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Multiphoton Microscopy with Ultrafast Lasers

Jeff Squier Department of Physics, Colorado School of Mines, Golden, CO USA Michiel Mller University of Amsterdam, The Netherlands Virginijus Barzda Department of Physics, University of Toronto, Mississaugua, ON, Canada

Why image with ultrafast lasers?


One of the challenges in microscopy is simply generating image contrast. A typical cell can range in size from 5 m to 100 m. (For scale, consider the width of a typical human hair is 30-50 m.) Needless to say, objects with these dimensions have small optical scattering and/or absorption path lengths (typical image contrast generation mechanisms) rendering individual cells relatively transparent. To compound the challenge, to truly examine the structure and function of a cell it is necessary to resolve the organelles within the cell the image contrast generated by these bodies further reduced by their exceptional small size. It doesnt stop there of course important clues to biological function require the resolution of bodies such as vesicles that are only 50 nm in diameter! At the opposite extreme is examining how groups of cells function. This is a central problem in the neurosciences - how do networks of neurons function? The challenge here is that while an individual cell may scatter or absorb little light, imaging through tissue results in significant scattering. Traditional widefield optical microscopy can be rendered useless by the scattered light in many tissue types for depths as small as 50 m. The absolute value in using an ultrafast laser as a light source for imaging lies in the fact that the ultrafast laser can effectively address this range of imaging issues. With a femtosecond laser, it is straightforward to generate intensities on the order of 100 GW/cm2 with a modest focus. At these intensities, optical nonlinearities are driven quite efficiently, and it is these nonlinearities that can be exploited to generate image contrast. Notably, the fact that these nonlinearities are driven efficiently over very small path lengths (~1 to 6 m) is actually a strength and is routinely used to generate highresolution, three-dimensional imaging capability. For this application, the typical wavelength of the ultrafast laser is in the nearinfrared (IR): 750 nm to 1300 nm. This can be significant considering the fact that scattering in many tissue types scales linearly with wavelength (shorter wavelengths typically exhibiting increased scattering). Imaging with an excitation source in the nearIR results in increased penetration depth when compared to imaging with an excitation source in the visible. Coupling this characteristic with the fact that nonlinear optical imaging is actually a non-imaging modality (more on this later), it is possible to image with high-resolution (~1 m3) at great depth (100s of ms) through significantly scattering media.

The simple fact that femtosecond lasers are inherently broadband devices can also be usefully exploited in linear imaging processes. For example, a technique known as optical coherence tomography uses this bandwidth and the coherence of the light (as the name implies) as a means to obtain high resolution imaging through scattering media such as tissue. The bandwidth of the laser determines the axial image resolution, the coherence is used to gate the non-scattered light enabling imaging with exceptional signal-to-noise even in the presence of large amounts of scattering. Finally, one of the most exciting aspects of imaging with ultrashort pulses, is the capability of molecular specificity. It has been estimated that up to 85% of the chemistry that occurs within the cell occurs across the cell membrane. Ultrashort pulse molecular specific imaging will provide an important window for visualizing and studying this activity. This chapter is organized as follows. First, we start with a brief introduction into how optical nonlinearities can be used to generate image contrast. Next, we examine the mechanics of imaging what are the laser sources, how is scanning accomplished, what are the signal levels we expect? Having developed the basic system architecture some simple methods for quantitatively characterizing the pulse (in space and time) at the focus of a typical high numerical aperture (NA) microscope objective are presented. Remember, for this modality of imaging to be successful it is necessary to efficiently excite an optical nonlinearity in a diffraction-limited volume. For this reason it is desirable to be able to measure the intensity that is produced at the focus of the imaging system. Finally, with these characterization tools, it quickly becomes apparent that the multi-element optical systems used to deliver the light can significantly broaden the ultrashort pulse, and impact the imaging efficiency. Thus, a few strategies for compensating for the dispersion produced by the microscope are discussed. With a solid understanding of all the microscopy basics, several examples of how multiphoton imaging is used are explored at the end of this chapter. This includes multimodal multiphoton imaging of photosystems, and CARS imaging.

Optical nonlinearities how they generate image contrast


To understand how image contrast is generated in a multiphoton microscope it is instructive to start with the basic imaging parameters. For example, it is quite typical to use a 100 fs pulse, 100 pJ of energy per pulse, and a pulse repetition rate of 80-100 MHz. This is an average power of 8-10 mW measured at the specimen. If focused with a 0.65 numerical aperture (NA) objective, the focal spot size is given by (Rayleigh criterion)
FWHMlateral r0 = 0.61 NA 2 n z0 = (NA )2

FWHMaxial

where FWHM denotes Full Width at Half Maximum1. Note that the lateral and axial distances differ in size the three-dimensional interaction volume is ellipsoidal, not spherical. For an excitation wavelength of 800 nm, r = 0.75 m. The intensity at focus is then on the order of 50 GW/cm2. This is sufficient intensity to induce a charge

separation, or polarization, of the specimen at the image plane. The induced timedependent polarization serves as a driving term to the wave equation. It is in fact a highly nonlinear driving term (not hard to imagine at such high intensity), and as a result acts as a source of new frequency components. Through careful detection, we can isolate these various new source terms. The nonlinear optical signals we detect are particularly advantageous for several reasons. First, since they scale as some power of the excitation intensity, they are highly localized and efficient signal generation only occurs within the focal plane of the laser beam. Thus, sectioning of the specimen occurs quite naturally. By rastering the beam in a two-dimensional plane, and measuring the nonlinear optical signal intensity as a function of excitation beam position, a cross-sectional image of our three-dimensional specimen is generated. Notably we dont need a camera for this task because the position of the laser beam is known, and the detected signal is inherently generated at the focal plane, a photomultiplier (or photodiode) is all that is needed for a detector. This is why multiphoton imaging is referred to as a non-imaging modality and is one of the strengths of the technique. In this case, photons that might be scattered by the specimen as they make their way back to the detector still contribute to our image. By contrast, scattered photons in a traditional imaging system create a background fog that reduces image contrast and degrades the resolution. In figure 1, a multiphoton imaging system that detects three-different nonlinear optical signals simultaneously is depicted2-4.

Figure 1. A multicontrast imaging system. Second and third harmonic are detected in the forward direction, multiphoton absorption fluorescence is detected in the back direction.

In the backward direction, the two photon absorption fluorescence signal is detected. The two photon absorption fluorescence signal scales as where I is the excitation intensity, and 2 the two-photon absorption cross section. The emission is incoherent, and emitted into 4. The emission wavelength is shorter than the excitation wavelength. For example, if you were imaging a photosystem at an excitation wavelength of 800 nm, you might be detecting two photon absorption fluorescence at 670-680 nm5. In the forward direction, two signals are detected: the third harmonic signal, and the second harmonic signal. As with two photon absorption fluorescence the second harmonic scales as the square of the excitation intensity. It differs from the two-photon absorption signal however, in that it is a fully coherent signal and centered at exactly twice the frequency of the fundamental6-8. The third harmonic signal scales according to
2 ITPA 2 I

I3 P (3 ) 3 (3 = + + ) E ( ) E ( ) E ( )
3 = 3 I 2

Thus it exhibits a third order dependence on our excitation intensity, is a coherent signal, and centered at three times the fundamental frequency9, 10.

Figure 2. Multiphoton image of a cardiomyocyte. Left second harmonic generation image, Right Third harmonic image. Figure 2 is an image of a heart cell or cardiomyocyte from just such a multicontrast imaging system. Note the difference in structure that the two contrast mechanisms bring out for this cell orientation2.

Basic multiphoton microscope system architecture


Laser Source. The heart of every multiphoton imaging system begins with the laser. To date, the optimal ultrafast laser source is Ti:sapphire. Ti:sapphire has multiple important characteristics that make it optimal for this type of imaging. The average system produces 1 W of average power, necessary for imaging through scattering media. It is tunable commercial systems tune from 700 to 950 nm on a single mirror set. Next, the pulsewidth is 100 fs or less throughout this wavelength range, making it possible to efficiently excite nonlinearities throughout this tuning range. Finally, all of this is achieved in a single platform. Scanning. In order to create an image the specimen and excitation beam must be rastered relative to one another. The predominant method for creating a cross-sectional image involves beam scanning as opposed to rastering the specimen. Common beam scanning techniques typically employ any one of a variety of mechanisms that can be used to angularly deflect the beam about two orthogonal, and independent axes. Galvanometric scanners are a typical example. They are inexpensive, provide excellent angular resolution, are linear in their deflection and employ first surface mirrors that add little or no dispersion to the system with very high reflectivity. These systems can be used to achieve frame rates on the order of 10-Hz without too much difficulty. It is possible to achieve higher frame rates, but you do pay a price in terms of ease of implementation and complexity. For example, rotating polygonal mirrors can be used to increase the sweep rate along a single axis. Facet-to-facet variations can complicate the image calibration and the timing electronics are more sophisticated than those required for a pair of packaged galvanometric scanners. Similarly, resonant galvanometers can be used, and indeed have been successfully employed in video rate multiphoton imaging systems. These, however, are nonlinear scan devices that create multiple challenges in terms of synchronization and image scaling. Recent progress has been made in implementing acousto-optic beam modulators as a high-speed scanning system. These systems are capable of arbitrary beam deflection at very high speeds. An added challenge with this type of scanner is they are not 100% efficient, and they result in a spatially chirped beam profile. Image relaying the scanners. No matter what type of system you decide to use to steer the excitation beam, ultimately an imaging system will be required to convert this angular deflection into a linear translation of the focal spot across the specimen plane. The imaging system may also serve a second function: enlarging the beam to appropriately fill the entrance pupil of the objective.

Here we want to exam the basic case of how we convert an angular deflection to a linear translation. This is accomplished through the telecentric system shown in figure 3. For simplicity only a single deflection axis is shown. Telecentric means that the marginal and principle rays for the system are parallel. If the scanner is located at one telecentric plane, and the entrance pupil of the objective at the other, you can see we achieve our imaging goal. As shown in the figure, in this configuration a collimated beam of light remains collimated, and completely fills the entrance pupil of the objective independent of the scan angle. It is important the beam remain stationary at the entrance pupil to insure that the full NA of the objective is used across the entire scan field.

Figure 3. The back focal aperture is the aperture for an infinity corrected objective. The scanner will be located at the input aperture plan of the scan lens, which is conjugate to the back focal aperture plane. The principal ray is the central ray shown in the diagram, and it is surrounded by two marginal rays. For the system shown in 4 an exact ray-tracing is required to locate the correct plane for the scanner location for a given excitation wavelength. In particular, we have designed around the requirement that the objective manufacturer has specified that the 160 mm singlet tube lens be located 100 mm from the entrance pupil of the objective. We have selected an identical 160 mm singlet as the scan lens. This particular system is used for 1.05 m light. The spacing between the singlets is optimized for this wavelength to be 313 mm. The scanner is placed at 214 mm in front of the scan lens. Figure 5 is a plot from the exact ray-tracing for a scan range of 3 degrees. Notice that the scanned beam is relayed perfectly to the entrance of the objective in a collimated beam. For clarity only the tube lens and entrance plane into the objective are shown.

Figure 4. Location of scan plane for a system that uses a tube lens and a scan lens of focal length f=160 mm. The back focal aperture of the system is required to be 100 mm from the tube lens. The scan mirror location was determined by ray-tracing.

Figure 5. Output of ray-tracing of the system from figure 4, only the tube lens is shown for clarity. The scanned beam remains collimated throughout the scan range as shown, and is centered on the entrance aperture of the objective. A second scan lens (relay lens) could be added for the other axis. However, for most applications it is usually adequate to place both scanners close together, and image

at the intermediate plane between the scanners. A second simplification used in a number of home-built microscopes is to simply place the scanners a distance fs from the scan lens, and place the tube lens a distance fTL from the entrance pupil of the objective. This is often quite adequate but it does result in using the tube lens in a position that was not necessarily intended by the microscope objective designer and may have implications in terms of aberrations. Detecting the signal. An important consideration in the design of your system is the signal levels you can expect to encounter. For quantitative imaging it is important to have a reasonable knowledge of the number of photons you expect to be able to detect. This will have important implications in terms of the type of detector you select, the frame rate, field-of-view, resolution, etc. To get a general feeling for the signal levels, lets assume a repetition rate of 80 MHz for our laser (most commercial systems operate in the range from 50 MHz to 100 MHz) and that we produce an image photon per laser pulse. Thats 80 million photons per second. We can then use this number to estimate a photocurrent. For a bi-alkali pmt that has good response in the 300 nm to 700 nm range, we can expect a gain on the order of 106 and a quantum efficiency of ~40%. This gives us an estimate of the photocurrent (80 106 photons/sec) * (106) * .4 electrons/photon *1.6 10-19 C/electrons = 5.12 A of current. For detectors with high quantum efficiency this can exceed the maximum range of the anode current. Thus, in many instances the image acquisition will occur under conditions where less than a single signal photon per laser pulse is generated. Damage thresholds Obviously disrupting the specimen through damage is a major concern in multiphoton imaging especially considering the intensity levels we are exposing it to! It is very difficult to make general statements concerning specimen damage, and in general every time you study a new system, its worth investing some time in trying to identify those mechanisms that your particular specimen is most sensitive to. For example, in studying photosystems, we have found that we could significantly enhance imaging time by decreasing the repetition rate of the laser. In other words by lowering the pulse repetition rate from 82 MHz to 1 MHz the viewing time could be significantly extended. For other biological systems, this effect may not be so dramatic, and the loss in repetition rate is just that a loss.

Characterizing femtosecond laser pulses at high numerical aperture


Microscopy with femtosecond lasers is generally performed with multi-element, high numerical (NA) aperture optics. It is important to be able to characterize the intensity at the focus of the high NA optic for several reasons. The multiphoton processes used to generate image contrast have a nonlinear dependence on excitation intensity. Thus, a quantitative measure of the focused intensity enables the researcher to optimize the imaging conditions creating the most efficient excitation under the tightest possible focusing conditions. A quantitative measure of the intensity also provides important measures of the exposure conditions at the sample particularly important when imaging living specimens.

The most meaningful measurements are performed at the full NA of the excitation optic replicating the imaging conditions. This means that collinear pulse characterization techniques are the most suitable. Most multiphoton microscopy is preformed over a broad wavelength range the laser source is tuned to optimize viewing conditions. This necessitates that the characterization technique also be applicable over a broad wavelength range. There have been multiple techniques developed to measure the pulse at the focus of a high NA microscope objective. We only review two here. Notably these two methods provide a measure of the pulsewidth and provide spatial characterization of the focus as well. Interferometric Autocorrelation Methods A straight-forward, and inexpensive pulse characterization method that meets the requirements of pulse characterization for microscopy (collinearity and broad wavelength range) is 2-photon absorption, interferometric autocorrelations performed at focus in a The system for performing this measurement is shown in figure 6. GaAsP diode11. The pulse from the laser is first directed through the balanced interferometer. The interferometer output is then imaged to the specimen plane. The full NA of the excitation optic is used, the pulses travel through the system completely collinear. The GaAsP diode is placed at the focus of the excitation objective. By scanning one arm of the interferometer, the temporal characteristics of the pulse at focus are recorded.

Figure 6. Scanning system for autocorrelation measurements. By blocking one arm of the interferometer, and scanning the GaAsP diode along z, the nonlinear excitation volume can be spatially characterized11. Thus, this single tool can be used to characterize both the spatial and temporal characteristics of the pulse at focus under conditions identical to those that will be used to image. Notably, the GaAsP diode has an exceptional bandwidth, capable of being used to perform this sort of nonlinear characterization over an excitation wavelength range from 600 nm to 1360 nm.

A second method that matches these requirements is third harmonic spatialtemporal pulse characterization12, 13. In this case the nonlinear media is something already available with every microscope a glass coverslip! In this instance, the set-up is identical to that shown in figure 6, except the GaAsP diode is replaced by the glass coverslip. A third harmonic signal (three times the frequency of the fundamental) is generated at either face of the glass coverslip, depending on where the excitation beam is focused. It is this third harmonic signal that provides the necessary optical nonlinearity for pulse characterization. The third harmonic signal is most readily detected in the forward direction. Figure 7 shows a third-order intensity autocorrelation generated in this manner to measure the temporal characteristics of the pulse. Again the axial focus can also be characterized by simply scanning the coverslip along the z-direction. Third harmonic characterization performed in this manner is extremely achromatic it can be performed over essentially the entire tunability range of Ti:sapphire for example.

Figure 7. Interferometric third order intensity autocorrelation measurement for a 20 fs pulse at the focus of an objective. The excitation wavelength was 800 nm. Collinear background free methods for phase retrieval In general, this level of intensity measurement (autocorrelation of pulse in time, and z-spatial characterization) is suitable for most multiphoton imaging conditions and provides sufficient information enabling the researcher to optimize the pulse width, and focusing. However, it is clearly lacking in information all information of the pulse phase is lost for example. This can be rectified by spectrally resolving the third harmonic autocorrelation signal. In principle, the resulting spectrally resolved signal can be used to retrieve the phase of the pulse. However, this measurement is decidedly complicated by the interference fringes that are a result of the collinear nature of the measurement. Accurate pulse retrieval requires the generation of an exceptionally large data set that effectively renders this method impractical. Several methods for pulse characterization have been developed to circumvent this difficulty. For example, Amat-Roldan et al14, have recently shown that small starch particles can be used to produce a second harmonic signal that can be effectively used to characterize the pulse. The spectrally resolved second order intensity signal still has fringes but is under sampled, and software is used to filter the remaining fringes. This

method enables practical data acquisition and is particularly nice as it incorporates a broadband, inexpensive nonlinear media. The fringes can also be removed experimentally through several methods. One method involves using orthogonal polarizations in the interferometer. Orthogonal polarizations cannot interfere, and thus background-free, fringe free autocorrelations can be produced. Frequency resolved, second order intensity measurements have been made in this manner for pulses as short as 20 fs at the focus of 1.2 NA objectives15. This method requires the introduction of type II phase-matched crystals as the nonlinear mixing media, making it less attractive as a general pulse characterization method. A second, more generally applicable experimental approach is to use circularly polarized light and once again use the third harmonic signal generated from a glass coverslip. Pure circularly polarized light will not produce a third harmonic signal. This is readily accomplished by the addition of a single quarter wave-plate at the output of the interferometer (figure 8).

Figure 8. Collinear, background-free, fringe-free system for characterizing pulse intensity and phase at the focus of a high NA objective. The output of one arm of the interferometer is now right circularly polarized, the other arm is left circularly polarized. Neither arm will independently generate a third harmonic signal. When the pulses are overlapped in time, the region of overlap results in a linearly polarized signal that can produce third harmonic. As one pulse is swept past the other (in time) and the third harmonic signal is recorded a background-free autocorrelation measurement results. If the detector is polarization insensitive (such as a pmt) the signal is also fringe-free. Thus, at this stage we have a collinear, background-free, fringe-free third order intensity autocorrelation that can indeed be used to characterize the pulse width. To extract the pulse phase we need to spectrally resolve this signal. However, spectrometers often act as excellent polarizers. When we spectrally resolve the third harmonic signal we see the fringes emerge once again! Experimentally we can easily resolve this dilemma. By the addition of a quarter waveplate (which is centered at the third harmonic wavelength) at the entrance of the spectrometer the fringes are removed background-free, fringe-free traces result. Traces taken in this manner are shown in figure

9. By experimentally removing the fringes it is now only necessary to sample a modest grid (64 x 64) for reliable retrieval of the pulse phase information.

Figure 9. Right -experimentally measured FROG trace at the focus of a 0.65 NA objective. Horizontal axis is wavelength, vertical axis is time. Left extracted intensity and phase from the FROG trace. The pulse measures 100 fs, and is essentially transform limited as can be seen from the flat phase. Methods for Measuring Pulses in Space and Time Resolution in multiphoton microscopy is not limited solely by how tight a spatial focus we are able to produce. It is also strongly correlated to how efficiently we drive the optical nonlinearity that is responsible for generating our image contrast. The resolution ultimately relates to the number of photons that we are able to collect. Thus, in this form of microscopy optimal resolution is a four dimensional problem: we are concerned with how well the pulses are focused both in space and time. As you might imagine there is a strong coupling between these quantities spatial aberrations also manifest themselves as temporal distortions. In this sense the efficiency of the imaging process takes a double hit spatial aberrations distort our focus decreasing our intensity as a function of position, they also distort the temporal characteristics of our pulse, decreasing our intensity as a function of time. Up to this point we have only discussed how to characterize and extract the temporal phase of the pulse- is there a method for simultaneously extracting the spatial

phase so that we can examine, for our specific imaging conditions, the complete spatialtemporal behavior of our system? The answer is a resounding yes. Such coupling is conveniently quantified using spectral interferometry16, 17. This method has several practical advantages. It is a linear technique, and thus requires exceptionally low light levels. It is a single shot technique no scanning is required. An example of a spectral interferometer system for characterizing objectives is shown in figure 10a. The resultant interferograms are shown in figure 10b&c.
y
2D CCD

Spectrometer

Reference arm Ti:Sa oscillator 800 nm, 80 MHz Sample arm

y (in mm) Wavelength (in nm)

optics to characterize

(a)

(b)

(c)

Figure 10. a) Spectral interferometer system, b) measured and simulated spectral interferograms with chromatic aberration, c) measured and simulated spectral interferograms with spherical aberration. The vertical coordinate of the spectral interferogram is the spatial position of the entrance aperture to the objective. The horizontal coordinate is wavelength. The spectral interferogram can provide a measure of important aberrations such as spherical aberration or chromatic aberration as well as their impact on the temporal characteristics of the focus. In figure 10 both of these aberrations and the resultant spectral interferograms are shown. The spherical aberration manifests itself as a fourth order variation of the fringes as a function of position. The chromatic aberration is apparent from the relative change in the fringe spacing as a function of wavelength. Both of these aberrations impact the how the pulse is focusing in time. The complete spatial-temporal phase behavior is extracted through methods discussed elsewhere in this book. The extracted phase for the spectral interferogram shown in figure 10c is shown in figure 11.
1

100 90

Normalized intensity

Group Delay (in fs)

80 70 60 50 40 30 20 10 0

phase (in radians)

0.5

0 -3

-2

-1

-3

-2

-1

(a)

y (in mm)

(b)

y (in mm)

Figure 11. a) Spatial phase for the central wavelength extracted from 10c, b) is the extracted group delay.

Dispersion compensation for microscopy


As we have seen, to optimize the efficiency of a nonlinear optical process, it is desirable to maintain a transform-limited pulse at the focus of our imaging system. We have techniques to measure the pulsewidth what are the amounts of dispersion we are dealing with? If youre fortunate enough to have the exact lens design, you can use the techniques described in the previous chapter to explicitly calculate the amount of dispersion accumulated through the imaging system. More likely however, youll be using commercially available objectives, and that information is highly proprietary. In this case, it will be necessary to employ the pulse measurement technique of your choice to measure the pulse before and after propagation through the imaging system to empirically determine the dispersion of your imaging system. To provide a benchmark for the amounts of dispersion you can expect a table from Mller et al.18 is provided below. Typical amounts of dispersion from objectives of various numerical aperture Objective (All are from Zeiss) C-Apochromat 63x/1.2 W Corr C-Apochromat 40x/1.2 W Corr Neofluar 100x/1.3 Oil Neofluar 63x/1.25 Oil Neofluar 40x/1.3 Oil CP-Achromat 100x/1.3 Oil Measured Pulsewidth (fs) Full Width at Half Max 18 18.4 14.7 16.4 17.5 15.9 GDD (fs2) from a prism pair -1140 -1104 -778 -887 -1104 -579

For these measurements the input pulse was a 15 fs pulse centered at 800 nm. The GDD reported in the table is derived from the prism pair compensator spacing. The prism spacing was optimized based on the measured FWHM. Notably this is the dispersion of only the objectives no scan optics or tube lenses are included. Methods of dispersion compensation The net dispersion for an imaging system, collimation lenses, scan optics, and microscope objectives ranges on the order of 1000 fs2 to 5000 fs2. This is most typically compensated using a prism pair arranged to provide negative group velocity dispersion. Fused silica is the prism material of choice. While much more dispersive glass such as SF10 makes for a more compact prism dispersion compensator, these more dispersive glasses limit the compressed pulsewidth. For example, if youre trying trying to produce 20 fs pulses at the focus of an objective that has ~500 fs2 of dispersion, the pulsewidth will be limited to ~30 fs by the residual third order dispersion for a SF10 prism

compensator. Fused silica prism compensators have a significantly lower third order, and under the same conditions, make it possible to reach the 20 fs pulsewidth limit. One important point to consider in constructing a dispersion compensation system is throughput. Lets use the case of two photon absorption fluorescence imaging to illustrate why compensator throughput needs to be carefully considered. While the instantaneous signal scales as the square of the intensity, the actual detected signal scales only linearly with pulse width. This can be seen from the simple expression E2 E2 2 Pdet ected NI = N 2 2 = N 2 A A where P is the average power of the detected signal and is proportional to the product of N, the number of pulses per second, the pulsewidth, and I the intensity. If we evaluate the intensity in terms of the pulse energy E, pulsewidth , and area A, there is a cancellation of one of the pulse width terms: the net result is the detected signal exhibits only a linear dependence on pulsewidth. Thus if our compensator compresses our pulse by a factor of two, we detect a factor of two increase in our signal. Now consider the scenario where our pulse compensator measures a throughput of 70%. In this case, our pulse energy is decreased correspondingly, and our detected signal therefore decreases by 50%. If we consider both of these conditions simultaneously what is the net gain? If our pulse is compressed by a factor of two, and our pulse energy is reduced to 70%, any gain in signal due to the reduction of the pulsewidth is completely offset by the reduction in energy. Our net signal increase zero. In this instance, it is hardly worth implementing the pulse compensator.

Application: Imaging Photosystems


Photosynthetic organisms have highly organized cellular organelles, called chloroplasts, where light absorption harvesting and conversion of solar energy into chemical energy takes place. The chloroplasts consist of an elaborate membranous network with pigmentprotein complexes embedded in the photosynthetic membranes 19. The photosynthetic membranes have dynamic structure that is highly responsive to the illumination conditions 20. Therefore, there is a very close correlation between the photosynthetic activity and structural changes of the chloroplast. The functional activity of chloroplasts can be characterized via chlorophyll fluorescence, while the structure can be studied with optical microscopy. Thus, multicontrast non-linear microscopy opens a new opportunity for studying photosynthetic mechanisms simultaneously by investigating the functional activity with multiphoton excitation fluorescence (MPF) and imaging the structural dynamics with second and third harmonic generation microscopy 21. Higher plants and algae use chlorophyll and carotenoid molecules for light collection. Both chlorophylls and carotenoids have large transition dipole moment and high first and second order hyperpolarizabilies. Therefore, the photosynthetic pigments serve as native labels for fluorescence imaging. Moreover, the same pigments also serve as harmonophores. The harmonophores are used as labels for structural visualization with second and/or third harmonic generation microscopy. Each contrast mechanism gives complementary information about the photosynthetic structure. The multiphoton excitation fluorescence (MPF) is a non-coherent process; thus, MPF depends on the concentration of the pigments as well as on the fluorescence quenching properties of the photosynthetic systems. In contrast, the second and third harmonic generation is a

coherent process. The intensity of harmonic signals strongly depends on the spatial organization of the photosynthetic pigments. Depending on the organization, harmonic radiation from each pigment molecule will constructively or destructively interfere resulting respectively in a strong or weak far field harmonic signal 22, 23. Direct comparison of three images obtained with different contrast mechanisms enables to elucidate the structural organization of clustered pigment-protein complexes below the diffraction limited resolution. In the following, we will present the multicontrast nonlinear microscopy studies of several photosynthetic structures. Imaging aggregates of light-harvesting pigment-protein complex In higher plants, pigment-protein complexes are embedded in thylakoid membranes and laterally organized in highly ordered arrays 24. The thylakoids, vesicle like formations, are arranged into stacks providing a multilamellar organization of the pigment-protein complexes 19. When extracted out of the membrane with a detergent, one of the proteins, the major light-harvesting pigment-protein complex of photosystem II (LHCII) can be aggregated into multilamellar, semicrystalline or random aggregates 25. In particular, the multilamellar aggregates serve as a good model system for studying photophysical properties of the photosynthetic membranes. Figure 11 shows MPF, SHG and THG images of aggregated (LHCII) from higher plants. The fluorescence image reveals a heterogeneous structure of LHCII aggregate. The fluorescence intensity can not be directly assigned to the concentration of pigment-protein complexes. The quenching properties of the pigment-protein complexes also have to be accounted. None the less, fluorescence signal is a good reference point for determining the location of the pigmentprotein complexes. The fluorescence image can be directly compared with the SHG image (Figure 11 b). The SHG image from LHCII aggregate shows more distinct structural domains than fluorescence. The strength of SHG depends on the macroscopic arrangement of LHCII in the aggregate. For randomly aggregated pigment-protein complexes the SHG signal vanishes. However, when heterogeneities such as non-centrosymmetric lamellar formations, edges of the aggregate, or interfaces between differently arranged aggregate structures are introduced, the SHG signal arises. Strong SHG signal can be observed when semicrystalline three-dimensional aggregate with non-centrosymmetric order is obtained. In Figure 11b, SHG signal appears due to the heterogeneous structure of the aggregate. The domains of higher SHG intensity can be assigned to the ordered structures which give constructively interfering signals. By comparing the fluorescence and SHG intensities, it appears that most of the higher fluorescence domains show higher SHG as well. This might argue that concentration of the pigments is higher in the more intense SHG domains; however, even if the concentration of pigments is higher, the noncentrosymmetric order has to be present in the domain. Alternatively, highly ordered noncentrosymmetric aggregates show lower quenching than more disordered aggregates. It has been shown that aggregation induces significant fluorescence quenching 26. Therefore, the variation of order in the aggregate most likely influences quenching properties of the domains. The THG image of LHCII aggregates (Fig 11c) clearly reveals many structural domains. The structural domains are more pronounced and not always correlate with

SHG and MPF domains. The overall signal strength of THG appears to be higher than SHG or MPF. THG has no constrains on the symmetry of the aggregate, however the signal will not be generated in the homogeneous three dimensional media due to the phase anomaly or Gouy shift that the laser beam experiences when passing through the focus of the microscope objective 27. When heterogeneities are introduced in the focal volume such as interfaces between materials with different refractive index or third order non-linear susceptibilities, THG signal emerges in the far field 28, 29. Some parts of the aggregate, which are highlighted by the fluorescence, do not show appreciable THG. This indicates that areas with low THG intensity, as well as SHG intensity, are randomly organized. The LHCII domains that show high THG intensity and have low SHG are probably organized in a multilamellar arrangement with the lamellae planes oriented perpendicular to the laser beam. The multilamellar arrangement enhances THG signal, while absence of centrosymmetric order in the lamellae as well as lack of twisting of the membrane planes across the lamellar stack gives no basis for SHG. On the other hand, the areas highlighted by SHG but not THG have non-centrosymmetric domains arranged in a continuous three dimensional structure. Alternatively, the multilayer membranes with the planes parallel to the propagation of the beam may not produce THG, while giving strong SHG and fluorescence signal. The structures exhibiting high SHG, THG and fluorescence possess interfaces oriented perpendicular to the beam propagation as well as non-centrosymmetric organization in the domains. The results obtained from imaging LHCII aggregates are very helpful in understanding the structural organization of chloroplasts. Imaging chloroplasts Chloroplasts can be imaged in situ and in vivo with non-linear microscopy 28, 30-32. The photosynthetic membranes of chloroplasts are comprised of various chlorophylls and carotenoids containing pigment-protein complexes. One of such complexes is LHCII. Therefore, the results obtained from imaging LHCII aggregates can be used for understanding the multicontrast microscopic images of chloroplasts. Chloroplasts are comprised of complex network of photosynthetic membranes. The multillamellar stacks of photosynthetic thylakoid membranes are called grana19. The grana are interconnected via stroma membranes into large membranous network filling the space of the whole chloroplast. The fluorescence in chloroplasts originates both from grana structure as well as stroma membranes. Figure 12 shows a chloroplast imaged with multicontrast nonlinear microscope. The image was recorded using Yb:KGW laser radiating at 1030 nm 33. The Yb:KGW laser excites preferentially carotenoids via two-photon excitation process 34 . The excitation from carotenoids is efficiently transferred to chlorophylls that emit fluorescence at wavelengths peaking around 680 nm. The fluorescence image (Fig 12 a) shows relatively homogeneous distribution of intensity over the entire volume of the chloroplast. The image was recorded by collecting red fluorescence between 630 and 720 nm. At these wavelengths, the fluorescence from photosystem II residing in the grana and photosystem I found in the stroma were not resolved. The SHG images of chloroplasts often show relatively homogeneous intensity over the whole area except of high intensity spots that can be attributed to starch granules 35 (Fig 12 b). In the light chloroplasts synthesize starch molecules, which crystallize into

granules with concentric layered structure 36. The starch molecules lack inversion symmetry, therefore generate strong SHG signal. However, due to the concentric arrangement of starch, SHG vanishes at the center of the granule. The SHG radiation destructively interferes from the oppositely arranged starch molecules in the granule. Away from the center, the arrangement of starch in the focal volume changes into parallel ordered layers, which generate strong second harmonic. The starch granules appear as double spots in the SHG image (Fig 12b). The double spots of SHG can be readily observed at high resolution with oil immersion objective. If plants are kept in the dark for several hours, the starch granules get consumed. The SHG image of dark adapted chloroplasts does not show characteristic intense spots of starch granules (Fig 12 e). The SHG signal of dark adapted chloroplasts appears to be not very strong, somewhat similar to LHCII aggregates. While single membranes might produce strong SHG due to chiral organization of the pigments reveled by anomalous circular dichroism measurements 37, alternating arrangement of stacked thylakoid membranes diminishes SHG. The destructive interference of SHG from each thylakoid membrane in the grana appears when the planes of the membranes are oriented parallel with respect to the laser beam propagation direction (case of edge aligned chloroplasts). Therefore, SHG signal from the grana and stroma membranes appears to have very similar intensity. The most pronounced structural details inside the chloroplasts can be observed with THG contrast mechanism 21, 32, 38. Since starch does not produce appreciable THG, the structures of light and dark adapted chloroplasts appear very similar (Fig. 12 c, f). It has been shown that multilayer structures enhance THG as compared to corresponding monolayer interfaces 39. Therefore, the multilamellar structures of the grana in chloroplasts, similarly to the LHCII aggregates, enhance the intensity of THG. The orientation of membranes perpendicular to the excitation beam propagation direction renders the largest THG signal. Although for the edge aligned chloroplasts, the thylakoid membranes in the grana are oriented parallel to the beam propagation direction, none the less, appreciable signal of THG is observed (Fig. 12 c, f). This apparently is due to the interface between the grana structure and stroma region. The THG signal is resonantly enhanced by carotenoid molecules. Carotenoid molecules have high second order hyperpolarizability 40. The two-photon absorption of carotenoids at the excitation wavelength of 1030 nm is high; therefore resonance enhancement of THG signal is anticipated. The multicontrast imaging provides rich information about the structure of chloroplast. The structural organization can be deduced from the relative strength of the signal generated by different contrast mechanisms. By recording non-linear signals of chloroplasts at different orientations a three dimensional organization of the grana can be obtained. This allows deducing granum ultrastructure below the diffraction limited resolution of optical microscope. More over, the intensity variation of different non-linear responses provides information about the dynamic changes of pigment-protein organization in the photosynthetic structures. The naturally pigmented photosynthetic organelles give strong non-linear signals. Therefore, multicontrast imaging microscopy has a great potential for studying in vivo and in situ photosynthetic structures.

MPF

SHG

THG

2 m

2 m

2 m

Figure 11. LHCII aggregates imaged with multiphoton excitation fluorescence (a), second harmonic generation (b), and third harmonic generation (c) microscopy. The arrows indicate regions of interest where THG shows weak and strong signals corresponding to strong and weak SHG and MPF signals, respectively.

MPF

SHG

THG

2 m

a MPF

b SHG

c THG

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Figure 12. Light treated (a, b, c) and dark adapted (d, e, f) chloroplasts imaged with multiphoton excitation fluorescence (a, d), second harmonic generation (b, e) and third harmonic generation (c, f) microscopy. The intense double spots in SHG image (b) appears due to starch which is absent in dark adapted SHG image (e). The chloroplasts are edge aligned with thylakoid membranes oriented perpendicular to the image plane.

Application: CARS imaging


Studying the vibrational modes of molecules can provide a wealth of information about the specimen of interest. The vibrational frequency is determined by both the mass of the atoms involved and the bond strength. Weakly bound heavy atoms vibrate at low frequencies, whereas strongly bound light atoms vibrate at high frequencies. Thus the vibrational frequency provides information about the chemistry of the molecule. In addition, intermolecular interactions may affect the frequency and amplitude of specific vibrational modes. Molecular vibrational frequencies typically range from ~500 to ~4000 cm-1, where the energy is expressed is 1/ , with in cm. For biomolecules, the region around 3000 cm-1 is dominated by CH, OH and NH stretch vibrations. In the region between ~750-1800 cm-1 one finds for instance CC and C=O stretch vibrations, but also bending modes and vibrational modes that involve more than two atoms of the molecule. This region - also know as the "fingerprint" region - can be used as a spectral signature for the molecule. Unique to this area of spectroscopy is that many of the spectral features remain resolvable even at room temperature and in complex samples such as live cells and that it does not require any form of labeling. Traditionally, information on molecular vibrations is obtained using spontaneous Raman scattering or infrared absorption. Both of these methods have serious drawbacks for applications in microscopy. Raman scattering is typically very weak, thus requiring (very) long acquisition times. In addition natural luminescence from the sample may readily overwhelm the weak Raman signal. Infrared absorption requires long wavelengths (5-15 m range), which significantly limits the attainable resolution. Also water absorption of the infrared light may be a problem in this case. CARS - a non-linear optical analogue of Raman scattering The Raman signal can be enhanced significantly (typically by ~4 orders of magnitude) using a coherent non-linear interaction. Coherent anti-Stokes Raman scattering (CARS) is a third-order non-linear process in which three laser beams coherently interact with the sample to generate a coherent signal beam (see Fig. 1a). More specifically, a pump beam with frequency pu , and a Stokes beam with frequency s , set-up a grating off which a third laser beam (denoted by probe, with frequency pr ) undergoes a Bragg diffraction, generating an anti-Stokes signal beam at frequency as = pu s + pr . The non-linear process is resonantly enhanced when the difference frequency between pump and Stokes matches a vibrational energy level in the sample ( pu s = vib ). In a plane wave approximation and for a non-absorbing medium, the CARS signal strength is given by 41:

2 I CARS (3) I pu I s I pr sinc 2 k d 2

(1)

where I denotes the intensity of the lasers and CARS signal, d is the thickness of the scattering volume, sinc ( x ) = ( sin x ) x and k = kas k pu + ks k pr describes the phase mismatch between the wave vectors k. It has been shown 42, 43 that for high numerical aperture focusing conditions the phase matching condition is less stringent. A collinear beam geometry ( kas = k pu = ks = k pr ) is therefore commonly used to achieve maximum spatial resolution in microscopy applications. Also for practical reasons pump and probe are generally derived from the same laser source ( pu = pr and I pu = I pr ). (3) is the thirdorder non-linear susceptibility which is composed of two contributions (see Fig. 1):
(3) (3) (3) = R + NR

(2)

where the non-resonant term ( NR ) is independent of frequency and real, while the (3) resonant term - R = j R j ( as pu j i j ) - is a complex sum over all involved vibrational resonances j , with eigenfrequency j , oscillator strength R j , and linewidth j . Most often two picosecond lasers are used for CARS microscopy (see 44, 45 for recent reviews). The bandwidth of these lasers (~1-3 cm-1) is narrow compared to the typical linewidth of vibrational modes in the condensed phase ( 10 cm-1) and determines the spectral resolution. In this mode of operation - also known as single-frequency CARS - a single point in the vibrational spectrum is acquired for a specific setting of the lasers, and the Stokes laser has to be tuned to acquire the whole spectrum. Alternatively, in multiplex CARS (Fig. 13b) a narrow-band pump and probe laser is mixed with a broad-band (femtosecond) Stokes laser. In this case the vibrational spectrum is acquired simultaneously over a range of frequencies determined by the bandwidth of the Stokes laser. The spectral resolution is determined by the bandwidth of the pump and probe laser. The characteristics of CARS outlined above, result in a number of features that are especially suitable for high resolution vibrational microscopy: (i) It is a non-linear optical technique that provides signal levels that are many orders of magnitude (typically 3-4) stronger than attainable for spontaneous Raman scattering. (ii) Near-infrared wavelengths can be used that ensure sub-micron optical resolution and minimal sample heating and damage. (iii) The non-linear dependence of the CARS signal on input laser intensity provides inherent optical sectioning, i.e. three-dimensional imaging capability. Finally, (iv) the signal propagates coherently and is of shorter wavelength than all the input fields making it readily detectable in the presence of a fluorescence background. These features, combined with recent short-pulse laser enabling technology, have made CARS microscopy to what it is today: a three-dimensional microscopic tool with unique applications in biology, biophysics and the material sciences46, 47 48-57.

Figure 13. Energy diagrams for the resonant (a and b) and non-resonant (c and d) contributions to the CARS signal. Single-frequency CARS (a) uses narrow-band picosecond lasers and yields a single point in the CARS spectrum. Multiplex CARS (b) on the other hand combines a narrow-band pump and probe laser with a broad-band (femtosecond) Stokes laser. In this configuration the CARS signal is generated simultaneously over a significant range of the spectrum. The non-resonant background - suppress it or use it To generate contrast in microscopy the signal from the molecules or structures of interest need to be discriminated from other contributions to the signal. For low concentrations of the molecular species of interest, the purely resonant contribution to the (3) 2 (3) 2 (3) (3) << NR + 2 NR Re { R signal ( R } , see Eq. 1 and 2) becomes negligible and the CARS signal is dominated by the non-resonant contribution. Clearly, laser-induced fluctuations (such as pulse-to-pulse power fluctuations, timing jitter between pump and Stokes laser (3) (3) pulses, etc.) limit the detectability of the small resonant contribution ( 2 NR Re { R } ) on
(3) top of a large - and noisy - non-resonant background ( NR ). Therefore, various 2

approaches (using, for instance, specific polarization schemes 58, 59, time-resolved measurements 60 or through combined phase- and polarization control 61-63) have been developed to suppress the non-resonant background. Alternatively, the CARS signal can be detected in the backward (epi) direction. It has been be shown 64, 65 that for samples with a thickness smaller than the wavelength the resonant CARS signal is generated both in forward and backward direction. The nonresonant signal on the other hand, which generally results from the - much thicker - bulk of the sample - is generated primarily in the forward direction. In multiplex CARS microscopy 48, 52, 66 on the other hand, the total CARS signal is detected in the forward direction with a spectrum dominated by the cross term ( 3) ( 3) ( 2 NR Re { R } ) giving rise to the characteristic dispersive lineshape. In multiplex CARS the non-resonant contribution boosts the resonant contribution in a heterodyne fashion. Since the signal is detected simultaneously over a significant spectral range the spectral signal-to-noise ratio (SNR) is shot-noise limited. Single-frequency CARS generates the CARS signal most efficiently, because of the optimized temporal overlap of the two lasers at one particular Raman shift. A clear

advantage of this approach is that it permits very rapid image acquisition in CARS microscopy50, 53. In multiplex CARS the spectrum is acquired over a significant range of vibrational frequencies simultaneously. The signal-to-noise ratio of the spectra is therefore independent of laser-induced fluctuations in the signal. This generally results in high quality spectral data over a large spectral range 57, 67. CARS imaging - chemical and physical specificity without labeling In one of the first demonstrations of CARS microscopy 43 the feasibility to apply this technique to living cells was demonstrated. In this work - and in other work on (live) cells - contrast was based on C-H stretchng vibrations, which resulted mainly from accumulated lipid 50, 68, 69. In more recent experiments, it has been shown that CARS microscopy can achieve a sensitivity that enables detection of a single lipid bilayer 70 or even monolayer 52. Also other important cellular components have been observed using vibrational contrast, such as chromosomes 69, nucleic acids and proteins 65, 71, 72. It should be realized that since these objects show negligible linear absorption at the employed laser wavelengths, temperature induced damage is unlikely even at the substantial laser powers used 73, 74. An appealing demonstration of the potential of CARS microscopy for biological research is that by Nan et al. 50, which is shown in Fig. 14. In this application CARS microscopy is used to image the distribution of lipid droplets in time. Lipid droplets are the cellular stores of triacylglycerol and cholesterol esters, and represent the largest energy reserve in the body. These structures are densely packed with lipids, and therefore readily imaged by CARS microscopy. Fibroblast cells can be induced to convert to fat cells. The most obvious characteristic of this process is the accumulation of lipid droplets in cells. First a reduction of lipid droplets is observed 24 hours after adding the induction medium (Fig. 14B), and at 48 hours most of the cells contained few or no lipid droplets (Fig. 14C). After that, cells start to accumulate lipid droplets and eventually become adipocyte (fat) cells.

Figure 14. CARS images taken at 2845 cm-1 of the same a fibroblast (3T3-L1) cell culture at different times after adding induction media: 0 h (A), 24 h (B), 48 h (C), 60 h (D), 96 h (E), and 192 h (F). (Reproduced with permission from 50.) The properties of water within cells or in close vicinity to biomolecules are of great interest to both biology and biophysics. Using multiplex CARS microscopy, it has been shown 67 that interstitial water in between the bilayers of a multilamellar lipid vesicle shows a hydrogen bond structure which is significantly different from that of bulk water. Using the dependence of the CARS signal strength upon the relative orientation of the molecular Raman tensor and the polarization of the laser fields 54, the orientational order of the water molecules was also analyzed. The interstitial water molecules were found to be almost isotropically distributed 67. In another study, CARS microscopy was used to map the diffusion coefficient of water within a cell 75. Regions of severely restricted water mobility were clearly identified. Using a related technique - based on the nonlinear optical Kerr effect - Potma et al. also showed that the rotational mobility of water molecules is significantly lower within intact mammalian cells than in pure water 76 . Although most applications to date are within a biological or biophysical context, CARS microscopy also has clear potential in other areas of research, such as the material sciences. In recent experiments, Potma et al. have demonstrated the potential of the technique - in terms of chemical specificity, high spatial resolution ( 270 nm) and short acquisition time - to image lithographically imprinted patterns of polymer photoresist 51 (Fig. 15).

Figure 15. Epi-detected CARS images of a patterned TBOC film. The sample was scanned axially and laterally through focus yielding a xz cross-section of the polymer film at a Raman shift of (a) 880 cm-1 which is resonant with the tert-butoxyl vibration and (b) 925 cm-1 which is non-resonant. Images are 20.0 5.0 m (x,z) and consist of 256 64 pixels. The dwell time is 0.3 ms. The difference image (a-b) is given in (c), along with a one-dimensional integration of image (c) along the z axis, the result of which is given in (d). (Reproduced with permission from 51.) The full richness of CARS microscopy is explored when a full CARS spectrum is recorded at high SNR. Detailed chemical identification or physical characterization of the physical state of the sample often requires the analysis of spectral line shapes, relative peak heights of different vibrational features, etc. Resolving these features in singlefrequency CARS is limited by laser induced fluctuations. Spectra with unprecedented signal-to-noise can be recorded, however, using multiplex CARS microscopy. An example of this is shown in Fig. 16a which shows CARS spectra of a single lipid bilayer in the liquid crystalline (lower trace) and gel phase (upper trace) respectively. From these

spectra the lipid chain order - defined as the ratio of the signal intensity from the antisymmetric and symmetric methylene stretching modes respectively - can be determined to increase from 0.69 0.03 in the liquid crystalline phase to 0.79 0.04 in the gel phase. Since in multiplex CARS all spectral channels are recorded simultaneously, the resonant contribution can be determined quantitatively against the non-resonant background. This is demonstrated in Fig. 16b where the signal from a lipid bilayer shows a signal strength which is exactly twice that of a lipid monolayer.

Figure 16. (a) Multiplex CARS signal from a liquid crystalline (1,2-Dioleoyl-sn-glycero3-phosphocholine; lower curve) and gel phase (1,2-Dipalmitoyl-sn-glycero-3phosphocholine; upper curve) lipid bilayer. (b) Multiplex CARS signal from a lipid mono- (upper curve) and bilayer (lower curve) of 1,2-Dipalmitoyl-D62-sn-glycero-3phosphocholine. See 52 for experimental details. (Reproduced with permission in modified form from 52.) Vibrational contrast opens a unique window in three-dimensional microscopy. Since vibrational specificity remains even at room temperature and in complex environments, this approach can provide imaging contrast based on either the molecular chemical identity or the physical state of the sample. CARS permits the use of this spectroscopic approach in high resolution microscopy. It is safe to assume that the rapid development in ultrashort pulse laser technology will permit widespread use of this technique. Already researchers are combining CARS with methods known from fluorescence microscopy, such as near-field optical microscopy 77-79 and correlation spectroscopy 80, 81. This new technology opens exciting avenues for important research questions in biology, physics, biophysics and the material sciences.

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