Although a few questions can be answered with lower resolution by e.g. magnetic resonance imaging, here we would like to focus on the techniques that can be addressed with a microscopic setup in a broader sense. Hence, mainly three technologies are of relevance:
- Multi-photon imaging
- Fast volume imaging, such as light sheet or light field microcopy
- Electrophysiology
In the past, electrophysiology, which is based on measuring electrical potential at several contact points, was by far the most prominent method. Recently, propelled by the development of optogenetic tools and voltage-sensing fluorescent proteins, microscopy methods have started to replace the traditional electrophysiology approaches, building on a reduced invasiveness and increased versatility and specificity.
When considering microscopy modalities to address functional neuroscience questions, only light microscopy is suitable, since the specimen needs to be alive and intact. To visualize neuronal activity researchers turn to fluorescent reporter molecules, which act as local sensors which ideally allow quantifiable measurements of:
- Ion fluxes induced by neuronal activity for decoding inter- or intracellular messaging between neurons (however, usually slow and indirect)
- Changes of voltage/electric fields induced by neuronal activity for decoding inter- or intracellular messaging between neurons
- blood flow or energy consumption to determine metabolic activity of individual cells or entire regions of a nervous system (however, usually slow and indirect with limited spatial information)
Each of the methods listed above has significant constraints and drawbacks and additional limitations originate from the challenging nature and requirements of the living brain as a sample:
- Living brain tissue is very opaque. Thus, penetration depth and light scattering are often limiting factors.
- In addition, neuronal signals are very rapid and often spread over large volumes.
- Samples need to be kept alive and undamaged.
- Simultaneous observation of the activity of multiple neurons in the network is necessary to unravel the interdependence of neuronal activity within the network.
Hence, our understanding of brain functions would greatly benefit from a new method which overcomes these limitations.
The goal is to find a new imaging technology which could enable better access to functional information of neuronal activity in biological samples.
The performance of such a new method could be measured based on 5 criteria:
- A strong enough signal of the neural activity is obtained (ideally enough to detect neuronal activity in a single shot without the need of repeated stimulation)
- Spatial resolution is sufficiently high that individual neurons can be distinguished
- Temporal resolution is fast enough to capture the rapid dynamics of functional activity
- Acquired volume is large enough to simultaneously detected spread of information in the neuronal network
- Imaging needs to be possible in the context of a living animal or must support isolated living samples in the range of cm3.