To understand the impact of the latest developments in fibre delivery and turnkey multi-laser combiners, it is useful to briefly recap the history of lasers in instrumentation used in life sciences. Lasers are widely used in these applications, primarily to excite emissions from fluorescent antibodies, labels, recombinant fluorescent proteins, concentration indicators and other fluorophores.
For the first 25 years or so, most instruments relied on free-space (open beam) delivery of the laser beam to the interaction zone. But, starting around a decade ago, the maturation of fibre coupling and fibre delivery driven by the telecom boom enabled fibre delivery to begin to make inroads into the life-sciences market.
Today, fibre delivery now plays a major role in bio-instrumentation, with a key market trend being the integration of longer laser wavelengths in the same instrument to allow simultaneous detection of several fluorescent probes. In flow cytometry, for example, this allows multiple blood cell types to be counted in a single instrument run.
According to Matthias Schultze, director of marketing of OEM at Coherent, there are numerous advantages to fibre delivery that have driven this trend. He tells Electro Optics: ‘First it remotes the laser from the potentially congested interaction zone in the instrument. This reduces space constraints and simplifies thermal management in the most critical part of the instrument. It also simplifies the beam delivery by eliminating numerous turning mirrors, prisms and lenses that would otherwise be required and also eliminates the potential for any of these macroscopic optics to lose alignment due to ambient thermal or mechanical drifts. Where single-mode polarisation fibre is used, the fibre also acts as a spatial filter delivering a highly constant circular output with linear polarisation and other predictable and stable optical characteristics.’
E pluribus unum
For instrument builders, the biggest remaining opto-mechanical design and assembly challenge was how to integrate the output of multiple separate lasers in a single fibre. This has required the use of waveplates, dichroic filters for each successive laser and then re-coupling the combined output back into a single fibre. These custom beam combining assemblies were difficult to optimise and required considerable optical expertise – even adding a single new wavelength to an instrument meant a system re-design. And hot swapping, replacing a failed laser in the field or upgrading to a higher power, was not something the end user could realistically contemplate.
In response to this situation, engineers at Coherent set out to design a flexible plug-and-play combiner that preserves single-mode output and linear beam polarisation, requiring no mechanical adjustments whatsoever, and is completely passive, meaning it does not use any electronics.
In Coherent’s OBIS Galaxy, launched at Laser World of Photonics in Munich last month, the wavelength-dedicated laser input sockets use the latest FC/UFC connectors that feature maximum optical registration precision. Each input is then collimated as a macroscopic beam using a rigidly mounted lens. The beams are then combined in a novel, patent-pending manner using only one optic per wavelength.
Schultze continues: ‘The combined beams are then re-coupled into a single fibre terminating in a FC/APC (8o) connector. Now adding a laser, removing a laser, replacing a laser or upgrading a laser to a higher power is a simple plug-and-play task. To ensure internal alignment is permanently maintained, each OBIS Galaxy is also subjected to an extensive protocol of thermal cycling and mechanical shock testing.
‘Since this is a universal problem, we’ve focused on making the OBIS Galaxy a universal solution. Specifically, we are offering fibre adapter cables so that instrument builders and end users can use lasers from other manufacturers, not just Coherent lasers.’
Riding wavelength flexibility
Modulight, based in Tampere, Finland and San Jose, California, has recently released a compact OEM laser diode platform specifically targeting biophotonics and life science applications.
Once again, simplicity and flexibility are key; the Series 6500 laser systems house a driver and cooling solution inside the module and offer a USB based control interface and an SMA-905 fibre output connector for easy integration inside biophotonics equipment or equally to set ups using an external illumination source.
‘We have more than 10 standard wavelengths supported by this platform over the range from 450 to 1550nm and can also build in any custom wavelengths we manufacture in our own laser diode fab. This product has got a lot of interest specifically in fluorescence and microscopy applications,’ explains Petteri Uusimaa, president and CEO of Modulight, ‘We are just about to release new wavelengths on the shorter side of the spectrum by offering 405nm and 488nm that are also widely used in fluorescence and spectroscopy.’
One interesting domain Modulight is looking into is the use of fluorescence as an on-line efficacy tool in photo-medical treatments, like photodynamic therapy (PDT).
Uusimaa continues: ‘Modulight [is] looking into integrating a separate fluorescence unit to our systems to monitor progress of the treatment based on the fluorescence signature of the photochemical process in PDT.’
Filtering for fluorphores
Laser Components, based in Chelmsford, UK, and its partner Omega Optical work largely with researchers at universities to generate new and bespoke filters for use in biophotonics and that are compatible with specific fluorophores according to their absorption and emission capabilities.
Laser Components’ partners recently released a paper – High-speed Multispectral Confocal Imaging – at a San Francisco conference, essentially referring to the use of fluorescent filters to produce images of tumours and cancers.
Kimberley Loft, technical sales engineer, explains: ‘Whether a fluorophore is small or large it will emit photons in a certain manner – like a footprint, if you will – and from this we can produce a spectral curve, which will describe the probability of absorption and emission of photons across the entire wavelength. So there will be two overlapping spectral curves; the overlap, or shift, is called the Stoke’s shift.
‘This provides a spectral window, and what researchers try to do is separate the incoming excitation light from the emitted fluorescence. The overlap can be very small – sometimes less than 15nm, so our filters have to be very high-spec with a high transmission in the pass band but effective blocking in the outer band.’
Laser Components supplies single filters and filter-sets, as well as filter cubes that combine two standard filters with a dichroic filter in between, all mounted into a fixed ‘set’.
Loft adds: ‘Provided the customer can tell us what microscope they are using and the fluorophores they are looking at, we can manufacture a filter cube that can slot or screw straight into their microscope.’ She added that the very high transmission rates produced by the filters – up to 90 per cent – are essential for effective use in biophotonics.
A slice of the action
Japanese photonics firm Hamamatsu has recently been involved in a light sheet microscopy project, to produce images of whole-brain activity within the zebra fish, as reported in Nature Methods in May. This used the company’s ORCA Flash 4.0, a high speed, high sensitivity Gen II sCMOS camera.
With this method a thin slice of brain is illuminated by a laser light sheet, while a suitable camera is synchronised to the laser pulses.
Since the launch of the ORCA Flash 4.0 in December 2011, Hamamatsu has been working with research groups to understand what they need to further improve with the new imaging technology. Its high speed and high sensitivity is one of the features required for imaging breakthroughs – neuron activity happens at the millisecond level and needs to be viewed at cellular resolution.
Clearly the use of lasers in biophotonics, backed by improvements in filter- and camera-design, is moving forward and shows significant promise.
