early as 1968 [9]. In this context it is important to cite the recent comprehensive set of survey updates written by Stanley since 1992 (see [5] and references therein). These include not only instruments but reagents or kits for CL or BL. They constitute an excellent source of information about commercial instrumentation.

3.OVERALL IMPROVEMENT IN THE DESIGN OF IMAGING INSTRUMENTS

Until recently, most standard or imaging luminometers were a simple combination of existing parts, such as a dark cabinet, a photon-counting photomultiplier or a CCD camera, a computer with an acquisition board, and image-processing software. Generally, these components had not been specifically selected or tailored in view of their effective integration. Although good performance could be obtained, reproducibility was not always guaranteed and the analysis of samples and treatment of data were neither straightforward nor fast. The concept of high throughput was not yet important.

Starting in the mideighties, an increase in sensitivity and speed as well as the simplicity in the handling of reagents resulted in the fast development of luminometric tests at the expense of isotopic labeling tests. This move was later associated with an increased availability of molecular luminescence probes. In an almost parallel move, pressure grew due to needs originating from gene sequencing and comparative genomic hybridization (CGH), to name only two. As these techniques present strong requirements in terms of speed, sensitivity, and spatial resolution, the quasi-artisan aspect of instrumentation referred to above was bound to disappear. The design of recent instruments insures generally that

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the best performance from all components will combine to provide the highest sensitivity, reproducibility, speed, and ease of handling.

As a consequence of the optimization of performance, different performing instruments were first designed for one specific task. However, the best instruments from the latest generation correspond to universal instruments that make it possible to run several types of experiments such as fluorimetry, luminometry, and densitometry for instance. This has interesting consequences for users who work in different domains of luminescence for research and development purposes and do not have to buy a whole range of instruments.

However, many recent instruments are still not considered satisfactory, since professional developers in the field of high-throughput screening (HTS) want to use the full performance of the latest generation of robots and computers for automation. This results in new instrumental developments, like the possibility of reading not only 96, but 384 or even 1536 wells plates as well as DNA chips, very rapidly (in a minute or so) and repeatedly without any mechanical failures. Hence, in the eyes of company scientists developing new assays, many present-day instruments still correspond to an intermediate stage of development. For research laboratory scientists, on the other hand, the actual equipment offers excellent performance.

4.USER-ASSEMBLED SYSTEMS ARE STILL COMPETITIVE

As noted above, the number of integrated instruments has grown tremendously. However, the intrinsic quality of equipment that can be considered as subsystems or components for assembling, for instance, an imaging luminometer has also greatly increased. As a consequence, even today, an expert user may assemble a unit to carry out a specific job with good efficiency.

5.CCD CAMERAS: THE HEART (AND EYES) OF IMAGING SYSTEMS

Special CCD cameras are used to acquire high-resolution images for scientific work. They are also called HCCD cameras. They belong to the two following classes: cooled CCD cameras or intensified CCD cameras. Intensified cameras have been mostly used to follow spectroscopic fast events but they are now used in imaging to offer very high sensitivity. Cooled CCD cameras, however, offer better spatial resolution for luminescence work in the field of analysis and a broader combination of spectral ranges and sensitivity.

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Among the components or subsystems found in luminescence imaging, CCD cameras constitute the key element. There are two main reasons to use these devices. The first is that they can detect very weak light emission and thus provide high sensitivity. The second reason is that they have an extended linear range for the conversion of light intensity into an electric signal. For the user this means carrying out the quantitative determination of an analyte over an extended range of concentrations.

Of course, other components are necessary to assemble an instrument. However, they are generally less complex and have been optimized more rapidly or their characteristics were not so critical to the overall performance (at least before the CCD reached the present state of development). In contrast, the problems that faced the use of CCD cameras when they appeared on the market some 25 years ago were not fully appreciated at once. These problems have been solved one after another, leading to new hardware features and new concepts. Even now, the conception of a CCD camera for its integration in a specific instrument is not a straightforward process. Without delving into the hardware complexity, it seems interesting to describe—even in a simplified manner—the structure and basic characteristics of modern CCD cameras.

6. A SIMPLE DESCRIPTION OF THE HARDWARE

In a crude description, a CCD chip consists of a monoor bidimensional array of tiny elementary detectors (the size of which may reach less than 10 m by 10 m), formed on a silicon substrate and with an associated electronic circuitry (Fig. 1). Each of these detectors will define a pixel, that is, an elementary constituent of an image. For this reason, such an elementary detector is also often called a pixel and we will use this terminology in the rest of the chapter. For imaging purposes, which concerns the present chapter, only 2D arrays are used, with a pixel size in the vicinity of 50 m by 50 m. In short, during acquisition of a signal, each pixel of the array will convert the photons reaching its surface into electrons that drift toward a well (the depletion region) where they are stored. In the following step, called the readout, the trapped electrons will be counted and the count will define the value of the corresponding pixel in the resulting image.

At a more detailed level, one must mention the presence of elements called electrode gates, which overlap the pixel surface. During the readout process, a voltage is applied in sequential steps across the electrodes to shift the charge of each pixel row by row along one dimension of the array until the charge reaches the bottom row, which constitutes the serial or shift register of the chip (Fig. 1). The charge thus collected will then move along the serial register until it reaches

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Figure 1 Layout of the pixels of a CCD. (Reproduced by permission, from the Book of Photon Tools, Oriel, Stafford, CT.)

the end of the array and enters a low noise preamplifier (Fig. 2). The preamplified signal is then fed to an analog-to-digital (A/D) converter and the digital signal is sent to the computer. All pixels are treated sequentially.

Readout speed for a typical CCD camera is somewhere between 100 kpixel s 1 and 5 Mpixel s 1. Cameras with up to 4 Mpixel chips have been built. How-

Figure 2 Readout pattern of a two-dimensional CCD. The bottom row corresponds to the shift register. Its output is connected to the preamplifier input. (Reproduced by permission, from the Book of Photon Tools, Oriel, Stafford, CT.)

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ever, for imaging very low light levels, slow scan cameras are used that read out at less than 50 kpixel s 1.

A microprocessor set in the camera housing together with the preamplifier and A/D converter controls two clock drivers to precisely time the readout sequences. In some recent cameras the preamplifier is integrated on the chip. The computer sends the commands to control the acquisition step (setting the temperature and exposure to the source of light, acting on the shutter when reading continuous signals) and the readout step (counting the number of electrons stored in each pixel). Exposure and readout are normally triggered as two successive events. Note that in a CCD the process of reading a pixel is destructive.

Strictly speaking, the elements of hardware mentioned above are only those of the chip. A camera is built around a chip, adding ancillary equipment such as an optics or a cooling system. The simple description provided above allows, however, an analysis of the most important features to take into account when selecting or using CCDs.

7.KEY PROPERTIES FOR DEFINING THE PERFORMANCE OF A CCD CAMERA

7.1 Introduction

Some of the parameters defined below are interconnected and it is impossible to introduce all of them one by one independently of the others. The subject is complex and the presentation given here is simplified. More may be learned on the subject from the equipment catalogues of various companies [10–12] or from specialized publications [13, 14].

7.2 Resolution

Spatial resolution is limited by the size of the pixel of the array. Its choice depends on the intended use of the CCD camera (spectroscopy, luminometry, etc.). The pixels of a CCD chip form a flat bidimensional array and as such are conceived to acquire a 2D picture. The consequence is that to keep the benefit of high resolution, the object used to form an image must be as flat as possible (chromatographic plate, blot, microarrays, Petri dish, etc.). However, the associated optics may project a distorted image of the peripheral region of a large object on the chip. As the size of the chip is limited to a few square centimeters, it means that for given optics, the size of the object is limited so as not to degrade performance.

Some objects may have a large size (gels, autoradiographic films, or TLC plates, for instance) and special techniques must be used to obtain a global image while keeping the high resolution constant. The usual way to treat the problem is to scan the object.

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Figure 3 Inside view of the Wallac Arthur multiwavelength imaging system. (Reproduced by permission from EG&G Life Sciences, Evry, France.)

To illustrate the process, we describe the solution provided by the Arthur imaging system from Wallac EGG [15]. It is a general-purpose, CCD-based horizontal scanning imaging system that scans sample areas as large as 23 cm 28 cm to provide images with a constant resolution down to 50 m. Both luminescent labels and fluorescent dyes may be used. The general structure of this instrument is shown in Figure 3.

The sample is placed horizontally on a two-axis transport mechanism at a predefined focal plane. A cooled CCD camera scans an area previously defined by the user and takes multiple adjacent frames covering that area. These frames are combined in the computer into one single high-resolution image.

7.3 Quantum Efficiency

Quantum efficiency (QE) is defined as the ratio of the number of photoelectrons created by a given light signal to the number of photons in this light signal. This parameter is wavelength-dependent and is usually available as a graph of QE versus wavelength.

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7.4 Dynamic Range

The dynamic range factor is defined as the ratio of the largest signal that can be acquired to the signal corresponding to the limit of detection (LOD). In this definition, the signals correspond to the stored charges and are analogous signals. So the dynamic range is a measure of the range of signals, hence of the range of concentrations of the analyte, that can be measured simultaneously.

When working conditions have been defined for a given experiment, the signal corresponding to the LOD is the signal the level of which is equal to the noise associated with this signal. This definition of the LOD corresponds to a signal-to-noise (S/N) ratio of 1. Note that values of 2 or 3 for S/N are used in different subfields of analytical chemistry to define the LOD. The two most important parameters to set the working conditions are temperature and exposure time.

What is the largest signal that can be measured? It was noted earlier that when a signal is acquired during the exposure time, photoelectrons are generated and stored in each pixel. It turns out that each pixel cannot store more than a specific number of electrons, which is known as the well depth capacity or saturation charge. It is expressed in electrons or charge per pixel. The saturation charge is typically in the range 300,000–500,000 electrons for HCCDs.

The charge corresponding to a given signal varies from 0 (supposing for the moment that one does not have a dark current) to the saturation charge. As noted before, it is an analog signal that is converted after the readout step into a digital binary value by the A/D converter. The total range for the binary values is divided in n levels so that the converted saturation charge corresponds to the maximum value of 2n. Typical values for n are 12, 14, or 16. In the latter case this corresponds to 65,536 levels (gray levels).

It is clear that the n value should be such that 2n is at least equal to the dynamic range to benefit from the full performance of the chip.

Note that any signal may be conveniently measured in terms of electrons or of energy per unit surface (J cm 2). To carry the conversion between the two modes, one has to use the wavelength dependence of the QE of the chip and the surface of the pixel.

7.5 Dark Current

All CCD chips have a leakage current, also called dark current, which is of thermal origin as in photomultipliers. As a result of this phenomenon, a charge slowly builds up in each pixel even when the detector is shielded from light.

This means that any signal is contaminated to some amount by the dark current. As the latter is of thermal origin, it varies with temperature. In fact, the variation is exponential and cooling the chip results in reduction of the dark

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current by a factor of about 3 for each 10°C lowering of temperature. So many CCD devices are cooled to obtain a low dark count value (see Sec. 8.1). This also reduces baseline drift induced by temperature changes.

7.6 Noise

7.6.1Introduction

In Sec. 7.4, the word noise was used. The analysis of noise in CCDs is quite complex and a simplified analysis will be given here. True noise is also called pixel noise and results from the combination of several noise components. The latter, analyzed in the next three sections, does not add together directly to give the total noise [10, 12, 14].

7.6.2Shot Noise from the Signal

Any light signal is composed of discrete particles, the photons, which are emitted at random. The resulting fluctuation in the number of photons reaching a pixel in a unit of time is transferred to the photoelectron flux generated by absorption. The fluctuation in the associated current is the shot noise. Due to the nature of the photons, the associated shot noise for a flux of N particles is equal to the square root of N. It depends on the exposure time.

7.6.3Shot Noise from the Dark Current

The electrons associated with the dark current are also released in a statistical manner and are associated with shot noise. The latter is very sensitive to temperature and also depends on the exposure time. Its contribution to the total noise is significant only at low signal levels.

7.6.4Readout Noise

An electronic noise component is also generated by the transfer of charges and by the preamplifier. For each readout process, one readout noise is generated. This readout noise is not very sensitive to temperature but increases with readingout speed. Readout noise for a HCCD is about 10 electrons RMS or less.

7.6.5Fixed Pattern Noise

In the manufacturing process inhomogeneities arise in the silicon substrate and all the pixels in the array are not exactly identical. They may show variations in their response to the same photon flux. Even in the absence of a light signal, differences occur in the dark current in a group of pixels. However, these differ-

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ences remain constant from frame to frame if temperature is kept constant. As a result, this fixed pattern noise may be corrected for by simple subtraction.

7.7 Binning

Pixel binning is a technique used to increase the S/N ratio and, as a corollary, the dynamic range. It is a specific reading mode of the pixels allowed by the software: in the binning mode, a group of pixels (a superpixel) is read out together as if it were a single large pixel. As one readout noise is generated for each readout, whether it relates to a single pixel or to a superpixel, the S/N ratio is increased as a result of binning. Usually, the software allows the user to control the size of the superpixel. Note, however, that binning results in a loss of spatial resolution so that the user must trade off between dynamic range and spatial resolution.

8. IMPROVING PERFORMANCE

8.1 Dark Current Reduction by Cooling the CCD Chip

Practically, the chip is cooled by means of a cryogenic fluid (often liquid nitrogen)

or, more conveniently, by heat flow from the chip to a multistage Peltier thermoelectric device. By cooling at 40 to 50°C, dark current may be brought down

to less than a hundredth of an electron per pixel per second (e pixel 1 s 1) allowing a much larger exposure time, hence an increased sensitivity. For instance for the ultimate generation of the ORCA series, which use 1280 by 1024 chips, Hamamatsu claims 3.6 e pixel 1 h 1 [16].

8.2 Effect of Readout Rate on Readout Noise

As noted briefly above, the readout noise level is sharply decreased when readingout rate is low. Cameras allowing a slow readout are called slow-scan CCD cameras.

8.3 Back-Illuminated CCD Cameras

This class of CCD has a higher quantum yield than a front-illuminated similar CCD. The origin of this improvement is as follows. The silicon substrate for the chip is etched down, on the face opposite the electrodes, to reach a thin layer ( 15 m), and the light illuminates the array from the back, opposite from the usual way (Fig. 4). It was noted in Sec. 5 that electrode gates that are used for the readout process overlap the pixel surface. Since these electrodes have a given thickness and are laid on the front face, photoelectrons have to travel across a