Detecting and visualizing neuronal signals for life science

How the human brain works is one of the biggest unsolved question of our time. Deciphering how higher brain functions emerge from the activity of single neurons will give important insights into unravelling this mystery. To date, light as well as electron microscopy can be used to acquire structural information of neurons and their connections. However, functional information, the propagation of signals from neuron to neuron, and the characteristics of the neuronal networks are still challenging to obtain. Easier access to this kind of information will further our understanding of how complex structures such as the brain integrate and function, not only in health but also in disease. As a result neuroscience research could trigger significant breakthroughs in medical technology.

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.

	Multiphoton Microscopy	Fast volume imaging	Electrophysiology
Advantages	Highest penetration depth of available light microcopy techniques Relatively low photodamage Specificity is high in combination with optogenetics and fluorescent reporters	Neuronal activity in a large volume can be monitored Specificity is high in combination with optogenetics and fluorescent reporters High temporal resolution	Direct recording of electrical activity of neurons Real time recording of neuronal activity No reporters need to be introduced into the sample
Disadvantages	Scanning technique with limited temporal resolution Penetration depth still limited to more superficial brain areas Expensive equipment necessary Reporters need to be introduced into the sample prior to the experiment (see challenge on “New contrasts and label free imaging for life science” for limitations of Fluorescence Imaging) Disadvantages depend on the quantity which the reporters measure: Calcium ion flux is mostly a downstream process with slower dynamics Blood flow or energy consumption is only indirect and offers limited spatial information	Penetration depth is limited. Frequently only cellular resolution possible Reporters need to be introduced into the sample prior to the experiment (see challenge on “New contrasts and label free imaging for life science” for limitations of Fluorescence Imaging) Disadvantages depend on the quantity which the reporters measure: Calcium ion flux is mostly a downstream process with slower dynamics Blood flow or energy consumption is only indirect, relatively slow and offers limited spatial information	Intrusive technique, since electrodes need to be placed into the region of interest and thus the surrounding tissue is damaged. Electrophysiology is not an imaging technique as such, it provides only traces of electrical activity. Morphological information cannot be obtained. Spatial information is often limited. Targeting distinct neurons challenging
	Imaging modality & based on the use of reporters

Multiphoton Microscopy

Fast volume imaging

Electrophysiology

Advantages

Highest penetration depth of available light microcopy techniques
Relatively low photodamage
Specificity is high in combination with optogenetics and fluorescent reporters

Neuronal activity in a large volume can be monitored
Specificity is high in combination with optogenetics and fluorescent reporters
High temporal resolution

Direct recording of electrical activity of neurons
Real time recording of neuronal activity
No reporters need to be introduced into the sample

Disadvantages

Scanning technique with limited temporal resolution
Penetration depth still limited to more superficial brain areas
Expensive equipment necessary
Reporters need to be introduced into the sample prior to the experiment (see challenge on “New contrasts and label free imaging for life science” for limitations of Fluorescence Imaging)
Disadvantages depend on the quantity which the reporters measure:
- Calcium ion flux is mostly a downstream process with slower dynamics
- Blood flow or energy consumption is only indirect and offers limited spatial information

Penetration depth is limited.
Frequently only cellular resolution possible
Reporters need to be introduced into the sample prior to the experiment (see challenge on “New contrasts and label free imaging for life science” for limitations of Fluorescence Imaging)
Disadvantages depend on the quantity which the reporters measure:
- Calcium ion flux is mostly a downstream process with slower dynamics
- Blood flow or energy consumption is only indirect, relatively slow and offers limited spatial information

Intrusive technique, since electrodes need to be placed into the region of interest and thus the surrounding tissue is damaged.
Electrophysiology is not an imaging technique as such, it provides only traces of electrical activity. Morphological information cannot be obtained.
Spatial information is often limited.
Targeting distinct neurons challenging

Imaging modality & based on the use of reporters

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Detecting and visualizing neuronal signals for life science

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