CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2018/073093, filed on Aug. 28, 2018, and claims benefit to German Patent Application No. DE 10 2017 119 663.3, filed on Aug. 28, 2017. The International Application was published in German on Mar. 7, 2019 as WO 2019/042973 under PCT Article 21(2).

FIELD

The present invention relates to a method for counting photons by means of a photomultiplier, to a computing unit and to a computer program for performing it and also to a microscope system for carrying out the method.

BACKGROUND

Light detectors having the highest possible sensitivity are used for example in confocal microscopy to capture the fluorescence light emitted by the sample with the best possible signal-to-noise ratio and quantitatively as accurately as possible. Various types of photomultipliers are suitable herefor. It is in particular desirable to use semiconductor photomultipliers (SCPM) such as silicon photomultipliers (SiPM) for this purpose, since these offer advantages in terms of sensitivity, temporal resolution and robustness at relatively small costs.

In an SCPM, an analog pulse100is produced from each detected photon, wherein a typical signal profile (intensity over time) is shown inFIG. 1. The pulse shape is divided into a rapid increase101in the ns to sub-ns range and a drop102in the range of typically 20-100 ns. The signal height is given substantially by the bias voltage above the avalanche breakdown and the capacitance of the individual cell, and of course by amplification factors of the subsequent electronics. The rapid increase is a consequence of the breakdown, at which the capacitance of the biased diode is discharged to below the breakdown voltage, and the slower drop is the result of the RC time constants in combination with what is known as the “quenching” resistance. The area under the signal curve corresponds, as it were, to the charge liberated during the breakdown. An SiPM having dimensions that are reasonable for confocal microscopy (e.g. 1.3 mm×1.3 mm; 200-1500 individual cells connected in parallel) offers, in addition to great homogeneity, signals of equal size or the same charge for each cell impinged upon by a photon.

In principle, the data recording in photomultipliers can be effected either in the counting mode or in the integration mode (also referred to as digital or analog mode).

One possible approach for obtaining a signal that is proportional to the number of the incident photons is to count the events by comparing the analog signal to a threshold and by increasing a counter each time the latter is exceeded. As soon as pulses superpose, however, the measured counting rate is smaller than the actual number of events. Knowing the instantaneous photon statistics and the pulse shape, it is possible to draw conclusions with respect to the error and possibly correct it. In the case of pulsed excitation, it is also necessary to know the fluorescence lifetime to perform successful correction. That is to say, the correction must be effected in dependence on the sample, which entails additional outlay.

A further possibility to keep the errors as small as possible when counting pulses is to bring the pulses into as short a form as possible. This can be accomplished either by high-pass filtering of the output signal or by way of a capacitive tap between the diode and quenching resistor, such as for example shown in US 2013/0099100 A1. However this reduces the signal height.

The methods mentioned have in common that a saturation effect occurs during counting and that for example pulses with twice the height cannot be differentiated from simple pulses.

WO 2012/017762 shows a method in which a measurement signal is subjected to threshold value processing such that only signal values that lie above the threshold value are taken into account. The area under the pulses is then ascertained to determine the number of photons therefrom. However, due to the threshold value processing, the area and number of photons are not always proportional. For this reason it is necessary to calculate both a reference value and the threshold value.

It is therefore desirable to specify an improved method for counting photons.

SUMMARY

In an embodiment, the present invention provides a method for counting photons using a photomultiplier. A measurement signal is obtained from a raw signal produced by the photomultiplier by correcting the raw signal for a noise signal and/or an offset, wherein an incident photon produces a pulse in the raw signal. The measurement signal is integrated over time to form an analog integrated measurement signal. A number of photons that are incident in the photomultiplier is ascertained by comparing a value of the analog integrated measurement signal to an integral proportionality value which corresponds to a specific number of photons incident in the photomultiplier.

