New to two-photon imaging combined with optogenetics? This is what I learnt from my “failed” PhD project.

The intricate workings of the human brain bear striking resemblance to the harmonious coordination of an orchestra in full performance. Just as a conductor directs a symphony by stressing the tempo and the dynamics of soundwaves, the brain orchestrates spatiotemporal precise patterns of neural activity that shape what we see, feel, and identify with. This precise rhythmic composition is shaped by the interplay of different group of neurons with distinct morphological and physiological characteristics across multiple brain regions, just like different instruments within the strings and woodwind families play together to produce a beautiful well-balanced melody. Understanding how different neurons play together is the fundamental aspect of neuroscience research. In the last twenty years in vivo calcium imaging and optogenetic have revolutionised the way one studies neural circuits. While the first allows an indirect measurement of neural activity at different spatial scales, ranging from single spines to large areas of the cortex in rodents, the latter provides means to precisely stimulate or inhibit activity of single or large ensembles of neurons. This harmonious combination of targeted perturbations and dynamic readout of brain activity gives one the power to conduct precise changes in the melodies being played.

This romanticised view of all-optical approaches draws people’s attention and may even be considered “sexy” within the scientific community, and rightly so I would argue, as these tools are a product of great ingenuity and provide extremely valuable cutting-edge technology for unveiling the mystery behind how the brain works. However, several limiting aspects of all-optical approaches are not talked about enough and should be central in the experimental design of a research project. Here, I highlight some of the well-known technical limitations associated with the use of all-optical approaches, putting emphasis on issues I came across while navigating through failed experiments using optogenetics during my PhD. I also comment on how such limitations should be considered when interpreting the data collected and suggest (try to suggest) ways to tackle them.