GB2390900A - Large dynamic range detection system - Google Patents

Abstract

A large dynamic range light detection system includes an asymmetric beam splitter for directing a larger fraction of incident light to one photomultiplier and a smaller fraction of the incident light to another photomultiplier. Preferably the light is split in to 90% and 10 % fractions and the photomultiplier receiving the larger fraction is operated in photon counting mode and the photomultiplier receiving the smaller fraction is operated in integrating mode. The beam splitter may be uncoated glass and fast modulators attenuate incident light on the photomultipliers.

Description

1 2390900

LIGHT DETECTION SYSTEM

This invention relates to large dynamic range light detection, for example for use in fluorescence readers to accommodate large dynamic ranges while maintaining optimal signal-

to-noise performance.

Fluorescence readers are often used for re-sequencing or gene expression studies. In these systems, light such as that from a laser is directed onto a target which may include molecules capable of fluorescing. The emitted fluorescent light is then detected and analyzed.

Detection is often accomplished using a photomultiplier tube in which incident light falls upon a photocathode thereby liberating primary electrons via the photoelectric effect. These primary electrons encounter structures known as dynodes to release secondary electrons. The electrons migrate to an anode and produce a current pulse. The dynamic range of the photomultiplier tube (PMT) is the ratio of the strongest expected signal to the weakest expected signal. At the low end of the signal range it is advantageous to count photons while at the high end such counting may no longer be possible due to pulse overlap and for other reasons. A bruteforce approach to the large dynamic range problem is to increase measurement (averaging) time to extend the detection range toward lower signal levels.

While other solutions are available (compare, for example, a quantum photometer in "The Art of Electronics" by Horowitz and Hill, P. 998, ISBN 0-521-37095-7, Second Edition 1989), they do not permit the fast (pixel times on the order of microseconds) simultaneous measurement of current and fast photon counting. The present invention will increase dynamic range without increasing measurement or averaging time.

The present invention seeks to provide improved light detection.

In one aspect, the system according to the invention for large dynamic range light detection includes at least one asymmetric beam splitter for receiving incident light and to direct a larger fraction of the incident light to at least one other photomultiplier tube. In a preferred embodiment, the photomultiplier tube receiving He larger fraction of incident light is operated in a photon counting mode and the photomultiplier tube receiving the smaller fraction of the incident light is operated in an integrating mode. A suitable larger fraction is 90% of the incident light and a suitable smaller fraction is 10% of the incident light. A

suitable beam splitter is uncoated glass. A digital signal processor may be provided for operating on the signals from the photomultiplier tubes. It is also preferred that a fast modulator be provided to attenuate the incident light based on an actual signal thus resulting in dynamic compression.

The preferred embodiments can extend signal dynamic range to allow photon counting at the low end of the dynamic range and extend the range up to a maximum light load that the light detector can accommodate. The systems can allow covering dynamic ranges that are limited by the photon counting detection limit at the lower end and by the destruction threshold of the PMT at We high end. They also make it possible to achieve dynamic ranges of 104 and more.

British patent application no. 0011109.6, of which this is a divisional application, covers a system for large dynamic range light detection which includes a photomultiplier tube for receiving incident light photons and for generating an output electrical signal in response to the incident light. A discriminator/counter responds to the output signal from the photomultiplier tube to count photons for output signals below a first selected level. A charge integrator responds to the output signal from the photomultiplier tube to integrate the output signal for output signals above a second selected level. Control circuitry is provided responsive to the discriminator/counter and to the charge integrator so that dynamic range is increased. In one embodiment, control circuitry is provided to record outputs from the discriminator/counter and from the charge integrator. In another embodiment, the control circuitry selects an output either from the discriminator/counter or from the charge integrator or a linear combination of the two based on strength of the output signal and stores the selected output. The control circuitry may be a digital signal processor.

In another aspect covered by British patent application no. 001 1109.6, there is provided a system for increasing dynamic range which includes a photomultiplier tube for receiving incident light photons and generating output electrical signal in response to the incident light. An analog-todigital converter responds to the output signal to generate a digital signal, and a digital signal processor operates on the digital signal. The digital signal processor is programmed to analyze the signal to determine whether the signal is within a photon counting range or within an integrating range. The digital signal processor is further programmed to mimic photon counting when the signal is in the photon counting range or to

integrate the signal when the signal is in the integrating range and to generate an output. A photomultiplier tube preamplifier circuit may be provided to broaden pulses from the photomultiplier tube to cover several sampling intervals.

These aspects covered in British patent application no. 0011109.6 can be incorporated in the teachings herein.

An embodiment of the present invention is described below, by way of example only, with reference to the accompanying drawings, in which: Figure 1 is a block diagram illustrating one approach covered in British patent application no. 0011109.6.

Figure 2 is a block diagram illustrating an approach utilizing a digital signal processor also covered in British patent application no. 0011109.6.

Figure 3 is a schematic diagram illustrating an embodiment of the present invention.

Figure 4 is a schematic diagram illustrating an embodiment of the invention using uncoated beam splitters.

With reference to Fig. 1, a hybrid approach to increasing dynamic range will be described. Incident light illustrated by an arrow 10 such as light from fluorescing molecules is detected by a photomultiplier tube (PMT) 12. An output of the PMT 12 forms an input both to a discriminator/counter 14 and a charge integrator 16. The discriminator/counter 14 covers a range of low signals and eliminates most of the excess noise of the PMT 12. In a preferred arrangement, the output current of the PMT 12 is first converted into a voltage using an electrometer which may be considered part of the PMT 12 block in Pig. 1. The integrator 16 covers stronger signal ranges where excess noise is no longer a problem, up to the PMT's saturation/destruction limit. For a typical system, the low and high signal regimes will overlap by a factor of two or more and thus can be gauged to give a continuous transition from counting to integration. The outputs of the discriminator/counter 14 and integrator 16 are read out and reset by a control circuit 18. The control circuit 18 either records both results in storage 20 or chooses one of them based on signal strength and stores only that one in the storage 20. The control circuit 1 B may be a digital signal processor (DSP).

Fig. 2 is an example utilizing fast digital signal processors which can perform both the counting and integrating functions. In this example, the output from the P1dT 12 is digitized in an analog-toigital converter 22 and is processed by a digital signal processor 24. The

fast DSP 24 analyzes the output of the analog to digital converter 22 in a manner such that not a single photon even is missed if possible. This functionality can be achieved by having a PMT preamplifier circuit (not shown) that broadens the PMT pulses just enough to cover a few sampling intervals while not yet reducing pulse height excessively. The DSP 24 analyzes the signal (e.g., by looking at its integrated value first) to find out whether it is in the counting range or the integrating range and then either applies an algorithm that mimics photon courting (i.e., a pulse height discrimination and counting) or integrates the signal if not previously performed. In a crossover region between the high and low signal regimes either a transition point or a gradual transition using the two signals is possible. The DSP 24 can also compensate non- linearities of the signal-versus-light level response. This approach, too, gets rid of the PAT excess noise at the low end of the signal range.

A second cascaded approach to increasing dynamic range of a first embodiment of the invention is shown in Fig. 3. Incident light 10 encounters an asymmetric beam splitter 30 which directs most (e.g. 9096) of the incident light to a photomultiplier tube 32 (PMT). The remaining light (e.g. logo) passes through the bearnsplitter and may be directed to a last PMT 34 or be split up furler by additional beamsplitters that direct the larger fraction of the light passed on by the previous beam splitter to intermediate PMTs 38. The PMT 32 which receives the largest fraction of the signal is preferably run in photon counting mode while the PMTs 34 and 38 are operated in charge integration mode. As in the examples of Figs. 1 and 2, additional circuitry (e.g. a DSP) can be provided in the embodiment of Fig. 3 to choose the appropriate combination of output signals to be either combined into one output signal or to be recorded/stored in parallel.

The asymmetric beam splitters 30 would in this vase normally have to be coated plates. Also, their reflectivities may be different from one another for some designs. The angle of incidence shown serves for illustration purposes only. In the case shown, typical transmissions might be 109 and typical reflectivities might be 90%. If each of the PMTs has a dynamic range of 103, then the total dynamic range would be 104 for two PMTs and even more for additional PMTs.

Another embodiment is shown in Fig. 4. In this arrangement, the beam splitters 40 can be uncoated substrates which are less expensive than coated substrates. The uncoated substrate will reflect about 10% and transmit about 90% of the incident light. Again, the

PMT 32 gets the strongest signal and the PMT 34 gets the weakest signal with the PMTs 38 getting increasingly weaker signals as one moved from the PUT 32 to the PMT 34.

The embodiments illustrated in Figs. 3 and 4 may be limited by the damage threshold of the PMT 32 which sees the larger share of the signal. There are several ways to deal with the potential damage problem. First of all, one could modulate the illumination power by modulating the source of light directly or using an external modulator (e.g. for diode laser or LED source). Alternadvely,, the emitted fluorescent light can be modulated to reduce the amount of light going to PMT 32 which leaving the full signal on the PMT 34. This approach would protect bow the cathode and dynodes of the PhIT 32. Alternatively, the PMT bias voltage can be modulated for one or more electrodes, which will protect dynodes but not the photocathode.

Both the hybrid counting/integrating system and the cascaded detector system described above extend signal dynamic range by allowing photon counting at the low end of the dynamic range and extended up to the maximum light load the detector can handle. Both approaches allow covering dynamic ranges that are limited by the photon counting detection limit at the lower end and by the destruction threshold of the P1dT at the high end. Dynamic ranges well in excess of 104 and more are achievable with the designs of this invention.

The disclosures in United States patent application No. 09/313,102, from which this

application claims priority, in British patent application no. 001 1109.6, from which this application is divided, and in the abstract accompanying this application are incorporated herein by reference.

The teachings herein also provide for a system for large dynamic range light detection including: a photomultiplier tube for receiving incident light photons and for generating an output electrical signal in response to the incident light; a discriminator/counter responsive to the output signal from the photomultiplier tube to count photons for output signals below a first selected level; a charge integrator responshe to the output signal from Me photomultiplier tube to integrate the output signal for output signals above a second selected level, and control circuitry responsive to the discriminator/counter and to the charge integrator whereby dynamic range is increased.

Advantageously, the control circuit is operable to record outputs from the discriminator/counter and the charge integrator. Preferably, the control circuitry is operable to select output either from the discriminator/counter or the charge integrator or a linear combination of the two based on strength of the output signals and stores the selected output.

Advantageously, the control circuitry is a digital signal processor.

The teachings herein also provide for a system for large dynamic range light detection including: a photomultiplier tube for receiving incident light photons and for generating an output electrical signal in response to the incident light; an analog-to-digital converter responsive to the output signals to generate a digital signal; and a digital processor for operating the digital signal, the digital signal processor being operable to analyze the signal to determine whether the signal is in a photon counting range or in an integrating range, the digital processor being operable to mimic photon counting when the signal is in the photon counting range or to integrate the signal when the signal is in the integrating range and to generate an output.

The system preferably includes a photomultiplier tube preamplifier circuit operable to broaden pulses from the photomultiplier tube to cover a few sampling intervals.

Claims (9)

1. A system for large dynamic range light detection including: at least one asymmetric beam splitter for receiving incident light and operable to direct a larger fraction of the incident light to one light detector and to direct a smaller fraction of the incident light to at least one other light detector

2. A system as in claim 1, wherein the light detector receiving the larger fraction of incident light is operated in a photon counting mode and wherein the light detector receiving the smaller fraction of the incident light is operated in an integrating mode.

3. A system as in claim 1 or 2, wherein the larger fraction is approximately 90% and the smaller fraction is approximately 10%.

4. A system as in claim 1, 2 or 3, wherein the beam splitter is uncoated glass.

5. A system as in any preceding claim, including a digital signal processor operable to choose an appropriate combination of output signals to be combined either into one output signal or to be recorded/stored in parallel.

6. A system as in any preceding claim, including a fast modulator operable to attenuate light incident on the sample.

7. A system as in any one of claims 1 to 5, including a fast modulator operable to attenuate incident light on at least one of the PMT's.

8. A system for large dynamic range light detection substantially as hereinbefore described with reference to an as illustrated in Figure 3 or 4 of the accompanying drawings.

8. A system as in any preceding claim, wherein each light detector is a photomultiplier tube.

9. A system for large dynamic range light detection substantially as hereinbefore described with reference to an as illustrated in Figure 3 or 4 of the accompanying drawings.

Amendments to the claims have been filed as follows 1. A system for large dynamic range light detection including: at least one asymmetric beam splitter for receiving incident light and operable to direct a larger fraction of the incident light to one photomultiplier tube and to direct a smaller Faction of the incident light to at least one other photomultiplier tube.

2. A system as in claim I, wherein the photomultiplier tube receiving the larger fraction of incident light is operated in a photon counting mode and wherein the photomultiplier tube receiving the smaller fraction of the incident light is operated in an integrating mode.

3. A system as in claim 1 or 2, wherein the larger fraction is approximately 90% and the smaller fraction is approximately 10%.

4. A system as in claim 1, 2 or 3, wherein the beam splitter is uncoated glass.

5. A system as in any preceding claim, including a digital signal processor operable to choose an appropriate combination of output signals to be combined either into one output signal or to be recorded/stored in parallel.

6. A system as in any preceding claim, including a fast modulator operable to attenuate light incident on the sample.

7. A system as in any one of claims 1 to 5, including a fast modulator operable to attenuate incident light on at least one of the PMT's.