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.

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