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Brain research boosted by microscopy breakthroughs, Photonics West hears

A microscope technique able to image every neuron in a fruit fly brain, and some of the remarkable healing properties of light for treating neurological conditions, were two topics discussed during a neurophotonics plenary session at Photonics West. Greg Blackman reports

Imaging techniques are coming to the fore that are faster and cover larger volumes of neural tissue, and which will ultimately improve our understanding of how the brain works, attendees at a neurotechnologies plenary session at SPIE Photonics West heard.

‘We’ve never had a modality before where we’ve been able to image every single neuron in the [fruit fly] brain in a freely behaving animal,’ commented Dr Elizabeth Hillman from Columbia University, speaking at the event in San Francisco on 28 January.

Hillman, who was honoured by SPIE with the 2018 Biophotonics Technology Innovator Award, was referring to a new microscope technique her team has been developing called swept confocally aligned planar excitation (SCAPE) microscopy.

‘This [SCAPE] is a really exciting new platform for us to use to understand the brain in behaving animals,’ she added. The technique has been licensed to Leica Microsystems for commercial development.

The plenary session covered work from Ed Boyden at MIT on nanoscale resolution imaging with expansion microscopy, along with Na Ji at the University of California, Berkeley, who spoke about using a Bessel beam to scan brain tissue over a large field of view – the group scanned neurons in a mouse brain over a volume of 1.5mm x 3mm x 600µm.

Hillman’s group has experimented with fruit flies using SCAPE microscopy, whereby they puff odours at the fly, or make it hungry or scared, and image every neuron in its brain at 10 volumes per second. This isn’t possible with other microscopy techniques.

The team has also studied free-crawling fruit fly larvae, imaging neurons labelled with fluorescent dyes to understand how neurons fire to enact particular movements. They’ve also worked with mice, and are able to make video-rate volumetric imaging of the neuro-vasculature in the brain.

Hillman described SCAPE as a hybrid between light sheet microscopy and spinning disk confocal microscopy. It builds on light sheet microscopy by, instead of illuminating the sample with a plane of light and imaging through a separate objective, SCAPE illuminates obliquely from the edge of the primary objective lens, and then detects the light back through that same lens.

The system uses a simple galvo mirror to scan through the sample and create a 3D image. Hillman explained that the technique doesn’t need a complicated mechanical setup to keep the alignment accurate. To create an image at 20 volumes per second requires the galvo to move at 20 lines per second.

‘I can use a simple galvo and get high-speed volumetric imaging with no other moving parts,’ she said. ‘We only need to read between 100 and 200 rows of the camera, which means we can collect images at about 2,000 frames per second, which allows us to sample volumes according to how we would want.’

Conventional light sheet microscopy suffers from immersion media issues and numerical aperture issues, and means the technique can only image at one or two volumes per second. Two-photon microscopy is also limited in speed and is ‘not fast enough to capture the kind of activity from neurons’ required, explained Hillman.

‘When you have this new speed you are not worried about movements being motion artefacts; the movements are the behaviour,’ she said.

SCAPE can look at responses to optogenetics within an entire network, as well as image vasculature. It is very high speed, gives low phototoxicity and therefore good signal-to-noise ratio even though it is fast imaging; it uses stationary, simple objective lenses and no image reconstruction is needed.

Hillman’s group is also developing a two-photon version of SCAPE. ‘We’ve discovered that this works beautifully for imaging clear tissue except we can go really fast compared to conventional confocal resonant scanning,’ she said – the group was able to image an entire hemi-section of the brain 400µm thick in just four minutes.

Light treatment

Along with new methods for studying the brain, Dr Michael Hamblin of the Wellman Centre for Photomedicine spoke about the benefits of photobiomodulation, or treating diseases with light.

Near-infrared light is often used as it penetrates deep into tissues. ‘The kinds of diseases we can treat are all over the body, head to toe,’ Hamblin observed. ‘It’s remarkable how well it works and what a wide variety of diseases and conditions you can treat.’

The mechanism by which light is thought to stimulate a response in cells is by being absorbed by chromophores – mitochondria being a major chromophore – which then leads to cell signalling and activation of transcription factors.

Hamblin’s team has observed neurogenesis – new neuron generation – in mice brains suffering from traumatic brain injury after exposure to near infrared light at 810nm, with fluences of 20-40J/cm2.

He said that other studies have shown greater blood flow, more mitochondrial activity, and less inflammation in the brain after illuminating with NIR light. ‘There are a lot of things that go on in the brain when you put NIR light on it, and they’re pretty much all beneficial,’ he said.

Near infrared light has been shown to promote neurogenesis after brain damage from a stroke or a traumatic brain injury. It is able to reduce inflammation and oxidative stress, and increase blood flow to alleviate neurodegenerative diseases like Alzheimer’s and Parkinson’s.

Psychological tests have also shown that exposure of NIR light to the forehead has had beneficial effects on depression and anxiety.

In one trial, 11 people suffering from traumatic brain injuries were exposed to relatively brief and modest doses of NIR light, eight of which significantly improved following treatment.

A 12-week trial for Alzheimer’s disease saw symptoms in patients improve significantly after a course of three NIR treatments per week illuminating the brain’s default mode network, an intrinsic brain network. At their height, these improvements from NIR light were seven times larger than those from a clinical trial for Aricept, which is the only FDA-approved drug for Alzheimer’s.

At the end of the trial the improvement declined, but because the light is delivered via an LED array patients could potentially be given a device to take home for continued treatment. A chronic degenerative disease like Alzheimer’s will probably need repeated treatments with NIR light, Hamblin noted.

Putting light directly into the brain with a fibre has even been proposed for treating Parkinson’s – one of the existing treatments is to use a fibre for deep brain stimulation with an electrical contact.

Brain helmets with LED arrays have been built. These deliver a radiance on the scalp of around 10mW/cm2. At 400cm2 that’s 4W of optical power, which generally needs fans to keep it cool. ‘There is a lot of scope for optical engineers to construct LED devices to put on your head,’ Hamblin commented.

A whole range of neurological disorders can be treated in this way, from neurodegenerative diseases to psychological conditions. ‘It’s possible that the solution to all these intractable brain disorders could be as simple as shining a light on the head,’ Hamblin concluded.

The plenary session gathered together some of the best researchers in neurotechnologies, a lot of which will have funding from the US Brain Research through Advancing Innovative Neurotechnologies (BRAIN) initiative. Edmund Talley from the US National Institutes of Health (NIH) said that funding to date is only about 12 per cent of the $4.2 billion expected to be available until 2026.


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