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Figure 4 Principle of a back-illuminated thinned CCD chip. (Reproduced by permission from EG&G Life Sciences, Evry, France.)

larger thickness of silicon to reach the well and have a lower probability of reaching it. Furthermore, in a front-face CCD, light must penetrate the surface layer of silicon oxide, which is on top of the array and absorbs UV radiation. For this reason the QE is much higher in a back-thinned chip. It changes from about 40% for a front-face CCD to 70–80%, in the 600-nm region. However, the manufacturing of these chips is delicate and cameras built around a back-illuminated chip are more expensive.

8.4 Other Improvements

Some cameras allow multiple-mode readout. The user may switch modes between ultralow-readout noise for a high dynamic range and high-speed frame capture at reduced resolution. This is the case, for instance, for the Hamamatsu Orca II (high sensitivity at 14 bits vs. 5.3 fps at 12 bits).

QE may be increased selectively in different wavelength ranges. For instance, a layer of a UV-to-visible converter may be deposited on the front of a standard chip to boost sensitivity in the UV region. One example is Metachrome II from Photometrics [17]. This is only one of the solutions and users should carefully specify their requirements to be sure they get exactly what they need. QE may also be increased in the orange-red and NIR region of the spectrum. Special chips are made with an increased thickness to compensate for the low absorption of the material in that region.

Background subtraction is used to improve contrast. In this technique, a background pseudoimage is obtained with the shutter closed and is subtracted from the actual image, which of course must be acquired under the same condi-

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tions as the background. Most dark current and fixed pattern noise is removed and sensitivity is enhanced.

In video microscopy, for instance, background is normally subtracted using differential interference contrast (DIC) [18]. This technique, which requires a number of manipulations from the user, may now be automated using a new method called polarization-modulated (PMDIC) [19, 20]. It requires the introduction of a liquid crystal electro-optic modulator and of a software module to handle difference images. PMDIC has been shown to bring improvements in imaging moving cells, which show a low contrast, as well as thick tissue samples.

When reading a gel, a thin-layer chromatography (TLC) plate, or a DNA chip, different spots may give rise to signals that differ greatly in intensity. As a result, one pixel has not yet accumulated a sufficient number of charges to give an acceptable S/N ratio than another one is filled and overflows to neighboring pixels. The latter phenomenon is called blooming. Its consequences are a loss of resolution, errors in appreciation of signal intensity, and loss of time to dissipate the charge to start a new measurement. Some CCD chips are made with an antiblooming drain attached to each pixel. They avoid the overflow of charges to neighboring pixels.

An operating mode of a CCD called multipinned phase (MPP) allows a hundredfold dark current reduction at room temperature [21].

9. FILL FACTOR AND THE HANDLING OF COLORS

High-resolution CCDs have another interesting property. The fill factor of an imaging detector is the fraction of the array actually used for image acquisition. In these HCCD cameras, the fill factor is 100% as there is no blind zone in the array that would lower the resolution. In color video cameras for production line or mass market, each pixel is a compound unit made of three adjacent pixels, which are respectively sensitive to red, green, and blue (RGB system). Any pure R, G, or B light reaching the array will activate one of the three pixels only, the other two being blind. For any other color, the three pixels will each build a charge and a color will be affected to the set. So the fill factor for this type of camera is 1/3, with a corresponding decrease in resolution.

To obtain color images with a HCCD camera, filter wheels will be used, in which each filter will be chosen specifically for one fluorescent label, in emission and in excitation. For CL or BL work, one filter wheel is sufficient. For fluorescence, two are necessary (see, for instance, in Fig. 3 the positioning of the filter wheels in the optical path). If, for instance, a three-color experiment is performed, an image will be acquired for each label with the adapted filter(s). The three gray-level images are sequentially acquired. They will then be ‘‘colored’’

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Figure 5 Crosstalk between pixels due to multiple reflections between the window and the surface of the CCD chip. A photon coming from a initially should hit the pixel labeled i. If it is reflected off the surface, it bounces back toward the window and will be absorbed by pixel j as if it were coming from b.

individually by the computer in function of the specific filter/dye combination before being combined into a global multicolored image. So the high resolution is preserved and at the same time, each component image may be acquired with an individually optimized exposure time [22].

An interesting feature of the technique is that the photosensitivity of each dye may be taken care of individually.

10.OTHER PROBLEMS LINKED TO HARDWARE OR OPTICAL DESIGN

Only one example will be given. It concerns a specific form of stray light that is observed when the entrance face of the CCD is protected by a transparent window. This situation arises mostly for cooled CCDs. A small fraction of the measured light is reflected off the surface of the CCD chip, hits the window, and bounces back toward another pixel of the CCD chip (Fig. 5). So the reading on the first pixel is lowered and that on the second is increased, the overall effect distorting the true distribution in intensity.

11. CHARGE INJECTION DEVICE

A charge injection device (CID) is very similar to a CCD as it consists also in a 2D grid of pixels on a semiconductor substrate. The main difference between the devices is that the readout process in a CID is a nondestructive readout

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(NDRO). It is possible to interrogate any pixel of the array at any time to follow the buildup of the charge and the S/N ratio may be selectively optimized for a pixel by increasing its allotted collection time. However, the surface-mounted complex electronic circuits associated with the NDRO absorb a larger amount of the signal light than the electrode gates of CCD chips. This results in lower quantum yields, lower readout speed, and higher readout noise. So CCDs are preferred for luminescence work.

12. COMMERCIAL INSTRUMENTS

Following are some examples of cameras presently available. Some recent cooled intensified cameras compete with cooled back-illuminated cameras. For instance, Cooke DiCam-PRO [23] is an intensified cooled imaging system, with a 12-bit dynamic range and exposure down to 1.5 ns, conceived for low-level light and high-speed imaging applications. From the designers’ data, one may infer that it would have a global performance comparable to that of back-illuminated models such as the PixelVision Bioxight 14 bits modular camera [24]. The latter has a resolution ranging from 512 512 to 4096 4096 pixels. When configured for low-noise operation (10-Mhz system bandwidth), this camera can operate 1024 1024 CCDs at better than 15 e rms noise at 10 frames per second (fps). However, comparisons of a limited set of parameters may be misleading and potential users should clearly rank their needs to make the best choice.

Looking at systems that incorporate CCD cameras, the Berthold NightOWL [25] is a good example for a polyvalent imaging system for detection of ultraweak luminescent and fluorescent signals. Its technology is designed to increase the S/N ratio to the maximum possible, e.g., Peltier/air cooling down to 70°C of a back-thinned CCD. Both microscopic and macroscopic 2D or 3D samples could be measured. The camera is enclosed in a light-tight cabinet and may be moved vertically to fit the CCD detector into the actual sample size. Resolutions up to 80 m may be reached (Fig. 6).

Inside the dark box main sockets and automation port ensure integration of special probe requirements like incubators. There is provision to insert a fiberoptic-guided light source that accept filters for fluorescent work. The sensitivity of the NightOWL is claimed to enable direct detection of reactive oxygen species, even without enhancers like luminol.

The last generation of instruments make it possible to work faster as they offer an increased S/N ratio for the detectors. This results in shorter integration times. Furthermore, the computer-driven reading of microplates with 384 or 1536 wells rather than 96 increases the speed of analysis. Original and new design of the optical path of light and optimized and computer-driven selection of the optical elements and of the mechanical positioning of filters, detectors, or samples

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Figure 6 Schematic view of the NightOWL. (Reproduced by permission from Berthold EG&G Life Sciences, Evry, France.)

for a given experiment became important factors. These instruments allow new modes of excitation for fluorescent samples and/or handling of samples with large areas. They are generally built to cover genomics and proteomics applications, such as electrophoresis gels and various microarrays, and to analyze metabolites expressed as cells or organs.

In the field of DNA sequencing instruments, Perkin-Elmer [26] switched from the PMT-based model 373 sequencer to the CCD-based model 377, which allowed the simultaneous discrimination of four or more colors in emission, in a single lane of an electrophoretic gel. The CCD made possible the use of a specific fluorescent label for each type of base and throughput was improved more than four times [27]. Excitation uses an argon laser.

There are, however, many approaches to DNA sequencing, and even some recent instruments such as the sequencer made by LI-COR [28] do not use a CCD. The latter uses instead a very-low-noise silicon avalanche photodetector to excite near infrared-emitting dyes. The source is a laser diode emitting at 785 nm. It is a compact system that can be mounted on a focusing stage with confocal optics and it is meant for small laboratories that do not have HTS requirements.

The Wallac Arthur multiwavelength system, shown earlier (Fig. 3), is an example of a recent polyvalent CCD-based instrument. Three light sources make it possible to perform several types of excitation: top (fluorescence or reflectance),

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bottom (transmission), and a special edge illumination. The multiwavelength capability is given by six position excitation and emission filter wheels. The basic model uses a Kodak KAF-0400 CCD chip [29]. As a general-purpose imager, it allows measurements on various media, including membranes, blots, TLC plates, exposed autoradiography film, whole body sections, microscopic slides, and all kinds of documents. Kodak distributes CCD chips but also produces systems for CL detection and quantitation of proteins. Its Digital Science Image Station 440CF [30] uses a 752 582 pixel CCD equipped with Peltier cooling. The S/N ratio reaches 1600 for a single frame and rises to 6000 for multiple images. The images generated by the 12-bit camera, which is isolated from the sample to fight contamination and humidity, are linear over 3.0 orders of magnitude [31]. Remember that photographic films have a dynamic range of 1.5–2.0 orders of magnitude. Similar instruments are made by NucleoTech with its NucleoVision 920 instrument [32].

IF CCD-based detectors are the mainstay for luminescence imaging, PMTs are used in many instruments owing to their very high gain and low dark current. Their performance is still being improved in two areas. The first one relates to the nature of the photocathode or dynode design. Such intrinsic improvements lead to the availability of new UV or extended near-infrared PMTs with intrinsically lower noise. The second area of improvement is the integration of a PMT in a convenient module that fulfills a specific function. For example, Hamamatsu produced a side-on cooled PMT, the R6060, with a dark noise 10 times less than for normal PMTs [16]. The R6060-12 version has a built-in heatsink and fan to reduce initial cooling time. This device is convenient for measurements on samples such as living tissues for which it is not possible to control the temperature. Hamamatsu also produces the H7467 series, which is an ultracompact sensor that includes the necessary electronics for photon counting and a high level of functionality. It is an ideal device when a compact unit is required for luminescence work.

The Analyst from LJL Biosystems [33] is specially designed for HTS and runs with robots. It presents analogies to the Arthur described above, but is a different type of instrument as it is not an imager and relies on a PMT to detect light emission. It is dedicated to reading microplates. It allows homogeneous assays using time-resolved fluorescence and fluorescence polarization. Its specially designed read-head, which limits crosstalk between adjacent wells, and a selected PMT make it an efficient tool in luminescence. Each element is optimized for maximum reliability. It is claimed that it delivers the same performance for 384as for 96-well microplates due to a specific focusing system. It has a fully automated optical system, which reconfigures the system when changing the method of analysis to eliminate downtime.

Other manufacturers of PMT-based luminometers designed to read microplates include Anthos [34] and Tecan [35], which manufacture a wide range

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of instruments as well as adapted robotic processors and manipulators. The Lumicount from Packard [36] is also a plate luminometer designed to detect kineticbased CL. It also uses a PMT, which may run in a mode called digital photon integration in which high voltage, hence gain, is controlled to optimize the S/N ratio for each assay.

13. SOFTWARE

All recent instruments are controlled by personal computers and come with software applications. For luminescence work, the intrinsic performance and ease of operation of the instrument are the most important features. However, the full potential of a good instrument may be partly offset by an inadequate or insufficient software package.

In the field of imaging, present-day software should use mouse-driven or menu-selectable commands to control the camera and allow real-time image capture. The latter feature allows the users to monitor the image buildup and trigger the capture at the best moment. Temperature control and exposure times should be under software control.

For all instruments, the software should allow users to program a number of buttons for frequent assays, to run different types of calculation (examples: background subtraction, statistical analysis, curve fitting, and/or storage of raw data for quality control). Almost all instrument productors include a software with their instruments. The latter display specific features that are added to the basic modules: binning for CCDs in the X and Y directions, negative as well as positive viewing of images, image analysis module, band or lane detection, colony counting, etc. Many software packages dedicated to specific applications and running independently from a given instrument have appeared on the market. A typical example of this type of software is Kalcium PC [37]. It is an integrated package for image analysis of the cellular dynamics of calcium ions.

14. CONCLUSIONS

We have given a brief overview of the present state of instrumentation for CLbased analysis. Technological improvements have resulted in ultrasensitive, reliable, and user-friendly system components and instruments. As will be appreciated, the range of instruments offered has become very broad and the purpose of this chapter was not to be comprehensive and to describe all existing instruments or techniques. Readers interested in more details should read technical journals dedicated to clinical, chemical, or biotechnological laboratories or re-

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quest leaflets about specific instruments. Note that a host of instruments for very specific applications appeared recently on the market.

To illustrate this point and to close the chapter, three examples will be given. A compact analyzer, the FastPack from Qualisys [38], was designed for use at the point of care, at a doctor’s office or in a small laboratory to carry a prostate-specific antigen test (PSA). It is based on chemiluminescent technology and is as accurate as a large laboratory instrument. Vilber Lourmat [39] released the Photo-Print, a photodocumentation system for users interested in the printingout of photos. It does not require a PC for the printout but it is equipped with a software and floppy disk drive for transfer to a PC if the user wishes to do so. Last, but not least, specific DNA chip readers are available, such as the ChipReader made by Virtek Vision [40]. Its modular design provides flexibility to read microarrays in any format, detecting up to five fluorescent dyes. It reads very quickly due to its high-speed scanning mechanism.

REFERENCES

1.SP Colowick, NO Kaplan. In: MA DeLuca, ed. Methods Enzymol, vol LVII. Bioluminescence and Chemiluminescence, section IX. Instrumentation and Methods. New York: Academic Press, 1978, pp 529–559.

2.R Johnson, JH Gentile, S Cheer. Anal Biochem 60:115, 1974.

3.RJ Ellis, AG Wright. Luminescence 14:11–18, 1999.

4.P Helle, F Brau, JP Steghens, JC Bernengo. In: A Roda, M Pazzagli, LJ Kricka, PE Stanley, eds. Bioluminescence and Chemiluminescence: Perspectives for the 21st Century. Proceedings of the 10th International Symposium. Chichester: Wiley, 1998, pp 195–198.

5.PE Stanley. J Biolumin Chemilumin 12:61–78, 1997.

6.PS Fullam. In: A Roda, M Pazzagli, LJ Kricka, PE Stanley, eds. Bioluminescence and Chemiluminescence: Perspectives for the 21st Century. Proceedings of the 10th International Symposium. Chichester: Wiley, 1998, pp 207–210.

7.D Lapota, S Paden, D Duckworth, DE Rosenberg, JF Case. In: AK Campbell, LJ Kricka, PE Stanley, eds. Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. Chichester: Wiley, 1994, pp 127–130.

8.DH Leaback. In: AA Szalay, LJ Kricka, PE Stanley, eds. Biolumin Chemilumin Status Report. Chichester: Wiley, 1993, pp 33–37.

9.EW Chapelle, GV Levin. Biochem Med 2:49, 1968.

10.The Book of Photon Tools. Oriel Instrument, Stratford, CT, 1999.

11.Guide for Spectroscopy. ISA Jobin Yvon Spex, 1995.

12.The Photonics Handbook. Book 3, Laurin Publishing Co, 1996.

13.EL Dereniak, DG Crowe. Optical Radiation Detectors. Wiley Series in Pure and Applied Optics. Chichester: Wiley, 1984.

14.GC Holst. CCD Arrays, Cameras and Displays. Co-published by SPIE and JCD Publishing Co. SPIE Press, Vol. PM57, 1998.

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15.EGG, 9238 Gaither Road, Gaithersburg, MD.

16.Hamamatsu Corporation, Bridgewater, NJ.

17.Photometrics, Tucson, AZ.

18.ED Salmon, P Tran. In: G Sluder and DE Wolf, eds. Video Microscopy. New York: Academic Press, 1998, Chapter 9.

19.G Holzwarth, B Moomaw. Application Report. Hamamatsu Euronews 99 1:13, 1999.

20.G Holzwarth, SC Webb, DJ Kubinski, NS Allen. J Microsc 188:249–254, 1997.

21.Janesick, Proc SPIE 1071:153–155, 1996.

22.SJ Sternberg. J NIH Res 5:79–83, 1993.

23.Cooke Corporation, Tonawanda, NY.

24.Pixel Vision, Beaverton, OR.

25.Wallac Oy, EGG Life Sciences, Turku, Finland.

26.Perkin-Elmer Corporation, Applied Biosystems Division, Foster City, CA.

27.MD O’Neill. Laser Focus World 135–142, 1995.

28.LI-COR, Lincoln, NE.

29.Kodak Digital Science, Rochester, NY.

30.The IS440CF is exclusively distributed by NEN Life Science Product Inc., Boston, MA.

31.PC Mayer, JP Masucci. Intl Biotech Lab 1, April 1999, p. 12.

32.Nucleo Tech Corp, Hayward, CA.

33.LJL Biosystems, Sunnyvale, CA.

34.Anthos Labtec Instruments, Wals/Salzburg, Austria.

35.Tecan, Austria AG, Groedig, Austria.

36.Packard Instruments Corporation, Meriden, CT.

37.Kinetic Imaging, Liverpool, UK.

38.Qualisys Diagnostic Inc, Minneapolis, MN.

39.Vilber Lourmat, Marne la Valle´e, France.

40.Virtek Vision Inc, Woburn, MA.

5

Applications of Chemiluminescence in Organic Analysis

Yener Rakiciog˘lu

Istanbul Technical University, Istanbul, Turkey

Joanna M. Schulman and Stephen G. Schulman

University of Florida, Gainesville, Florida

1.	INTRODUCTION		105
2.	CHEMILUMINESCENT ORGANIC MOLECULES OF
	HISTORICAL SIGNIFICANCE		106
	2.1	Lophine and Other Indoles	106
	2.2	Luminol and Related Acylhydrazides	108
	2.3	Peroxyoxalate Chemiluminescence	110
	2.4	Lucigenin and Other Acridines	112
	2.5	Miscellaneous Organic Compounds	114
3.	RECENT DEVELOPMENTS IN ORGANIC
	CHEMILUMINESCENCE		115
4.	ULTRAWEAK CHEMILUMINESCENCE		118

1. INTRODUCTION

Intense and analytically useful direct chemiluminescence (CL) has been observed from a rather limited group of organic compounds. These include diacylhydrazides, indoles, acridines and acridans, polydimethylaminoethylenes, anthracenes, and aroyl peroxides. A substantial number of other kinds of compounds, when

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