Next Generation Light Sources for Imaging Fibre Lasers - Compact, Cost-Effective, Turnkey Solutions
- Fig. 1: The all-fibre MOPA (Master Oscillator Power Amplifier) – flexibility to meet the demands of a huge application space.
- Fig. 2: The visible spectrum of the SC400 spatially dispersed through a transmission diffraction grating demonstrates the continuous nature of the spectrum from violet to near Infra-Red.
- Fig. 3: Example high-spec. flow cytometry development platform showing up to 8 discrete laser lines and associated beam combination optics – can be replaced by a single supercontinuum laser. Image courtesy of Dr. William Telford, National Institute of Health / National Cancer Institute, USA.
- Fig. 4: Image courtesy of the Laser Analytics Group, University of Cambridge, UK2-D Fluorescent confocal imaging of the rhizome of Convallaria Majalis (Lily-of-the-Valley) stained with Safranin and Fast Green dyes (peak excitation wavelengths of 530 nm and 620 nm respectively).
- John Clowes
Next Generation Light Sources for Imaging Fibre Lasers - Compact, Cost-Effective, Turnkey Solutions. Lasers continue to be increasingly important components within biological imaging and analysis systems - from Argon-Ion and HeNe lasers used within flow cytometry and confocal microscopy, to femtosecond Ti: Sapphire and DPSS femtosecond sources in multi-photon microscopy. However, many commercial imaging systems are still limited in performance by the availability of suitable laser sources - both in the limited availability of wavelengths and in the size, cost and reliability of conventional laser technologies. Ultrafast fibre lasers offer turnkey, compact and reliable solutions for next generation biomedical imaging systems. Here we introduce the basic concept of the ultrafast fibre laser and describe some of the applications and benefits of this technology within biophotonics research and imaging systems.
Ultrafast fibre lasers, with their compact form, inherent reliability and low cost of ownership are becoming the laser of choice for many biomedical imaging applications, challenging the Ti:Sapphire laser within multi-photon excitation microscopy and diode, HeNe and Ar-Ion lasers within fluorescence imaging. The Master Oscillator, Power Amplifier (MOPA) architecture of Fianium's ultrafast fibre lasers provides simplicity and flexibility by design [fig. 1]. The MOPA comprises two independent modules - a low-power femtosecond or picosecond fibre laser followed by a high power diode-pumped fibre amplifier. By independently changing the parameters of the oscillator or the amplifier modules, one can achieve very different performance parameters from the laser, tailored to a given application. This approach has quickly enabled ultrafast fibre lasers to meet the growing demands of a wide range of applications.
The master oscillator is an all-fibre, passively modelocked laser which is both turnkey operated and self-starting without the need for any adjustment. The pulse repetition rates of the laser (from less than 1MHz to several hundred MHz) are ideally suited to quasi-cw laser applications and also for lifetime imaging applications. For applications within microscopy, ultrafast lasers are typically associated with multi-photon fluorescence microscopy, where femtosecond Ti:Sapphire lasers have been the historic laser of choice.
While not offering the broad wavelength tunability of the Ti:Sapphire, Fianium's FP1060-s femtosecond lasers do offer a low-cost, compact solution at discrete wavelengths from 980 nm to 1100 nm and offer potential for incorporation into any existing microscope system. Delivering average powers up to 5 Watts and with pulse durations shorter than 250 femtoseconds, the long wavelengths of the FP1060, extending beyond the tuning limits of most Ti:Sapphire sources, offer many benefits for Two Photon Fluorescence (TPF) and Second Harmonic Generation (SHG) microscopy [1, 2]. The FP1060 high power lasers from Fianium operate within the picosecond or femtosecond regime and can provide pulse energies from a few pico-joules to ten microJoules, particularly important for materials processing, both of devices and of tissues.
Extension of the FP1060 laser source from the near Infra Red to the visible and UV region of the spectrum, is achieved through nonlinear frequency conversion techniques including harmonic generation to 532 nm, 355 nm and 266 nm and in the generation of ultra broad band "supercontinuum" spectra - a phenomenon that will have a huge impact on next generation biomedical imaging systems. The supercontinuum fibre laser is based on a high power picosecond source (FP1060) and highly nonlinear photonic crystal fibre. The nonlinear interaction between the high intensity pulsed optical field within the tight confinement of a silica optical fibre waveguide, results in the generation of a continuous spectrum spanning from the visible (below 400 nm) extending in the IR to beyond 2 um. Fianium's SC400 and SC450 supercontinuum fibre lasers, utilising high pulse repetition rates in the MHz range, deliver high spectral brightness in the range of several milli-Watts per nm across the entire optical spectrum. Filtration of several nm of this spectrum can deliver tens of mW optical power at any wavelength required - a vast improvement over the discrete wavelengths offered by conventional diode and HeNe laser sources.
Furthermore, the repetition rates of the supercontinuum make them ideally suited to fluorescence imaging applications requiring either a quasi-continuous wave or a pulsed regime for time-resolved measurements. The remainder of this article focuses on examples of the use of Fianium SC400 and SC450 supercontinuum fibre lasers within fluorescence imaging.
Single Source Solution for Fluorescence Imaging
Flow cytometers (or fluorescent activated cell sorters) are critical tools for biomedical research. These complex instruments measure the properties of individual cells, often by detecting fluorescent molecules (fluorophores) attached to their surface. The fluorescent molecules act as probes for analyzing the identity of cells for studying the immune system, identifying cancer cells, and diagnosing disease.
Flow cytometers rely almost exclusively on lasers for exciting fluorophores. While the coherence and power level of lasers makes them ideal light sources for illuminating individual cells, their discrete wavelengths limits the types of fluorescent probes that can be analyzed by flow cytometry.
Although solid state laser technology has increased the variety of discrete laser wavelengths available, there are still significant gaps in the excitation capabilities that put limitations of the fluorescent probes used for biomedical analysis. Confocal fluorescence microscopy has similar laser source requirements to those of flow cytometry and is one of the most powerful techniques for probing a wide range of phenomena within biological sciences.
Almost all commercially available confocal microscope systems use a combination of lasers to excite fluorescence at a few discrete wavelengths within the visible region of the spectrum. As with flow cytometry, only a small number of excitation wavelengths are available by using fixed wavelength lasers. Many fluorophores are therefore unusable because of this limitation.
A recent development, which further aids fluorophore excitation, is the combination of multi-channel AOTF's (acoustooptic- tunable-filters) with the supercontinuum fibre laser. An SC400 supercontinuum laser in conjunction with an 8-channel AOTF, enables the delivery of up to 8 laser lines within the visible region of the spectrum, each individually tunable across the entire spectrum and all within the same, diffraction limited collinear beam.
The supercontinuum and AOTF provides the flexibility required for optimal excitation and detection of a wide range of fluorophores within flow cytometers or fluorescence confocal microscopes. The laser can be tuned precisely to the excitation peak of the fluorescent probes needed for analysis.
Furthermore, the supercontinuum not only improves performance but is also cost-effective. For example, with the exception of the 355 nm frequency tripled Nd:YVO4 laser, all laser sources and beam combination optics installed within the flow cytometer platform shown in fig. 3 can be replaced by a single SC400- AOTF. Furthermore, with continued advances in supercontinuum fibre laser technology, it is only a matter of time before the spectrum is further expanded down to the UV, enabling the replacement of all lasers within even the highest specification imaging systems. While commercial vendors of confocal microscopes will soon incorporate supercontinuum fibre lasers within their imaging systems, it is often research groups who are first to investigate the technology. Dr. Clemens Kaminski and his group at the Laser Analytics Group in Cambridge (UK) have recently incorporated a fibre supercontinuum laser within an Olympus Fluoview microscope scanning unit to demonstrate 2-D, 3-D and livecell imaging.
Figure 4 demonstrates an example of 2-D high resolution fluorescent confocal imaging using an SC450 supercontinuum fibre laser and AOTF system. In this example a Convallaria Majallis (Lily-of-the- Valley) specimen is stained with Safranin and Fast Green, having peak excitation wavelengths of 530 nm and 620 nm respectively. Since the two dyes have affinities to different regions of the sample, scanning the excitation wavelength highlights different regions of the sample. Fast Green stains the cellulosic cell walls present over the whole sample (620 nm and 640 nm images) where the Safranin dye stains lignin - present within the endodermis and xylem, highlighted within [figure 3] at 540 nm to 560 nm excitation wavelengths.
The need for suitable illumination sources across a wide range of biomedical applications including Flow Cytometry, Optical Coherence Tomography, CARS Microscopy and various guises of Confocal Fluorescence Microscopy continues to drive the development of laser technology.
Over the past 5 years, ultrafast fibre lasers have started to challenge solidstate, diode and gas lasers across numerous markets and we expect the ongoing rapid development in performance and reduction in laser cost to further establish this technology as the illumination source for next generation imaging systems.
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