Ultrafast Pulse Laser and its Applications in Microscopy Jing Wang Short pulse lasers have become increasingly important in recent years for many technologies, including microfabrication, thin film formation, laser cleaning, and medical or biological applications. 1. Introduction to Ultrafast Pulse Laser Systems The 1999 Nobel Prize in Chemistry was awarded to Ahmed H. Zewail for using ultrashort pulses to observe chemical reactions on the timescales they occur on, opening up the field of femtochemistry. In optics, an ultrashort pulse of light is an electromagnetic pulse whose time duration is of the order of a picosecond (10−12 second) or less. Such pulses have a broadband optical spectrum, and can be created by mode-locked oscillators. They are commonly referred to as ultrafast events. Although "ultrafast laser" is a misnomer, since the speed of light is constant in a given medium. An ultrashort pulse is a wave whose field amplitude follows a Gaussian envelope, as shown is Fig. 1, and whose instantaneous phase has a frequency sweep. The time-averaged intensity of such a pulse shows as a Gaussian shaped pulse.

Fig.1 Ultrashort pulses are generated by mode-locked lasers. By constructive interference, a short pulse is formed when many longitudinal modes are held in phase in a laser resonator. Various techniques have been employed, usually grouped under the terms “active” or “passive” mode locking and descriptions of these can be found in many standard texts and review articles. Active mode locking uses a modulator in the laser cavity whereas passive schemes use a saturable absorber, often a thin semiconductor film, to lock the relative phases. Modern solid-state mode-

locked lasers use a different scheme called self-mode-locking and titanium-doped sapphire (Ti:sapphire) has become by far the most common laser material for the generation of ultrashort pulses. Developed in the mid-1980s, Ti:sapphire has a gain bandwidth from 700 to 1100 nm peaking around 800 nm, the broadest of the solid-state materials yet discovered, high-gain crosssection and extremely good thermal conductivity. Mode locking is achieved through the action of an instantaneous nonlinear Kerr lens in the laser rod. The peak fluence of the laser approaches 10 Wcm2, which is enough to focus the beam as it travels through the gain medium on each pass. This Kerr lens then couples the spatial and temporal modes and maintains phase locking.

Fig.2 A basic oscillator cavity configuration is shown schematically in Figure 2. The laser is pumped by about 5 W from a continuous wave (CW) laser source, usually now an intracavitydoubled diode-pumped neodymium laser. This light is focused into the Ti:sapphire rod, collinearly with the laser axis, through the back of one of the mirrors. The cavity consists of a Brewster-angle cut Ti:sapphire rod, 5 mm or less in length, doped to absorb about 90% of the incident pump radiation, two concave focusing mirrors placed around it, a high reflector and an output coupler. A pair of Brewster-cut fused-silica prisms are inserted to control the spectral dispersion (chirp) introduced in the laser rod. Dispersion arises from the variation of the refractive index of the material across the gain bandwidth of the laser, which can lead to a temporal separation of the resonant wavelengths and place a limit on the generated bandwidth. The cavity dispersion, coupled to the Kerr lens effect, is an intrinsic part of the pulse-formation process. Ordinarily, the Kerr lens in the rod would contribute to the overall loss but this is overcome by a small adjustment to the resonator. Displacement of one of the curved mirrors by only ca. 0.5 mm pushes the cavity into pulsed mode. Here the cavity is corrected for the nonlinear lens effect and CW operation is restricted. Pulsing is established by perturbing the cavity to introduce a noise spike, literally by tapping a mirror mount. This configuration delivers 12-fs pulses centered at 800 nm with 5 nJ energy at an 80-MHz repetition rate. Using a shorter rod, shorter pulses have been obtained as the dispersion is better compensated and space time focusing effects are controlled. The laser repetition rate can be adjusted by the insertion of a cavity dumper

in a second fold, without prejudice to the pulse duration. The use of mirrors that have dispersion opposite to that of the rod obviate the need for prisms entirely. Alternatively, a mixture of prisms and mirrors can be used to generate pulses as short as 5 fs. At the time of writing, the use of mirrors with well-defined chirp characteristics is complicated by obviates the demand for extreme tolerances in the manufacturing process. Many schemes have been proposed for self-starting oscillators, perhaps the best of which is the use of a broadband semiconductor saturable-absorber mirror in the cavity. Advances in these areas will surely continue. The latest ultrafast laser from Spectra-Physics, InSight DeepSee, features an unprecedented 680 nm to 1300 nm continuous tuning from a single source, short 100 fs pulse widths and highest peak power levels into the infrared where imaging penetration depth is maximized. A detailed specification is listed below in Fig. 3.

Fig.3 2. Application of Ultrafast Pulse Laser in Biological and Medical Imaging “Seeing is believing” is an idiom first recorded in this form in 1639 that means "only physical or concrete evidence is convincing". It is the essence of St. Thomas's claim to Jesus Christ, to which the latter responded that there were those who had not seen but believed. This golden rule becomes a big bottleneck for biological and medical field because most of essential reactions happen at nanometer scale which is too small for our naked eyes. Microscope is invented and developed to visualize these biological samples. Ever since the development of first optical microscope in late 1500s, microscopy has been expanding very fast and been widely used in lots of research fields. However, most of microscopy studies in biology studies have been limited to the surface (cell membrane) or biological sample. To study the cytosolic reactions deep inside cells, people have to freeze down samples and then cut them into thin sections. These sectioning procedure is complicate and sensitive, accompanies with all type of potential contaminations and artifacts. Optical section can be used to avoid the complicated sample processing procedure, while it comes

with out-of-focus background and photobleaching of fluorescent probes. As a potential solution towards three-dimensional microscopy, ultrafast pulse laser showed exciting and promising advantages comparing to conventional light sources (lamp or cw laser), such as lower absorption / heating, low out-of-focus background and deep penetration depth inside sample. Multi-photon excitation microscopy, based on ultrafast pulse laser, has showed its superior power towards pushing microscopy from biological lab-bench sample to real medical / clinical samples. Here I will show two applications of ultrafast pulse laser in biology field. 2.1 Two-Photon Fluorescence Microscopy The invention of two-photon fluorescence light microscopy by Denk, Webb and co-workers (Denk et al., 1990) revolutionized three-dimensional (3D) in vivo imaging of cells and tissues. In 1931, the theoretical basis of two-photon excitation was established by Maria GoppertMayer, and this photophysical effect was verified experimentally by Kaiser and Garret in 1963. Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by the simultaneous absorption of two photons. The familiar one-photon fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination, as shown is Fig.4. This nonlinear process can occur if the sum of the energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Since this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance. Under sufficiently intense excitation, three-photon and higher photon excitation is also possible and deep UV microscopy based on these processes has been developed.

Fig.4

Confocal microscopy utilizes a pinhole to exclude out-of-focus background fluorescence from detection. Thus, this technique allows three-dimensional sectioning into thicker tissues. However, the excitation light generates fluorescence, and thus produces photobleaching and phototoxicity throughout the specimen, even though signal is only collected from within the plane of focus. This large excitation volume can cause significant photobleaching and phototoxicity problems, especially in live specimens. Furthermore, the penetration depth in confocal microscopy is limited by absorption of excitation energy throughout the beam path, and by specimen scattering of both the excitation and emission photons. The considerable advantages of using two-photon excitation in laser-scanning microscopy arise from the basic physical principle that the absorption depends on the square of the excitation intensity. In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope optics. As the laser beam is focused, the photons become more crowded (their spatial density increases), and the probability of two of them interacting simultaneously with a single fluorophore increases. The laser focal point is the only location along the optical path where the photons are crowded enough to generate significant occurrence of two-photon excitation. Figure 5 illustrates diagrammatically the generation of two-photon excitation in a fluorophore-containing specimen at the microscope focal point. Above the focal point, the photon density is not sufficiently high for two photons to pass within the absorption cross section of a single fluorophore at the same instant. However, at the focal point, the photons are so closely spaced that it is possible to find two of them within the absorption cross-section of a single fluorophore simultaneously. As a comparison, focus cw laser will excite all the area where the beam propagates, causing both out-of-focus background and photobleaching, as demonstrated in the left half of figure 5.

Fig.5 In practice, two-photon excitation microscopy is made possible not only by concentrating the photons spatially (by focusing of the microscope optics), but also by concentrating them in time (by utilizing the pulses from a mode-locked laser). The combined effect allows generating the necessary photon intensities for two-photon excitation, but the pulse duty cycle (the duration

of the pulse divided by the time between pulses) of 10-5 limits the average input power to less than 10 mW, which is just slightly greater than that used in confocal microscopy. Although the laser pulse durations are considered ultra-short, typically ranging between approximately 100 fs and 1 ps (10-13 to 10-12 seconds), in comparison to the fluorophore absorption event of about 10-18 second, they are relatively long in duration. The narrow localization of two-photon excitation to the illumination focal point is the basis for the technique's most significant advantages over confocal microscopy. In a confocal microscope, although fluorescence is excited throughout the specimen illuminated volume, only signal originating in the focal plane passes through the confocal pinhole, allowing background-free data to be collected. By contrast, two-photon excitation only generates fluorescence at the focal plane, and since no background fluorescence is produced, a pinhole is not required. This dramatic difference between the excitation regions of confocal and two-photon excitation microscopy can be demonstrated by imaging the photobleaching patterns of each method. A typical two-photon microscopy setup is showing below (Fig. 6). Ultrafast pulse laser were directed into the microscope via an epiluminescence light path. The excitation light is reflected by a dichroic mirror to the microscope objective and is focused in the specimen. Two-photon induced fluorescence is generated at the diffraction-limited volume. Images are constructed by raster scanning the fluorescent volume in three dimensions using a galvanometer-driven x-y scanner and a piezo-objective z-driver. The emission signal is collected by the same objective and transmitted through the dichroic mirror along the emission path. An additional barrier filter is needed to further attenuate the scattered excitation light.

Fig.6 The image showed in Fig.7 is anesthetized Rat neural cell of cerebral cortex expressing EYFP. Superior tissue penetration enables researcher to monitor neural activity and morphological changes of cells in a brain of a living mouse. Two-photon microscopy can detect

fluorescent signals from deeper layers than 0.9 mm from the surface of the brain cortex, so that neurons in all the layers of the cortex in a living mouse can be visualized.

Fig.7

2.2Coherent Anti-Stokes Raman Spectroscopy (CARS) Confocal and multiphoton imaging techniques are still the methods of choice for performing sophisticated studies on biological samples. These techniques visualize typical structures or dynamic processes in biological samples and depend on existing autofluorescent matter in the specimen or the availability of suitable fluorescent dyes. The drawbacks of conventional staining methods are evident: labeling is time-consuming and dyes bleach over time. Furthermore they lose intensity and alter the sample. Dyes often cause phototoxicity, harm the specimen and consequently influence the result of the experiment. CARS microscopy is a dyefree method which images structures by displaying the characteristic intrinsic vibrational contrast of their molecules. The crucial advantage of this method is that the sample remains almost unaffected.

Fig.8

CARS is a third-order nonlinear process that involves a pump beam at a frequency ω p and a Stokes beam at a frequency of ωs. The signal at the anti-Stokes frequency of ωas= 2ωp- ωs is generated in the phase-matching direction. The sample is stimulated through a wave-mixing process. The vibrational contrast in CARS is created when the frequency difference Δω=ω pωs between the pump beam and the Stokes beam is equal to the frequency of the molecular vibration of a particular chemical bond and oscillations of molecules with that bond are driven coherently. A sketch demonstrating the different mechanism of two-photon microscopy and CARS are shown in Fig. 8.

Fig.9 A typical experimental setup of CARS is shown in Fig.9. The overall microscope configuration is similar to a confocal system. By integrating CARS technology into a confocal system, the drawbacks of conventional microscope techniques can be overcome. The latest commercial developments result in an easy-to-use and efficient imaging microscope for a variety of biological and non-biological samples. Two infrared laser beams which are adjusted exactly in terms of spatial and temporal properties generate brilliant CARS images at different wave numbers. The combination of a couple of CARS filters and non-descanned detectors allows the detection in the forward as well as in the backward (epi) direction. Recording of the second harmonic generation (SHG) can be done simultaneously. Combinations of conventional and highspeed scanners support the analysis of dynamic processes at video rate as well as morphological studies at high resolution. Implementing single-particle CARS microscopy also requires improvement in signal-tonoise ratio (S/N) over that found in traditional spectroscopy so that a response from a single nanostructure is resolvable. There are two main sources of noise in these experiments: laser fluctuation noise and electronic noise from the detection system (the detector and lock-in amplifier, for instance). Noise due to laser intensity fluctuation can be effectively eliminated by

using heterodyne lock-in detection with megahertz modulation, whereby the intensity of excitation beam is modulated by an acoustic-optical modulator. Subsequently, a lock-in amplifier referenced to this modulation frequency can sensitively extract the induced signal. The fluctuation of laser intensity (1/f noise) usually occurs at low frequency of dc to 10 kHz. When f is in the megahertz range, the laser intensity noise nears the quantum shot noise limit, which is always present because of the Poissonian distribution of the photon counts at the detector. The pixel dwell time should be significantly longer than the modulation period to allow for reliable demodulation for each pixel. Such a modulation scheme has been successfully applied to pumpprobe microscopy and stimulated Raman loss and gain microscopy, and shot noise–limited detection has been achieved.

Fig. 10 In vivo imaging of a larvae of a fruit fly (Drosophila melanogaster), shown in Fig. 10. Fat cells are shown in red at a wave number of 2,850 cm–1 (at 816 nm). Two different structures are displayed in green autofluorescence (at 1,064 nm): Long tubes are parts of the tracheal system, striped matter in the background are muscles. 3. Discussion & Conclusion As discussed above, the most powerful advantage of two-photon excitation microscopy, using ultrafast pulse laser as the excitation source, is its ability to provide superior optical sectioning at greater depths in thick specimens than is possible by other methods. Three physical mechanisms exist that function in combination to allow the increased depth of penetration into thick specimens:  Absence of out-of-focus absorption allows more of the excitation light photons to reach the desired specimen level.  The red and infrared light employed in two-photon excitation undergoes less scattering than light that is bluer in color (shorter wavelengths).

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The effects of light scattering are less detrimental to two-photon microscopy than to confocal microscopy. These features provided ultrafast pulse laser a bright future in biological and medical field. Every technique has both sides. The short board of two-photon excitation microscopy and CARS are also obviously.  Slow speed comparing to common microscopy techniques. This made it impossible to image fast cellular dynamics in real time. The speed also limited the potential applications in medical field, especially clinical applications.  The cost of ultrafast laser is more than two levels higher than diode lasers or fluorescence lamp. It also requires frequent maintenance performed by professional stuff. Considering the continuous contribution from academia and industry sides, these technical problems will be solved sooner or later. By that time, ultrafast pulse laser will be able to show its full power and bring revolution change to our world. 4. References 1. 2. 3. 4. 5. 6. 7.

http://en.wikipedia.org/wiki/Ultrashort_pulse http://www.rp-photonics.com/ultrafast_lasers.html http://en.wikipedia.org/wiki/Coherent_anti-Stokes_Raman_spectroscopy http://www.leica-microsystems.com/science-lab/an-introduction-to-cars-microscopy/ http://bernstein.harvard.edu/research/cars.html http://en.wikipedia.org/wiki/Two-photon_excitation_microscopy http://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.ht ml

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