Two-photon mini microscope
New two-photon miniaturised microscope enables visualization of brain neurons in freely-moving and behaving animals
At this year’s meeting of Optics in the Life Sciences there was one abstract  that simply stated ‘We have developed a fast, high-resolution, miniaturised two-photon microscope (FHIRM-TPM) with a headpiece weighing 2.15 g. It is capable of hour-long recording of neuronal activities down to the level of spines in mice experiencing vigorous movements.’ The design and application of this miniaturised microscope has since been published in the journal of Nature Methods  and is discussed in more detail in this article.
The human brain weighs 1.4 kg and contains tens of billions of neurons and tens of trillions of neuronal synapses. Yet the inner workings of the brain remain a mystery as current brain imaging techniques cannot record neuronal activity whilst a body is freely-moving and behaving. The research team, led by Professor Heping Cheng, applied for funding from the National Natural Science Foundation to develop the FHIRM-TPM to record the neuronal activity at the level of single dendritic spines whilst animals were freely-moving and behaving, which was initially met with scepticism from some based on its practicality and feasibility. No doubt this was based on the team’s lack of experience in the neuroscience area and past research where recordings of neuronal activity were achieved in awake, constrained and head-fixed animals. However, the physiological relevance of such findings can be questioned as the behaviour of animals is not self-determined (i.e. caregiving, mating and fighting). With funding in place the team set about addressing this technical challenge.
Dr Weijian Zong explains the improvements in the miniaturised microscope
Many improvements to existing approaches were required and the team identified that in order to be successful the design features should: 1) efficiently excite GFPs and GCaMPs and be capable of detect emitted fluorescence signals; 2) have an imaging resolution high enough to resolve GCaMP emission in dendritic spines of in freely behaving animals; 3) be robust and capable of imaging the same animal for many hours; and, 4) be easy to install and disassemble for routine application.
In discussing the technological improvements required, Dr Weijian Zong identified the most important improvement as the design of a hollow-core photonic-crystal fiber (HC PCF) which enables the transmission of a 920 nm laser as opposed to previously used 800 nm lasers. The use of this laser allowed the imaging of calcium indicators (i.e. GCaMPs) which is not achievable with 800 nm laser.
Dr Weijian Zong stated that, “Secondly, we used a miniature objective with the highest numerical aperture that is commercially available so far. The NA of this objective is 0.8. It can achieve sub-cellular-level sub-micron-resolution imaging.” . Features that give FHIRM-TPM a high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels, and a field of view of 130 × 130 μm2).
There was also an update to the microelectromechanical systems (MEMS) scanner which gives a first resonant scanning frequency of 6 KH which helped the miniature microscope to achieve 40 Hz high-speed two-photon imaging.
The team also designed and manufactured a new supple fibre bundle (SFB). Its benefit being that it has a very high collection efficiency of fluorescence and its softness allowed the animal to move-freely and not impede its natural behaviour. The SFB also dramatically increased imaging stability.
Exploring and optimizing the new technology
Mr Runlong Wu was involved in the exploration and optimization of these new technologies and devices and points out that “…simply assembling them together cannot make a high-performance, robust miniature two-photon microscope successful…” . Indeed, the process involved the assembly and debugging of the microscope with the team continually trying and testing the set-up.
One-stop imaging platform for researchers
The purpose of this research was not simply to construct a miniaturised microscope; it was to use it to observe natural animal behaviour. This means the miniaturised microscope is integrated into an imaging platform that is equipped with multi-colour light-source, a micro-wide field camera and a fluorescence collector. This collector can be directly connected to the miniaturised microscope and placed with the animal in a free-moving area. This means that fluorescence can be collected at the same time as the animal is freely-moving around and that movement of the animal is no longer a limitation.
Testing the new technology
Dr Weijian Zong explained that even when the technology had been optimized, it still needed testing to show its capability under normal physiological conditions. Testing involved a number of behavioural paradigms such as the tail suspension test, social interaction experiments and stepping down from a stage. Dr Weijian Zong stated that, “In these paradigms, we can get very high-resolution and stable neural activity. This proved that our miniaturised two-photon microscope had achieved a very high-standard imaging performance.”
Next steps for FHIRM-TPM
The current research findings document the results from real-time and prolonged recordings of neuronal activity from single-spine neurons in animals that were allowed to freely-move and behave. Professor Heping Cheng’ team are already working hard on the next breakthrough by improving the optics of the current technology as well as increasing the depth and field of view, resolution and speed of their miniaturised microscope.
 Liangyi Chen. Fast high-resolution miniature two-photon microscopy for brain imaging in freely-behaving mice at the single-spine. Presented at the Optics in the Life Sciences Congress, April 2-5, 2017. Source: https://doi.org/10.1364/BODA.2017.BoTu2A.1
 Weijian Zong et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nature Methods 2017. Source: http://dx.doi.org/10.1038/nmeth.4305
 Weijian Zong. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Source: https://www.youtube.com/watch?v=AED0SJVAlp0