Optics: small, light and fantastic

It’s the International Year of Light and, to mark the occasion, Australian National University (ANU) optics engineer Dr Steve Lee and several of his students visited Sydney’s King’s School in July.

Taking with them a few bottles of clear liquid silicone and a dozen 3D-printed DIY lens fabrication kits, they proceeded to wow students, staff and parents by showing them how they could make small, high-precision magnifying lenses directly from highly transparent polymer droplets.

The do-it-yourself lens kits, a lens mount and adaptor for a variety of smartphone cameras could be on the market in the not-too-distant future for any smartphone owner interested in transforming their new iPhone or Samsung Galaxy into a powerful, portable microscope capable of producing spectacular still images and video of microscopic subjects.

Lee, group leader and lecturer in biomedical optics at ANU's Research School of Engineering, and Dr Tri Phan, of Sydney's Garvan Institute of Medical Research, shared 2014's Australian Museum Eureka Prize for Innovative Use of Technology for their simple, cheap technique for making the tiny, high-powered lenses.

Dr Lee will describe their invention and other promising applications for the tiny droplet lenses in an invited address to the 24th Australian Conference on Microscopy and Microanalysis at the Melbourne Convention and Exhibition Centre, which will run from 31 January to 4 February 2016.

His team's idea for an inexpensive, DIY smartphone microscope is being refined as it moves towards commercialisation. For once, the marketer's melancholy question — who will buy it? — answers itself.

Who wouldn't want to own one?

What child, parent or photographer — professional or amateur — would pass up the opportunity to own an inexpensive device capable of creating unprecedented, high-definition images and video of the micro-universe beyond the reach of the naked eye?

In the hands — or pockets — of millions of smartphone owners, Lee's revolutionary device would open a magical new window into a micro-universe previously inaccessible to the naked eye.

As happened with the advent of mass-produced, camera-equipped smartphones a decade ago, a torrent of new images and video would gush into popular internet-based sharing services like Flickr, Instagram, YouTube and Vimeo, transforming our view of the world around us.

Lee's original lens-making process employs two natural forces — gravity and surface tension — to form near-perfect perfectly spherical lenses of great optical clarity, but still have distortions at the edge of the lens due to non-asphericity.

Lee and his colleagues have since developed a new generation of tiny, aspheric polymer lenses, using a third natural force: capillary action. Their technique is currently under patent application.

They are experimenting with combs of multiple, inexpensive lenses to develop a working prototype optical endoscope (4 to 5 mm in diameter) that would use multiple, inexpensive lenses to obtain stills or video of living tissues biopsies at a resolution of only 2 µm.

In a related project, Lee is working to construct a high-speed laser scanning system that will be compatible with the micro-endoscope.

The real power of the ANU prototype constructed by Lee lies in its use of ultrafast pulses of infrared laser light that routinely captures video sequences of 480 frames per second at 512 x 64 pixel size, with a pixel resolution of 1 µm.

The high-speed imaging system yields blur-free, motion-freezing video of cells and mobile cellular traffic — for example, T-cells swarming to the site of a bacterial infection or natural killer (NK) cells attacking a tumour.

Lee's system will even make it possible to visualise a neuron's response to an action potential propagating along a neuron. An action potential triggers an influx of calcium ions that releases neurotransmitter molecules into the narrow synaptic gap between a projecting dendrite and the body of the next neuron in the communication network.

Lee says the pulse micro-endoscope laser imaging technique employs a technique called multiphoton imaging, which provides some significant advantages over laser confocal imaging microscopy — particularly for imaging deep inside delicate brain tissues, which can suffer damage from pulses of laser light if the exposure is too long.

In contrast, two-photon imaging greatly reduces the risk of tissue damage while enhancing image quality. Lee says it uses an infrared laser which is tuneable to the invisible, longer IR wavelengths between about 700 and 1300 nm.

He says tissues are more transparent to long-wavelength infrared light, allowing deeper imaging of the tissue sample.

The two-photon imaging technique bombards the fluorophores in the tissue sample with photons of relatively low, non-damaging energy. Under the laws of quantum physics, there is a probability that a single fluorophore will absorb two low-energy photons simultaneously to excite it to an energy level at which it will emit a single photon at a higher energy.

Lee says the use of long-wavelength, infrared light greatly reduces backscattering of inbound photons by cellular turbidity while maximising signal strength.

“Faster laser pulses are not always an advantage, because it means fewer photons per emission point," Lee said. “The two-photon technique is very inefficient compared to single-photon laser microscopy, so you need to maximise detection.

“Optimising signal detection [exposure] with pulse duration provides the best the possible image at the highest speed."

Lee and his colleagues are also looking at ways to enhance image quality by borrowing a technique from astronomy, called adaptive optics.

Adaptive optics has revolutionised ground-based astronomy. It employs a reference laser beam directed up through the atmosphere to detect temperature-induced deviations in starlight as it travels down through the atmosphere. Based on the deviation data, actuators beneath the reflector mirrors to bend the mirror in near real time to compensate for transient temperature changes in the atmosphere.

“A lot of the scattering of light passing through living tissues is similarly deterministic, so adaptive optics is increasingly being used in microscopy," Lee said.

“Our system removes distortions introduced both by the optics and the tissues. We precompensate the light path, before it enters the tissue sample, by computing the trajectory of the beam through the various layers of tissue, which have different scattering coefficients.

“Knowing how each layer scatters the beam, we can calculate the aggregate scattering coefficient of the tissue sample, which allows us to look much deeper," he said.

Lee's team has also begun to experiment with a technique called optical clearing that would complement adaptive optics in reducing backscattering.

It involves using chemicals that 'retune' the backscattering coefficients of the individual layers by reducing or removing materials, to make them more uniform. Combined with adaptive optics, it effectively makes the tissue more transparent across the full scanned depth of the sample.

Rather than treat each layer with a different chemical, Lee's team is looking for a single treatment that can provide an optimal, aggregate result, and has identified half a dozen chemicals capable of fulfilling the role.

Lee says his team aims to develop a range of strategies for imaging cellular activities deep into tissue.

Pictured: Dr Woei Ming (Steve) Lee. Born and educated in in Singapore, Lee later travelled to the UK and US, where he studied electronics, physics, biomedical sciences, nanotechnology and materials. He then moved to Canberra to head his own research laboratory at the Australian National University. He is an avid runner and martial arts devotee.