Fluorescence Measures with SiPM

Abstract: in this article, we present the design of a fluorimeter based on a SiPM photomultiplier. The excitation pulses are produced by an LED driven by a pulser that generates very short current pulses. The signal produced by the SiPM is amplified and acquired using a multichannel analyzer. The signal spectrum corresponds to the intensity of the fluorescence pulse, which, in turn, is proportional to the fluorophore concentration. The high sensitivity of the SiPM allows for precise measurements even at very low fluorophore concentrations.

Introduction

The project we propose in this work consists of a fluorimeter based on a solid-state photomultiplier (SiPM). The light produced by fluorescence is generally quite weak and is always related to the concentration of the molecule that emits the fluorescence light (fluorophore). Quantitative measurement of fluorescence can therefore be a way to determine the fluorophore concentration.

Since the SiPM works well in pulsed mode, the fluorescence excitation light is produced by an LED driven by a pulser (Fast LED Light Pulser & SiPM). This allows us to obtain very short light pulses. The figure below shows the setup, which consists of a “dark box” containing the LED, SiPM, and a cuvette containing the fluorophore solution. The front-end electronics are also located in the dark box. The rest of the setup consists of the pulser that generates the current pulse sent to the LED and the shaping amplifier that amplifies the pulse to be sent to the multichannel analyser.

As shown in the image below, the front-end electronics utilise a charge amplifier for signal energy/amplitude measurements and a trans-impedance amplifier for timing/coincidence measurements. In this application, we primarily used the charge amplifier.

The images below show the details of the cuvette holder and the supports for the LED and SiPM. The LED is equipped with an excitation filter, while the SiPM is equipped with an emission filter. The excitation filter selects the wavelength (in the blue range) needed to excite the fluorochromes. The emission filter, on the other hand, transmits only the light emitted by the sample (in the green range), blocking stray excitation light.

The image below shows test tubes containing fluorescein solution at different concentrations.

Il segnale amplificato viene poi acquisito dalla scheda Red Pitaya ed elaborato con l’applicazione multichannel analyzer (Improved Alpha Spectroscopy with Red Pitaya). Il risultato è mostrato nel grafico seguente. In esso sono sovrapposti, sullo stesso grafico, gli “spettri” di segnale corrispondenti a diverse (crescenti) concentrazioni di fluoresceina.For each measurement, we performed a Gaussian fit, thus determining the signal intensity corresponding to the peak channel. The signal intensity was correlated with the fluorescein concentration, predetermined during solution preparation.

The result is a linear relationship, as shown in the next graph.

Conclusions

The fluorimeter we described in this article has proven to be an efficient instrument, especially suitable for measuring small fluorophore concentrations. The most critical aspect is the LED pulser, because the linearity of the measurement relies on the consistency and repetitiveness of the light pulses produced by the LED. A certain degree of spreading of the pulse intensities is inevitable. This can be partially remedied by taking a large number of measurements and applying a Gaussian fitting to the data to identify the statistical mean value.

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