Optogenetic Stimulation Array for Confocal Microscopy Fast Transient Monitoring

Autores de CIPF

• Beatriz Martinez Rojas

  Autor

• Maria Del Mar Sánchez Martín

  Autor

• 

  Esther Giraldo Reboloso

  Autor

• 

  M Victoria Moreno Manzano

  Autor

Participantes ajenos a CIPF

• Monreal-Trigo, J
• Terres-Haro, JM
• Fillol, MA

Grupos de Investigación

• Laboratorio de Regeneración Tisular y Neuronal

  

  Jefe/a de Grupo

  M Victoria Moreno Manzano

Abstract

Optogenetics is an emerging discipline with multiple applications in neuroscience, allowing to study neuronal pathways or serving for therapeutic applications such as in the treatment of anxiety disorder, autism spectrum disorders (ASDs), or Parkinson's disease. More recently optogenetics is opening its way also to stem cell-based therapeutic applications for neuronal regeneration after stroke or spinal cord injury. The results of optogenetic stimulation are usually evaluated by immunofluorescence or flow cytometry, and the observation of transient responses after stimulation, as in cardiac electrophysiology studies, by optical microscopy. However, certain phenomena, such as the ultra-fast calcium waves acquisition upon simultaneous optogenetics, are beyond the scope of current instrumentation, since they require higher image resolution in real-time, employing for instance time-lapse confocal microscopy. Therefore, in this work, an optogenetic stimulation matrix controllable from a graphical user interface has been developed for its use with a standard 24-well plate for an inverted confocal microscope use and validated by using a photoactivable adenyl cyclase (bPAC) overexpressed in rat fetal cortical neurons and the consequent calcium waves propagation upon 100 ms pulsed blue light stimulation.

Keywords

• Biological techniques; confocal microscopy; graphic user interface (GUI); in vitro calcium stimulation and acquisition; instrumentation; irradiance control; optical current transducer; optogenetics; real-time monitoring; stimulation array