Electron Multiplying CCD Technology: Application to Ultrasensitive ...

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Electron Multiplying CCD Technology: Application to Ultrasensitive. Detection of Biomolecules. Donal J. Denvir, Colin G. Coates. Andor Technology Ltd., Belfast, ...
Electron Multiplying CCD Technology: Application to Ultrasensitive Detection of Biomolecules Donal J. Denvir, Colin G. Coates Andor Technology Ltd., Belfast, UK ABSTRACT A novel Charge Coupled Device (CCD) has been commercially produced by Marconi Applied Technology, UK under the trade name of L3Vision, incorporating a solid-state electron multiplying structure based on the Impact Ionisation phenomenon in silicon. Here we review this technology, and evaluate the first electron multiplying CCD camera, in particular using it to image weak emissions from microtitre plates. A theoretical model was constructed to predict S/N and Z-factor performances, which were compared to actual measurements, verifying that a greater than one order of magnitude improvement can be achieved over conventional CCDs. The demonstrations of remarkable sensitivity enhancement presented here are discussed in terms of the EMCCD camera’s suitability for use in life sciences applications such as High-Throughput Screening (HTS), and other approaches requiring ultrasensitive detection of biomolecules, including Single Molecule Detection (SMD). Keywords: CCD, EMCCD, High Throughput Screening, Single Molecule Detection, Electron Multiplying CCD, Zfactor.

1. INTRODUCTION Image-based detection approaches are fast becoming the popular choice for High Throughput Screening (HTS) in drug discovery, as opposed to traditional plate reading methods, in which measurements are taken from isolated points of a microtitre plate. An imaging approach however, bears the inherent advantage that a complete plate may be quantitatively examined in one single exposure. Detector performance will significantly benefit High Throughput Screening (HTS) efficacy, for example in terms of the statistical viability of the measured data set. Further, from an analytical perspective, current trends towards single molecule measurements have ultimate sensitivity, breaching a new era of low concentration analysis, by which biological reactions and tests can be carried out with minute samples, which are normally insufficient for measurement purposes. To this end, optimal Signal to Noise (S/N) standards must be achieved at significantly lower input signal levels. An advanced CCD camera design, offering unsurpassed sensitivity performance, will be shown here to yield markedly improved Z-factors under low-light conditions, which will facilitate both assay design and operation. We must first however, critically examine the variety of imaging detectors available: • Charge Coupled Devices (CCDs)1 have become ubiquitous for scientific imaging application. This is not surprising when their many attributes are considered: (i) they have the highest and broadest Quantum Efficiencies (QE) of any detectors available; (ii) they have excellent resolution, limited only by the pixel size; (iii) they have virtually no cross talk or other resolution artefacts such as halo or chicken-wire; (iv) CCD darkcurrent can essentially be made negligible through effective cooling. In fact the single main weakness of CCDs are their readout noise. Scientific CCDs can achieve 2-4 electrons of readout noise but only at slow readout speeds. However, at the more practical speeds of 1MHz pixel rates or above, this noise is typically 10 electrons or more. In applications where raw sensitivity is required, particularly at high readout rates, either Intensified CCDs (ICCDs) or Electron Bombardment CCDs (EB-CCDs) are favoured. But these detectors have their own drawbacks, as examined below. • ICCD (GEN II and III) The high electron gain from the Micro Channel Plate (MCP) bestows ICCDs their sub-electron readout noise, and this is their single biggest advantage (where ns-order gating is not required). They have reasonable QE (particularly Gen III

intensifiers) but still lag considerably behind that of CCDs. They also have a poor Noise Factor, typically 2 to 3.5 (vide infra). Resolution figures appear good on paper compared to CCDs, but their cross talk has a long low-level tail and they also suffer from lag, chicken-wire, scintillation, and halo effects. Further, they are complex and expensive, and suffer from a finite lifetime and damage effects from over-exposure. • Electron Bombardment CCD (EB-CCD) These were developed from ICCDs to overcome some of their shortcomings; the CCD is placed inside the vacuum tube, allowing direct detection of the photoelectrons from the photocathode. The photoelectrons are accelerated through several thousand volts resulting in high gains, giving them similar sensitivities to ICCDs, but this gain is achieved with near unity Noise Factor. Further, since they have no MCP or fiber optics they have better resolution and no chickenwire effect compared to ICCDs, but they still suffer from halo, a finite lifetime and damage effects from over exposure. Like ICCDs they are complex and expensive and because the CCD must be built into the vacuum this severely limits the available range of CCD formats. The QE limitations of ICCDs, pertain also to EB-CCDs. • Electron Multiplying CCD Marconi Applied Technology (UK) has developed a new CCD architecture2-5 that unites the sensitivity of an ICCD, or an EB-CCD, with the inherent advantages of a CCD. This technology is sold under the trade name L3Vision, and is covered by a patent (EP 0 866 501 A1). Presently only one CCD format is commercially available under the part number CCD65, however, more formats will be released imminently. At the time of writing, scientific cameras based on this device are available only from Andor Technology, under the trade name EMCCD (Electron Multiplying CCD). These cameras are characterised in the current study. Similar technology and patents (US 4912536 Mar. 27, 1990 and 5337340 Aug. 9, 1994) have also been developed by Texas Instruments, TX under the trade name Impactron6, but no devices were available for testing at the time of writing.

2. EMCCD - PRINCIPLE OF OPERATION

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Figure 1. Schematic of Marconi CCD65 showing the position of the extra gain register.

The L3Vision CCD65 is manufactured utilising standard CCD fabrication techniques. The camera is a front illuminated frame transfer device with an image size of 11.52 mm horizontally x 8.64 mm vertically, consisting of 576 active horizontal pixels x 288 active vertical pixels with a pixel size of 20 um x 30 um. The unique feature of this device is an electron multiplying structure that has been inserted between the end of the shift register and the output amplifier, and is referred to as the gain register, see figure 1. This structure is similar to the shift register except that the R2 phase (of the usual 3 phases, R1, R2 and R3 – see Fig. 2) of this section is replaced with two electrodes, the first held at a fixed Transfer direction R3

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potential and the second clocked as normal, except that a much higher voltage amplitude is used (between 40V and 50V) than is necessary for charge transfer alone. The design of the fixed voltage electrode and the clocked electrode, and the relatively large voltage difference between them, results in an intense electric field which is sufficiently high for the transferring electrons to cause Impact Ionisation7. Impact Ionisation generates new electrons, i.e. multiplication or gain, as illustrated in Figure 2. Only the multiplication transfer is shown as it is assumed that the reader is familiar with normal charge transfer in CCDs. The multiplication per transfer is actually quite small, only around X1.01 to X1.015 at most. This may seem quite insignificant but when executed over a large number of transfers, a substantial gain is achieved. For example, with X1.015 multiplication per transfer over 591 transfers (in the case of the CCD65), a gain of 1.015 to the power of 591, equalling X6630, is achieved. Considering that there are no other loss mechanisms, these gain levels signify that EMCCDs possess sensitivities every bit as good, and better than, the very best ICCDs and EBCCDs. To perceive how this gain register can be used to best advantage, one must first understand the origins of CCD noise. The Charge Transfer Efficiencies (CTE) of modern CCDs are very high, typically 99.9999%, meaning that photoelectrons generated in any pixel are transferred to the output amplifier of the CCD without loss, and without mixing with neighbouring pixels. It turns out that the only significant source of noise is the electronic noise of the output amplifier itself. As mentioned earlier, this readout noise will increase with readout frequency, but it is independent of the signal level (consequently readout noise can be ignored at high signal levels where the shot noise of the signal dominates). Therefore, inserting the electron amplification before the output amplifier (see Figure 1), the signal may be increased above the readout noise, hence effectively reducing the readout noise. In fact, a gain level equal to the readout noise in electrons should reduce the effective noise to one electron, and higher gains will result in sub 1 electron values, which for any practical purposes means that readout noise may be ignored. Importantly, through using higher gains at higher readout speeds, this noise performance can be achieved at any speed. This is not the first attempt at harnessing Impact Ionisation for electron multiplication, but this particular design of the gain electrode structure and placing just one gain section immediately before the output amplifier, represents a significant advance over previous attempts by Hynecek8. This earlier design suffered from excessive spurious charge generation and large pixel-to-pixel gain variations.

3. EXPERIMENTAL DESCRIPTION In this paper we review the L3Vision technology and examine its use in life science applications, in particular the low light level imaging of luminescent signal from microtitre well plates. To achieve this, we employed the Andor Technology DV465 EMCCD camera, which is based on the Marconi CCD65. The camera holds the CCD in a hermetically sealed vacuum and can be thermoelectrically cooled down to –70oC to reduce or remove CCD dark current. This arrangement also thermostatically controls the CCD temperature, so removing any errors due to the temperature dependence of the Impact Ionisation process. The clock voltage amplitudes are accurately controlled and could be adjusted via the computer software along with the other CCD parameters typical on a scientific camera. Gain adjustment was possible from X1 (unity), meaning no electron multiplication, to > X1000. This set-up made the measuring of gain dependencies on clock amplitude and temperature very straightforward. Single pixel noise was measured by making repeated measurements of a stable light source. These data points are plotted in Figure 3 as noise and S/N vs. signal for different gains, shown overlaid on the theoretical curves. The noise is actually the effective noise since the value measured has been divided by the gain, such that different gains can be compared. It is this parameter that is plotted in Figure 3, in units of electrons. To simulate luminescent signal in a High Throughput Screening experiment, a 96 well plate was imaged using a F2 macro lens, such that all 96 wells were visible and comfortably filled the field of view, resulting in each well filling approximately 200 pixels. These pixels were clearly derived from inside the well and do not include any signal from the well walls. All measurements and calculations were executed in terms of photoelectrons in the silicon, in order that the results are independent of the optical arrangement and of QE, and hence, can be easily related to other optical set-ups and wavelengths. In this approach a stable luminescent source was used, but the results would be equally applicable to fluorescence or other emissions, since essentially it represents an activation/agonist assay. All measurements were made

by reading out an image and co-adding all 200 pixels in a well. In keeping with the nomenclature of the paper by Zhang et al9, repeated measurements of the control were made (in this case these produced no optical signal), and from these ERWK WKH PHDQ EDFNJURXQG LH QHJDWLYH FRQWURO VLJQDO  c and the background standard deviation (control standard GHYLDWLRQ  1c were derived. SimiODUO\ UHSHDWHG VDPSOH PHDVXUHPHQWV ZHUH GRQH WR JLYH WKH PHDQ VDPSOH VLJQDO s, and WKH VWDQGDUG GHYLDWLRQ RI WKH VDPSOH VLJQDO 1s. Both the control and sample measurements were done for different gains and the results are shown in Figure 6, corrected fRU JDLQ DV H[SODLQHG HDUOLHU 7KH DFWXDO VLJQDO s – c (which is the actual optical signal since the control has no light) is plotted against the Z-factor for different gains where, Z = 1 -  1s  1c  s – c) Alternatively, to present results in terms of S/N a separate set of measurements were performed where repeated single FRQWURO DQG VDPSOH PHDVXUHPHQWV ZHUH PDGH WR \LHOG D PHDQ DFWXDO VLJQDO

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Figure 3. Expected and measured single pixel performance for different gains, assuming 20 electrons readout noise.

Finally, the EMCCD camera was employed for imaging low concentration of luminescent biomarker within microtitre wells. For this demonstration, serial dilutions of anti-Dig-HRP conjugate [Dig = digoxigenin; HRP = Horse Radish Peroxidase] (Roche) in tris buffer, pH 7.0 were used to prepare a series of solutions of the conjugate, ranging from

approx. 125 pM to 3.6 pM (using 50/50 vol. dilution steps). 10 µl amounts of each solution were combined with 50 µl of Supersignal Femto (Pierce) solution, a luminol/enhancer/peroxide mixture, in separate wells within a 384 well microtitre plate. A F-1.3 Nikon lens was used to image the ten sampled reaction wells centrally within the field of view.

4. RESULTS AND DISCUSSION Figure 3 shows the expected effective decrease in readout noise and increase in S/N of a single pixel for different electron multiplications. The ‘Ideal’ line corresponds to a detector with no readout noise, limited only by signal shot noise, whereas the ‘Theory X1’ line represents the performance of the CCD with no electron multiplication and assuming a readout noise of 20 electrons RMS. The ‘Theory X100’ in fact lies very close to the ‘Ideal’ line. Impact Ionisation is the acceleration of a conduction band electron in an electric field within the crystalline silicon. The electrons must gain sufficient energy in the electric field to excite a secondary electron by collision, therefore we would expect that the electron multiplication or gain should have a strong dependence on the clock amplitude of R2. Since a main energy loss for the electron is scattering by phonons, we would also expect the effect to increase with lowering temperature10. A full modelling of Impact Ionisation is beyond the scope of this paper but Figure 4 and 5 show measurements that verify both these dependencies. Clock amplitudes for gains above approx. X10,000 resulted in excessive charge generation due to breakdown. Prolonged operation in this regime may cause permanent changes to the device.

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The data shown in Figures 3, 6 and 7 clearly demonstrate the benefit of having gain. As is evident from Figure 6, an order of magnitude less light is required to achieve a 0.5 Z-factor when using gain. (A minimum of 0.5 Z-factor is considered to be required for robust assay ‘hits’.) In the theoretical curves, the ‘Ideal’ line shows the performance of an ideal detector that would only be signal shot noise limited, and the ‘Theory X1’ line is the expected CCD performance in the absence of electron multiplication. The theoretical curves take into account the gain, the shot noise of the optical signal, and the readout noise of the CCD. Darksignal was not modelled because it was insignificant at the sub-second

exposures and –70oC CCD temperature employed in the measurements. The theory curves for a single pixel, Figure 3, agree with the intuitive idea that once the gain exceeds the readout noise in electrons, there will be little further benefit from increased gain. To this end, the data agree well. In the case of Figure 6 and 7 it should be remembered that the noise from 200 pixels must be added in quadrature, in keeping with the nature of noise addition, therefore we expect a similar effect to occur at a gain of root 200 times the readout noise in electrons, giving X283. Both Figures 6 and 7 show that this simple model works well at low gain settings. However as the gain is increased, the data and the model diverge, in fact there appears to be little benefit for gains above about X50 or X100. Presently, we assume that this is due to the fact that we have not included the Excess Noise Factor in our model. The Excess Noise Factor arises from the statistical nature of Impact Ionisation, which introduces an uncertainty or noise into the amplification process. A similar noise source exists for ICCDs as a result of the statistical nature of the electron multiplication in the MCP. This extra noise source is quantified through the Excess Noise Factor, F, a scaling factor which the signal shot noise should be multiplied by to arrive at the actual noise. Ideally it should be unity, which is the case if no gain is used, but from a simple statistical model2,3 it should be approximately 1.4 for gains above approx. X10. From our data this model appears too simplistic; F is near unity for all but the lowest level signals, which is very desirable, but rises to 2 or 3 at single electron signal levels. This Noise Factor, while undesirable, is still a small price to pay when measuring weak signals that are being degraded by the readout noise, as can be clearly seen from the presented data. Attempts at developing better models are underway6.

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While Figures 3 to 7 yield good quantitative data, Figure 8 has been included to give some visualisation to this data. It shows an image of part of a 384 well plate where each well covers around 5000 pixels. Two rows of five wells contain a serial dilution of low concentration (25 – 0.75 pM) anti-Dig-HRP conjugate in tris buffer mixed with luminol/peroxide, the higher concentration wells located on the left hand side of the image. The fuzzy base observable on the G=X1 image is the readout noise of the CCD, around 20 electrons RMS. Camera exposure times were kept sufficiently short in order that the lowest concentrations should be undetectable under this X1 operation. Indeed, it can be clearly observed that the lower concentration wells on the right hand side of the image are indistinguishable from the noise at X1 gain setting. However, increasing the EMCCD gain results in these wells becoming clearly distinguishable, as is readily apparent from the associated 3D-surface plots. The lowest intensity well (RHS of second row) corresponds to approximately 7 electrons per pixel, which from Figure 3, corresponds to a S/N of only 0.3 at a gain of X1, a level completely lost in the CCD readout noise. G=X9 and G=X45 show the signal being multiplied out of the readout noise and at gain X500 it can be clearly seen above the background. From Figure 3 we see that the S/N has been increased from 0.3 to 2. The fuzzy base seen in G=X500 are actually individual spikes from single darksignal and induced clocking charge electrons (see below), because of the high gain ranges each of the four images are not on the same scale, so these spikes are actually well clear of the readout noise floor seen in the G=X1 image.

Figure 8. Images captured with the EMCCD camera of wells within a 384 microtitre plate, containing serial dilutions of luminescent biomarker, at gain factors of X1, X9, X45 and X500. 3D surface intensity plots are shown for each image in order to facilitate visualisation of signal to noise variation.

Similar experiments (results not shown) have also been successfully carried out for serial dilutions of the conjugate spotted onto a derivatised glass slide, demonstrating that the electron multiplying camera may also be used to great effect for imaging weak signal from well-optimised biochips, i.e. in situations where ‘chemical phenomena’ such as nonspecific binding of protein or oligonucleotide is not the background limiting factor. The above observations point not only to the inherent benefits of the EMCCD to HTS assay development and operation, but also underscore the suitability of the camera to the rigorous demands of single molecule imaging studies11-13, utilising for example, a total internal reflection (TIR) microscopy configuration. Extremely weak signals emitted from single fluorophores can be amplified above the readout noise of the camera, without sacrificing the high and broad QE of the system (which incorporation of an intensifier invariably does). As with an ICCD, the degree of amplification (gain) is readily adjustable, to provide the optimum conditions for single molecule detection. With unity gain the EMCCD is just like any other CCD, but with the gain turned on it can out-perform an ICCD. Where speed is a requirement within dynamic single molecule imaging studies, the camera’s fast pixel readout rate, combined with sub-binning options, enable suitably rapid frame rates to be achieved. Moreover, single molecule microscopy using CCD detection has the capability to effectively investigate many spatially separated individual molecules in a parallel fashion. We must finally consider some other issues important to EMCCD camera operation: • Dynamic Range At some point increasing gain will result in a decrease in Dynamic Range. To counter this effect, the CCD65 has been designed with a large output amplifier capacity. In practice, the number of bits of the A/D used in the camera electronics will set the maximum dynamic range. This dynamic range will be maintained up to gains equal to the readout noise, in electrons; above this and dynamic range will deplete. For the DV465 EMCCD camera the maximum dynamic range is 43,000:1. • Darksignal Darksignal or darkcurrent is just the same as in conventional CCDs. Traditionally, when judging the optimum operating temperature to eliminate the darksignal contribution, a temperature at which the darksignal shot noise is comfortably below the readout noise is selected (taking into account the desired exposure time). In this case, further cooling provides no real benefit. For the EMCCD, however, where there can be essentially no readout noise and single electron events can be detected, ideally no darksignal is desired. This does not mean that more extensive cooling is needed to see the benefits of the electron multiplier, rather that yet more sensitivity through yet longer exposures (should the application allow this) can be obtained with further cooling than usual. • Clocking Induced Charge Impact Ionisation can occur even under normal clocking in any CCD, but when properly set-up it is very small; only one in 10 or 100 transfers will produce an electron. This phenomenon is referred to as Clocking Induced Charge, or Spurious Charge, and is usually lost in the CCD readout noise in even the lowest noise CCDs. However, for the EMCCD at high gain, even individual electrons can be seen as sharp spikes in the image. The effect is similar to a darksignal, but unlike darksignal, it is independent of exposure and will actually increase with lower temperatures (just as the electron multiplication does, since they both use Impact Ionisation, see Figure 5). This charge will set the absolute limit on the sensitivity of the EMCCD, however, this should not be over-stated as it is only about 1 electron in 10 pixels of any image at most. To keep clock induced charge to a minimum, careful attention should be paid to the clock amplitudes and edges.

5. CONCLUSIONS • The solid-state electron multiplier of the L3Vision CCD gives it sensitivity better than an ICCD, considering that it is not constrained by intensifier QE limitations. Such unsurpassed sensitivity, combined with the other advantageous attributes of a CCD format, renders it ideal for any low light level application and makes it a substantial threat to ICCDs and EB-CCDs.

• Even modest EMCCD gains result in the effective readout noise being reduced to sub 1 electron values, essentially rendering this otherwise limiting parameter negligible at any readout speed. • Application of EMCCD gain facilitates statistical optimisation in HTS operation. An order of magnitude less light is required to achieve a 0.5 Z-factor when using gain. • The EMCCD sensitivity enhancement will also benefit single molecule detection experiments, in which the noise sources associated with the CCD camera itself must be minimised as far as possible, such that they will not swamp the weak signal emitted by the single molecule source. • This remarkable technology is new and is hence still evolving. Larger arrays are being tested at the time of writing and Back Illuminated versions are under fabrication.

REFERENCES 1. J. R. Janesick, Scientific Charge-Coupled Devices, SPIE Press, Bellingham, Washington, 2001. 2. P. Jerram et al, “The LLLCCD: Low Light Imaging without the need for an Intensifier”, SPIE Vol 4306, 2001. 3. C. D. Mackay et al, “Sub-Electron Read Noise at MHz Pixel Rates”, http://www.marconitech.com/ccds/l3vision/technology.php. 4. S. H. Spencer and N. J. Catlett, “Low Light Level Solid State TV Imaging”, March 2000, http://www.marconitech.com/ccds/l3vision/technology.php 5. E. J. Harris et al, “Evaluation of a Novel CCD Camera for Dose Reduction in Digital Radiography”, http://www.marconitech.com/ccds/l3vision/technology.php 6. J. Hynecek, IEEE Trans. on Electron Devices, Vol. 48, No. 10, Oct. 2001. 7. Y. Okuto and C. R. Crowell, Phys. Rev. B, Vol. 6, pp.3076, 1992. 8. J. Hynecek, “CCM-A new low-noise charge carrier multiplier suitable for detection of charge in small pixel CCD image sensors”, IEEE Trans. On Electron Devices, Vol 39, No 8, Aug 1992. 9. Ji-Hu Zhang, T. D. Y. Chung, and K. R. Oldenburg, “A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays”, J. of Biomolecular Screening, Vol 4, Number 2, 1999. 10. C. R. Crowell and S. M. Sze, “Temperature Dependence of Avalanche Multiplication in Semiconductors”, Applied Physics Letters, Vol 9, No 6, Sept 1966. 11. W. P. Ambrose et al, “Single Molecule Fluorescence Spectroscopy at Ambient Temperature”, Chem. Rev. Vol 99, pp2929-2956, 1999. 12. R. M. Dickson et al, “On/off blinking and switching behaviour of single molecules of green fluorescent protein”, Nature Vol. 388, pp355-358, 24 July 1997. 13. T. A. Byassee, W. C. W. Chan, and S. Nie, “Probing Single Molecules in Single Living Cells”,Anal. Chem. Vol. 72, pp5606-5611, 2000.