In many laser-based types of bioinstrumentation, including flow cytometers, confocal microscopes, and array readers for proteomics, laser output is delivered to the system’s final optics regime via fiber coupling. Over the past few years, this fiber delivery has evolved from simple remote delivery to finally encompass plug-and-play use of multiple lasers with sub-micron beam positioning accuracy. This capability is supporting a new generation of instruments that combine state-of-the-art performance with ease of use.

Flow Cytometry

Figure 1. In flow cytometry, cells are counted according to their fluorescence signature. They can also be sorted in some instruments by pulsing a deflecting electric field in response to this fluorescence signature.
Flow cytometry is one of the most important laser-enabled techniques in life sciences. It is widely used in research and for common clinical tests, such as blood counts. In flow cytometry, a suspension of cells in buffer (e.g., from a patient’s blood) is treated with several fluorescent markers. These are fluorescently labeled antibodies or other fluorophores that selectively bind to target cell types. In the instrument, the cells flow rapidly through a narrow stream or flow cell configured to make them pass in single file through one or more focused laser beams (Figure 1). The resultant fluorescence is detected by one or more photodetectors, each equipped with a bandpass filter so that it only records light in a specific wavelength band.

In many instruments, another detector is configured to detect scatter, as this can give important parallel information on the size and shape of the cell crossing the focused laser beam. Additionally, by fluorescently labeling the cells’ DNA, they can also be sorted or counted according to their ploidy (essentially how much DNA they contain).

The first instruments used a single laser wavelength, typically an argon ion laser with output at 488nm. But, it was soon recognized that by using multiple lasers and detectors, and by employing an increasingly diverse choice of dyes with a sophisticated range of antigen affinities, multiple different target cell types could simultaneously be counted (and/or sorted). Furthermore, by taking ratios of the signals from multiple detectors, the total number of profiled markers can readily exceed the number of detectors, which usually exceeds the number of lasers. For example, a “12 Color” system uses 12 photodetection bands, and perhaps five lasers, to detect 20 or more markers in a single sample run.

Early Fiber Days

In the first flow cytometers to incorporate multiple lasers, each of the beams had to be appropriately conditioned and positioned using a set of beam delivery optics and a focusing telescope for each separate laser. This enabled the focused beam waists to be arranged as a closely spaced line of elliptical spots through which the cells flowed.

This brute force approach had several limitations which became worse as the number of lasers increased, eventually becoming untenable for today’s state-ofthe- art flow cytometers that can have several different lasers. Cost was one issue, since there is constant market pressure to lower the price of clinical lab tests, yet each telescope alone represents several hundred dollars. Size became a factor because it is difficult to package the numerous beam delivery optics close enough together. And, increasing system complexity reduced field reliability and created issues around restoring/maintaining alignment when even just one laser was added or exchanged. In addition, particularly with older lasers, the thermal budget couldn’t be increased unchecked within a sensitive instrument like a cytometer.

In response, instrument builders began to adopt fiber delivery in the 1990s, often purchasing the laser from one source and the fiber-coupling setup from another. Here, the key challenge was to launch (focus and align) the laser output into a polarization-preserving, single-mode fiber, with a core diameter of only 3.5μm. This was done by positioning the focusing lens relative to the fiber input facet using adjustable mounts with up to six degrees of adjustment. Achieving perfect launching alignment requires considerable expertise, but became somewhat routine for several high-end instrument suppliers. And, since the single-mode fiber acts like a spatial filter, this approach delivered the key goal of making each focused laser beam have an identical focused size and shape, no matter what the characteristics of the original laser beam (round, elliptical, astigmatic, etc.) were.

A New Generation of Fiber-Coupling

Figure 2. Coherent has developed beam shaping assemblies that couple directly to a laser’s fiber output.
After many years of virtual technology stasis in bioinstrumentation beam delivery, the situation is now changing quite dramatically. The market pressure for this is the need to build cytometers (and other fluorescence-based instruments) capable of simultaneously detecting and analyzing an even greater number of fluorescent markers. In the clinical analysis sector, this will enable diseases such as HIV and metastatic cancer to be followed and profiled in unprecedented detail. But at the same time, system builders want to expand their market by making instruments more affordable and easy to use. These instruments also need to be very simple to field service and upgrade, e.g., by exchanging or adding lasers. With the existing fiber setup, each beam exiting its delivery fiber still has to be combined and/or shaped using dichroic optics and/or prism pairs. Moreover, the six axis mounts are known to be vulnerable to long-term drift due to physical shocks and thermal changes.

At the same time, a new generation of smart lasers has become available, such as the Coherent OBIS series. These are compact lasers in which the controller and laser are contained in a package having a maximum dimension of only 3 inches. Moreover, by using both laser diodes and the wavelength-scalable, optically pumped semiconductor laser (OPSL), OBIS products are available at an ever expanding range of wavelengths and output powers, optimized for exciting the latest fluorophores across the UV and visible. Yet, all these different wavelength lasers are packaged with a common optical, mechanical and electronic interface. For many instrument applications, these modules finally delivered the true plug and play operation that had been desired for many years.

In the past year, Coherent also introduced fiber coupled (OBIS FP) modules. Here, the laser is pre-aligned into the single- mode fiber, whose ferule is then permanently welded onto the laser itself. There are no mount adjustments that can ever creep or shift. This alignment ensures all aspects of the coupling are perfectly optimized, including matching numerical aperture and fiber facet preparation, for example. In developing these products, Coherent leveraged the telecom background of their fiber optic group to address another important issue. Specifically, many fiber coupled laser modules suffer early failure because of the high power density at the input and output facets, which must be just a few microns in diameter for single-mode operation. To avoid this limitation, Coherent uses a proprietary fiber in OBIS FP which has a single mode core, but significantly larger input and output facets, while maintaining single mode performance.

Learning from Telecom

Figure 3. Example of a fully packaged BSO where the output of three separate fibers are precision aligned by the use of V-groove technology.
Engineers at both laser manufacturers and cytometry manufacturers recognized that even optically demanding instruments like flow cytometers, where alignments must be held to a few microns, were now on the verge of plug and play operation too. The only remaining obstacle was alignment and beam-shaping of the fiber outputs into the instrument’s interaction zone. Again fiber delivery provided the solution. Engineers at Coherent developed beam shaping assemblies, coupling directly to a laser’s fiber output and delivering a Gaussian or”top-hat” beam profile. Compact and robust, these assemblies come pre-aligned and can be easily directed into the flow cell (Figure 2).

Then, borrowing from telecom technology to permanently hold multiple fibers using V-groove technology or PermAlign™ assembly techniques, Coherent also developed passive optic assemblies with multiple fiber inputs and with a pattern of output beams that is locked in during manufacture to meet application requirements. These concepts have now been implemented in a series of both standard and custom Beam Shaping Optical assemblies (BSOs). In the case of flow cytometry, a single BSO is designed and assembled to deliver a specific pattern of wavelengths, stripe dimensions and spacing which can be aligned with the flow cell as a single module (Figure 3).

The combined advent of fiber-coupled smart lasers and these new BSOs means that, two decades after instrument companies began to use fiber delivery for laser sources, they can finally exploit all the inherent capabilities of using fiber. Whether they are using one laser or ten, the beam(s) is always perfectly aligned.

Summary

Fiber-coupling has certainly come a long way from the days of trained optical technicians carefully adjusting knobs on a fiber launching mount. Moreover, this new plug and play approach is more robust and compact, and will lower cost of ownership by reducing both downtime and service costs. Better performance and reduced costs — a combination both instrument builders and their customers are ready to embrace.

This article was written by Dan Callen, Product Line Manager, and Michele Winz, Ph.D., Manager: Photonics Engineering, Coherent Inc., (Santa Clara, CA). For more information, contact Mr. Callen at dan. This email address is being protected from spambots. You need JavaScript enabled to view it., Mr. Winz at This email address is being protected from spambots. You need JavaScript enabled to view it., or visit http://info.hotims.com/45603-200.

Photonics Tech Briefs Magazine

This article first appeared in the May, 2013 issue of Photonics Tech Briefs Magazine.

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