Speaker
Description
Calcium is an universal second messenger which regulates a plethora of cellular processes including cell survival, gene transcription, neuro-transmission and mitochondrial functions.
Several methods have been developed for measuring calcium intracellular concentrations ([Ca$^{2+}$]) mainly based on fluorescent or bioluminescent probes. The light emission is due to the chemical reaction between probes and calcium ions in biological environment and it is proportional to the calcium concentration. Among bioluminescent probes, aequorin represents a common tool for measuring a wide range of [Ca$^{2+}$], from sub-micromolar to millimolar, offering as well the possibility to target the different sub-cellular compartments involved in the process under study. The aequorin response is directly proportional to the [Ca$^{2+}$] and the cells are preserved by photodamage because external excitation is obviously not needed. The major drawback of this technique is the low level of emitted light, consisting in a sequence of single photons.
Silicon Photo-Multipliers (SiPM) are a valuable device for this application not only for their well known single photon counting capability but also in perspective to analyse simultaneously calcium signals generated in different cellular subcompartments, like cytosol, mithocondria or lysosomes. This talk reports the results of a serie of tests aimed at qualifying a flexible, portable, low cost SiPM-based system for calcium sensing.
In this set-up the biological sample plate (with an area of ∼1 cm$^{2}$) is placed directly in contact with the sensitive area via an index matching grease. The detector in use is a Hamamatsu S13360-6050CS SiPM 6x6 mm$^{2}$ operated at 2.5V above the breakdown voltage, coupled to a custom, versatile and compact front-end electronics with a pole-zero cancellation filter. A full signal time development of 30 ns is reached in order to to be compliant with a single photon counting rate up to 2 MHz with a 5% probability of pile-up events. The system has been operated both in counting and gated current integration mode (separately and simultaneously) to cope with signal frequencies ranging from MHz level up to ∼100MHz.
In our experiments, the external stimulus that triggers intracellular [Ca$^{2+}$] release in the cells consists in the administration of adenosine-triphosphate (ATP) and the information of interest consists in both the intensity and the time development of the induced chemiluminescence signal, where anomalies are known to be correlated to the onset of age-age-rated and neurodegenerative diseases.
The SiPM system has been qualified in terms of sensitivity and linearity of the response varying the number of cells on the plate, over the range of biological interest. A good linearity with both photon counting and gated current methods is obtained, allowing to investigate different [Ca$^{2+}$] concentrations over a range of 5 order of magnitude. Moreover it was shown to be sensitive down to few cells on the field of view. Finally, a preliminary test was performed looking at the concentration of [Ca$^{2+}$] in mithocondria proving sensitivity also to induced signals in cellular subcompartments.
This prof-of-concept clearly showed the potential of the technique and identifies the specifications for the design of a dedicated system, targeting as well the integration of filters on a segmented SiPM area to measure simultaneously the concentration of fluorophores emitting at different wavelengths. This will open the possibility to engineer assays targeted to the simultaneous measurement of the activity in different subcellular compartments, providing a novel technique for studying genetic and age-related metabolic and neurodegenerative diseases at an early stage.
