Recent Advances in Laser Technology for Laser Scanning Fluorescence Microscopy

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1 Recent Advances in Laser Technology for Laser Scanning Fluorescence Microscopy Gail McConnell, Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK BIOGRAPHY Dr Gail McConnell has a PhD in physics and applied physics from the University of Strathclyde, UK (2002). She was awarded an RSE Research Fellowship in 2003 and currently holds an RCUK Academic Fellowship at the Centre for Biophotonics, University of Strathclyde. Her interests involve novel laser technology for improved optical imaging and the applications of laser sources in controlling cellular function. ABSTRACT As the life sciences communities continue to embrace confocal and multiphoton laser scanning fluorescence microscopy (CLSM and MPLSM), the growing range of applications places greater demands on laser technology. For example, with powerful imaging methods such as video-rate MPLSM and ratiometric CLSM becoming available, appropriate laser technology must be ready to fully capitalise upon these sophisticated techniques. Additionally, robust, simple to use and (ideally) inexpensive sources are sought to improve the efficiency and ease of existing imaging techniques. Nonlinear optics and new laser gain media and geometries may hold the key to creating useful solutions. KEYWORDS light microscopy, confocal microscopy, multiphoton microscopy, sources, lasers, nonlinear optics, photonic crystal fibres ACKNOWLEDGEMENTS The author wishes to acknowledge funding from Research Councils UK. AUTHOR DETAILS Dr Gail McConnell, Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK Tel: g.mcconnell@strath.ac.uk. INTRODUCTION Confocal laser scanning fluorescence microscopy (CLSM) and multiphoton laser scanning fluorescence microscopy (MPLSM) are both widely used by life sciences researchers for imaging fluorescently labelled live cells and fixed tissue specimens. These powerful techniques enable the capture of high-resolution optical sections to depths in excess of 100 µm [1, 2]. From these lateral contrast images, three- or four-dimensional reconstructions can be generated to investigate the structural morphology of the sample, providing information on the nature of cellular functions or interactions. The success of CLSM and MPLSM is dependent upon the application of an appropriate laser source and in both cases there are a number of considerations when making the correct choice for the given sample. Firstly, one must choose a source which provides sufficient average power. Typically for live cell and tissue imaging, this will be <1 mw at the specimen for CLSM and <50 mw for MPLSM. Secondly, for high spatial resolution imaging, beam quality is important and ideally a TEM 00 mode should be used. Thirdly, the laser must emit at the correct wavelength to successfully excite single- or multiphoton fluorescence or autofluorescence from the specimen. Finally, the laser source output must also have the requisite stability, especially for three-dimensional imaging, time-lapse studies of live cells and quantitative studies and inter-experiment comparisons [3]. The technology associated with the design of lasers applied to CLSM and MPLSM is mature yet is unfortunately rather complex and this complexity can be daunting to the end user, often a life sciences researcher, who needs to optimize the system to obtain the best possible images. To overcome this, simple, reliable and easy to use laser systems dedicated to CLSM and MPLSM are becoming routinely available and these solutions can be considered as a black box. However, despite their ease of use, such systems are designed for reliability and not flexibility and therefore are often not well suited to the needs of the researcher. As a consequence, the search for novel and easy-to-use reliable laser technology to improve the efficiency and flexibility of CLSM and MPLSM constitutes a significant area of research in the field of biophotonics. MATERIALS AND METHODS The laser sources required for CLSM and MPLSM involve quite different technology. Historically, the laser sources most typically employed for CLSM are undoubtedly the aircooled krypton/argon ion mixed gas laser, otherwise known as a Kr/Ar laser, and the Ar + gas laser. The Kr/Ar source provides stable coherent continuous-wave radiation at discrete wavelengths in the visible region of the electromagnetic spectrum. Three such laser lines are typically employed for CLSM, namely at = 488 nm, = 568 nm and = 647 nm, with each spectral line averaging output powers of 3 to 5 mw when provided by a 15 mw source. This increases to average powers of 5 to 8 mw accordingly, where a 25 mw source is employed. This is more than ample average power for CLSM and neutral density filters are routinely employed in the beam path external to the Kr/Ar source to further attenuate the power to avoid photodamage to the sample [4]. High-quality optical interference filters are commonly used to separate the wavelengths, enabling simultaneous or sequential single, double, and triple fluorophore excitation. The stable Kr/Ar source delivers near-diffraction limited output, in a single transverse mode with a circular cross-section, which is ideally Figure 1: Compact vertical external cavity semiconductor lasers lasers offer the potential for high power, continuous wave output at visible wavelengths. Image courtesy of Dr Jennifer Hastie, Institute of Photonics, University of Strathclyde. Microscopy and Analysis 21(5):19-23 (UK), 2007 MICROSCOPY AND ANALYSIS SEPTEMBER

2 suited for CLSM. Another advantage is the relatively small size of the system, which is helpful where space is restricted. The Kr/Ar source may also be fibre coupled. In this instance, the laser is coupled into an optical fibre to relay the source from one place to another, often over several tens of metres. This provides a quasi-portable solution, allowing simple movement of the source from lab to lab, should this be required. However, the Kr/Ar source is not without problems. The source will likely require some amount of attention during the operational lifetime, even if there are no actual failures. Through routine mechanical drift, thermal fluctuations and the inevitable accumulation of dirt on the laser cavity optics, periodic adjustments to the source are required for optimum performance. Although the Kr/Ar source is the most frequently applied for CLSM, alternative gas-based sources such as the He-Ne laser exist, providing fixed wavelength output at other visible wavelengths [5]. These typically incur the same problems as described previously for the Kr/Ar laser. Semiconductor diode lasers also offer a potential solid-state solution for CLSM [6]. It is now relatively inexpensive to acquire a red emitting laser diode with an average power in excess of 100 mw. These diode lasers have very long operational lifetimes, often in excess of 1000 hours, and they can be controlled electronically and therefore can be configured to emit only when an image is being acquired. However, these otherwise robust systems have limitations for CLSM. Firstly, the beam quality is poor, typically suffering from astigmatism and different divergences in each axis. To some extent this can be overcome by coupling the output from the semiconductor laser diode into an optical fibre that supports single mode propagation at the emission wavelength [7]. This can, however, contribute towards a significant optical loss in the system and adds to the cost and complexity of the system. Anamorphic prisms [8] or cylindrical lenses [9] can also be used to correct the beam quality but these also contribute towards optical loss. The output wavelength of semiconductor diode lasers is also fixed to discrete wavelengths but the wavelength can drift by around 0.3 o C nm -1 and therefore temperature controlling is required. Discrete emission wavelengths from the ultraviolet through to the infrared are possible using semiconductor diode technology [10,11] and the particular emission wavelength is dependent upon the gain material used. There are, however, spectral ranges that are not currently covered by existing commercial semiconductor based sources. For MPLSM, the laser technology required is quite different. Key to the success of this technique is the application of an ultra-short pulsed laser source that contributes the necessarily high peak intensity required for simultaneous absorption of two or more photons by the sample [1]. To achieve this, the most common lasers are ultra-short pulsed infrared emitting sources. Indeed, the femtosecondpulsed laser has become a cornerstone of Figure 2: (a) A femtosecond-pulsed Ti:Sapphire laser anomalously pumping a <1 m section of PCF creates a white-light supercontinuum source. (b) The resultant spectrally dispersed white-light supercontinuum source extends across the entire visible wavelength range. MPLSM [12]. The commercial availability of Ti:Sapphire laser sources providing more than 50 mw average power at the sample with a wide wavelength tuning range from 690 to1050 nm has proven successful for a range of nonlinear imaging and spectroscopy methods, including harmonic generation microscopy and MPLSM [12]. Alternative neodymiumdoped and ytterbium-doped dielectric or glass laser sources with slightly longer emission wavelengths at around 1 µm have also proven useful for nonlinear microscopy [12]. Both the Ti:Sapphire and the 1 µm-wavelength systems can provide three-dimensional reconstructions of an imaged volume with submicrometre spatial resolution with improved specimen preservation over linear microscopy methods. Despite great efforts to engineer robust and simple to use systems, these ultra-short pulsed laser sources are undoubtedly complex and can still pose a technical challenge for the end user. Additionally, the complexity of the system is reflected in the price tag, with a lowend fs-pulsed Ti:Sapphire laser source with a broad wavelength tuning range costing in excess of $150k. Also, although the wavelength tuning range of the Ti:Sapphire is very broad, some applications would benefit from the availability of ultra-short pulsed sources at both shorter and longer wavelengths [13,14]. The wavelength tuning and pulse duration control mechanisms of the commercially available Ti:Sapphire systems vary from micrometer-screw gauge controls that must be mechanically adjusted by the end user to hands-free computer controlled systems. Both approaches have advantages and disadvantages which are largely dependent upon the confidence level and preference of the end user. Despite the relative ease of use of the computer controlled systems, however, not all applications are currently catered for with the existing technology. For example, quantitative ratiometric imaging that requires fast wavelength tuning of the source or multiple-wavelength video rate MPLSM is not possible with Figure 3: Sample CLSM images taken using a white-light supercontinuum source as excitation. (a) CLSM image of guinea pig detrusor labelled with anti-pgp 9.5 and Alexa 488 (green) and anti-smooth muscle myosin and Alexa 594 (red). (b) Merged fluorescence and transmission CLSM image of live guinea pig smooth muscle cell loaded with the calcium indicator Fluo MICROSCOPY AND ANALYSIS SEPTEMBER 2007

3 either the mechanically or computer controlled system. These limitations must be overcome using appropriate technology to meet the demands of MPLSM applications. To overcome some of these limitations, there is currently a significant research effort to create laser technology dedicated to CLSM and MPLSM applications. This includes nonlinear optical processes in bulk media and microstructured fibres as well as new laser gain media and cavity design. Vertical cavity semiconductor lasers (VCSEL) and vertical external cavity semiconductor lasers (VECSEL) may overcome the problem of the poor beam quality from conventional semiconductor diode lasers. Both systems involve a semiconductor gain media but the VCSEL employs a monolithic resonator whereas the VECSEL uses an external cavity [15]. In both cases, the devices can be designed to provide a TEM 00 beam output, enabling efficient fibre coupling to improve beam handling. Currently, the majority of VCSEL and VEC- SEL laser sources are optically pumped rather than electrically stimulated [16,17]. This adds to the cost and complication of the device. However, electrically activated sources will undoubtedly become available in the near future with emission properties that are suited to CLSM and MPLSM applications. The most common VCSEL and VECSEL emission wavelengths are currently in the infrared around 780 nm [18] and 850 nm [19]. These wavelengths depend upon the gain media used and are therefore fixed but these are useful wavelengths for many of the key MPLSM applications. Despite the fact that semiconductor media is notoriously poor at supporting ultra-short pulse generation, researchers have successfully demonstrated ultra-short pulsed output from VECSEL systems at GHz repetition frequencies [20]. With further work, it is anticipated that these lowcost systems could potentially replace the expensive Ti:Sapphire laser systems. Furthermore, visible wavelength devices are becoming available with sources around 660 nm and shorter wavelengths under investigation, as shown in Figure 1 [21]. VCSEL and VEC- SEL technology is new compared with conventional semiconductor diode lasers but efforts to develop shorter-wavelength emitting devices will undoubtedly impact upon the VCSEL and VECSEL communities. Due to the ease of use, reliability, long lifetime and relative low-cost of these compact VCSEL and VEC- SEL devices, they are also ideally placed to become a useful tool in CLSM in the longer term. Nonlinear optical frequency conversion processes have proven extremely successful in the development of laser sources specifically dedicated for CLSM and MPLSM. Indeed, nonlinear optics forms the basis for MPLSM and harmonic generation imaging. The simplest instance of nonlinear optical frequency conversion applied to CLSM is using an appropriate frequency doubling crystal in conjunction with an infrared emitting laser source to create a visible wavelength laser. Figure 4: Cartoon of digital micromirror device (DMD) arrangement for rapid wavelength selection and tuning. Other processes such as sum- and differencefrequency mixing can also be employed with similar conversion efficiencies but these are less common due to the requirement for more than one laser source. Optical parametric oscillators (OPO) and optical parametric amplifiers have also found applications in MPLSM thanks to their ultra-short pulsed output and potentially large wavelength tuning range [22]. Indeed, these systems have offered great promise, with fs-pulsed output from >1.2 µm for MPLSM [23]. However, OPO solutions are often based on expensive pump laser sources and despite extensive engineering they can be unreliable and can often require more maintenance than the typical end user is willing or able to perform on a frequent basis. Advances in optical fibre technology through the development of microstructured fibres, often known as photonic crystal fibres or PCF, have transformed the fields of nonlinear fibre optics and laser source development. PCFs are microstructured fibres and like conventional fibres, incident radiation is guided along a solid core. However, the cladding region of the PCF comprises periodically arranged air holes surrounding this solid silica core that extends the length of the fibre [24]. This design produces a photonic bandgap in the transverse direction that results, for instance, in fibres that are continuously singlemode throughout the visible range [25]. This improved guiding property also enables a reduction in the core diameter down to a few micrometres. This leads to a significant increase in the propagating peak intensity that is enhanced with the application of ultrashort pulsed radiation. This peak intensity increase is of obvious benefit in the study of nonlinear effects, but also significant is that the exact nature of the microstructure determines the group velocity dispersion (GVD) of the fibre. Typically, the zero-dispersion point, 0, in a 1-2 µm core diameter PCF is shifted from the bulk silica value of around = 1270 nm down to = nm. This means that it is now possible to have a fibre with anomalous dispersion at a convenient input wavelength to accommodate a wider range of commercially available laser sources, such as the Ti:Sapphire laser [26]. Easily the most impressive manifestation of the intrinsic high nonlinearity in an anomalously dispersive PCF is continuum and whitelight supercontinuum generation, which can extend well over an optical octave [27]. A PCFbased white-light supercontinuum and the resultant dispersed spectral output is shown in Figure 2. The specific mechanisms involved in the white-light supercontinuum generation are complex but the key is the ability to form solitons at wavelengths above the zero-point for the group velocity dispersion. The associated and well-known effects of soliton selffrequency shift and shedding of energy to shorter wavelengths due to third-order dispersion provide the broadening, while fourwave mixing tends to fill in any remaining gaps [28]. The notable feature of the PCF is Figure 5: Using the white-light supercontinuum source. Fluorescence (a) and transmission (b) confocal images of CHO cells loaded with 1 µm solution of the fluorescent Ca 2+ indicator Fura-2AM were obtained. Figure 4(c) shows the levels of fluorescence from the loaded cells in comparison with the control cells with no applied fluorophore. MICROSCOPY AND ANALYSIS SEPTEMBER

4 that the small mode area brings extreme prominence to these otherwise often subtle effects. The resultant white-light supercontinuum can extend continuously from the ultraviolet to the infrared. The wavelength range required to excite fluorescence from the sample can then be extracted from the white-light supercontinuum using one of a number of approaches including optical bandpass interference filters, a monochromator or a computer-controlled digital micromirror device [29,30,31]. The total average power across the spectral range of the white-light supercontinuum can easily exceed 200 mw and if we consider the ultraviolet or visible spectral range, it is possible to have an average power of around 1 mw nm -1 which is ample for CLSM applications. The stability of the white-light supercontinuum is suitable for CLSM experiments, with intensity correlation analysis indicating <1% fluctuation in fluorescence signal from pixel to pixel, thus enabling reliable quantitative CLSM imaging [32]. The source can easily be coupled into existing CLSM systems that can support the same spectral range and the TEM 00 output means that the resultant images are of comparable resolution to those obtained using alternative laser sources. Some examples are shown in Figure 3. An additional advantage of this source is that by using a fs-pulsed pump source to create the white-light supercontinuum, the resultant spectrally broad source is also ultra-short pulsed, with durations of a few picoseconds. The white-light supercontinuum source opens up opportunities for wavelength-flexible CLSM fluorescence lifetime microscopy (FLIM) and pump-probe imaging experiments [33,34]. The ultra-short pulsed output also offers possibilities for nonlinear imaging via multiphoton absorption or harmonic generation microscopy thus further extending the range of imaging applications that can be enabled [35]. The current drawback to this system is financial; an expensive pulsed laser is required to pump the PCF, often costing in excess of $150k. Additionally, the PCF is expensive when compared with conventional optical fibre. However, since the pump laser technology is routinely available in many labs for other experimental procedures, the relative cost in upgrade with the addition of the modular PCF accompaniment is small considering the power, reliability and wavelength flexibility afforded by the resultant white-light super continuum source. The immediately accessible wide spectral range presents an advantage for rapid CLSM and ratiometric imaging. By projecting the dispersed white-light supercontinuum onto a digital micromirror device (DMD), individually addressable mirror elements can be switched at high speeds to serve as a spectral filter, as shown in Figure 4 [31]. The white-light supercontinuum source is dispersed and applied at normal incidence to the DMD. When a discrete range of mirrors is activated (shown as the white strip), light is reflected from these elements at a 12 o angle (exaggerated above) whilst the remainder is rejected. By controlling the DMD through a PC, it is possible to control the mirror elements and hence extract disparate spectral ranges at speeds of around 100k Hz. Such rapid wavelength tuning is beyond the capability of the few other available wavelength-flexible laser systems. Also, many of the useful ratiometric indicators, such as the calcium indicator Fura-2, are optimally excited with ultraviolet wavelengths. Ultraviolet laser sources are currently limited to one or two fixed wavelengths from GaN-based systems or gas-based lasers whereas the white-light supercontinuum extends from 330 nm and can efficiently excite the full range of commercially available fluorophores. Sample CLSM images of CHO cells loaded with Fura-2 taken using the white-light supercontinuum source at ultraviolet wavelengths are shown in Figure 5 [36]. Modular PCF-based sources also offer improvements for MPLSM. As described previously, the high peak intensity of ultra-short pulsed laser sources is ideal for MPLSM but the ability to control the pulse duration can vastly improve the imaging process. For example, the main difficulty in using ultra-short pulsed laser sources for MPLSM is undoubtedly minimising the effects of positive dispersion to retain the necessarily short pulse duration at the focal plane within the sample [37-39]. As the ultrashort pulse propagates through optical elements, normal dispersion stretches and distorts the wavelength components of the broad (typically >1 nm) spectrum and stretches the pulse duration. As a consequence, the peak intensity of the source can be significantly reduced. The dispersion of a laser scanning microscope system can easily exceed fs 2 cm -1 [40]. In the case of a 100 fs pulse leaving the laser source, this value of dispersion can mean a factor of five increase in the pulse duration at the sample which clearly affects the peak intensity [41]. Schemes that compensate for dispersion of Figure 6: (a) Modular PCF-based pre-dispersion compensation system. Pulses of 250 fs duration from an = 820 nm Ti:Sapphire source were coupled into an 8 cm long section of photonic crystal fibre with 0 = 890 nm. Negative dispersion was controlled using a grating pair and the output then propagated towards a laser scanning system. Comparative contrast images of living smooth muscle cells from the rat pulmonary artery labeled with the voltage sensitive indicator di-8-anepps were obtained using a 60X 1.4 NA lens (Nikon). Using the same average power of 24 mw and repetition rate whilst only controlling the pulse duration of the source (b) corresponds to imaging using the 250 fs pulses while (c) used the PCF pre-dispersion system for temporal control. No muscle cells could be observed using the longer pulses while the structures were clearly visible when using the pre-dispersion compensation system. laser scanning microscope systems can provide a solution to the detrimental effects of pulse broadening. Such systems typically involve adding a variable amount of negative dispersion into the path of the excitation beam (normally before the laser scanning system, thus constituting pre-dispersion compensation) to counteract the positive dispersion experienced by the pulse. Several methods of pre-dispersion compensation systems have been reported. These include the prism pair compressor used with a standard Ti:Sapphire laser, but the pulse duration of the system is limited to >30 fs and requires substantial modification to the laser geometry [42]. It is also possible to use a step-index silica fibre-based compression scheme with an amplified Ti:Sapphire laser, but this is a rather expensive and photon-inefficient solution [43]. A simple PCF-based predispersion compensation system may be used to successfully overcome these limitations and to significantly extend the capability of MPLSM [44]. Whereas white-light supercontinuum generation uses the anomalous dispersion present within the PCF, pumping in the normal region creates substantial self-phase modulation effects which, when used in conjunction with a prism or grating pair, can be used for temporal control of the pulse duration not only immediately leaving the laser but also at the microscope stage. By minimizing the pulse duration at the focal plane using this approach, it is possible to increase the fluorescence signal from the sample [45]. In a study comparing conventional fs-pulsed output from a Ti:Sapphire laser and a pre-dispersion compensated system based on PCF, a factor of eight increase in fluorescence signal was measured in compressing a 250 fs pulse to 25 fs using this approach [45]. The system is presented along with typical MPLSM images in Figure 6. In the same way, the depth of imaging using 22 MICROSCOPY AND ANALYSIS SEPTEMBER 2007

5 MPLSM can also be improved through temporal control. Since sample dispersion is typically negligible, the shorter pulse duration is largely unchanged and hence the increased propagating peak intensity of the source is retained deeper within the sample. By employing the same PCF-based dispersion compensation method for temporal control, more than a three-fold increase in MPLSM depth penetration was measured when compared with a non-compensated system [46]. Sample images are shown in Figure 7. Using dispersion-controlled pulses to counteract the effects of positive dispersion in the imaging system enabled depth penetration of over >800 µm in a fluorescent dye solution, compared with only 240 µm using a non-compensated setup. Similarly, fluorescent tissue samples imaged using the pre-dispersion compensation system offered a three-fold improvement in depth resolution [46]. CONCLUSIONS As the life sciences communities continue to embrace CLSM and MPLSM, the range of applications and demands on the technology will continue apace. With powerful applications such as video-rate MPLSM and ratiometric CLSM now becoming a reality, robust laser sources and laser scanning microscope technology are well-placed to revolutionise life sciences research. Clearly, nonlinear optics and novel laser gain media and geometries will play an important role in this development and the successful integration of this technology. Laser development for CLSM and MPLSM applications is an exciting field and new advances are certainly on the horizon. REFERENCES 1. Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nature Methods 12: , Diaspro, A., Chirico, G., Collini, M. Two-photon fluorescence excitation and related techniques in biological microscopy. Quarterly Review of Biophysics 38:97-166, Girkin, J.M., McConnell, G. 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