Name: femtosecond laser filaments for use in sub-diffraction-limited imaging and remote sensing
Uploaded: 2019-04-25T12:30:00.000+00:00
Duration: 6 min 16 s
Description: Watch this Scientific Journal Video about Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing at JoVE.com

The research is supported by the Office of Naval Research (ONR) (Award N00014-16-1-2578 and N00014-16-1-3054), Robert A. Welch Foundation (Grant No. A-1547, No. A-1261), Air Force Office of&#160;Scientific&#160;Research (Award No. FA9550-18-1-0141), SMART Fellowship and a grant from King Abdulaziz City for Science and Technology (KACST).

1. Creation of the Femtosecond Laser Filament
<ol>
	<li>As femtosecond filaments require the output of a Class 4 laser, wear appropriate eye protection rated for the particular laser system in use and establish a clear and well-defined beam line with an appropriate beam dump. Follow all standard laser safety procedures.</li>
	<li>Begin with the output of a pulsed, amplified femtosecond laser whose instantaneous output power is greater than or equal to the critical power for self-focusing in air, about 3.2 GW for a Ti:Sapphire laser at 800 nm wavelength. Generate the amplified pulse in a commercial femtosecond laser amplifier system using manufacturer&#8217;s protocol. In practice, pulse energy of about 1 mJ for an approximately 35 fs pulse is sufficient. Good results are obtained with pulse energy of 2-4 mJ.</li>
	<li>Pass the laser beam through an iris that slightly clips the outer edges. It is observed to promote filament formation, since filament formation is known to be seeded by sharp gradients and inhomogeneity in the spatial intensity profile of the laser.</li>
	<li>Pass the beam through the converging lens that has a focal length of approximately 200 cm or greater, so that the geometric focusing is not so great that self-focusing is overwhelmed by optical breakdown or diffraction. Slightly tilt the lens with respect to the direction of propagation, since additional anisotropy is known to help seed the self-focusing process.</li>
	<li>Observe a filament at a location near the geometric focus of the lens. Diagnose filamentation by a diffuse (several-mm-sized) halo surrounding a bright (approximately 100-&#181;m-sized) core. The halo could be seen on a white paper and the bright cores usually flicker.
	<ol>
		<li>Additionally, observe a characteristic self-phase modulation process in the air, which produces bright, multi-colored conical emission rings that are visible beyond the filament. For lasers with energies which are several times the threshold for filamentation, multiple filaments are observed. These are visible as multiple bright spots in the conical emission pattern, and can be eliminated by attenuation before the iris.</li>
	</ol>
	</li>
</ol>
2. Remote Scanning of the Target Surface
<ol>
	<li>Put a two-axis motorized translation stage capable of moving the sample in the direction transverse to the propagation of the laser beam on the table. Ensure that the laser beam is incident on the center of the stage. Bolt the stage on the table with screws. For laboratory purposes, it is generally easier to keep the laser beam fixed in space while scanning the target under the beam.</li>
	<li>Place sand in a container (5 mm x 25.4 mm x 25.4 mm). The thickness of sand is around 2 mm.
	<ol>
		<li>Put the metals (copper, stainless steel, aluminum) on the top of sand (Figure 3a). Cover the metals with another 2 mm layer of sand (Figure 3b).</li>
		<li>With the laser off, put the container in the center of the translation stage. Make sure that the center of container is at the location where filamentation is observed for step 1.1-1.5.</li>
	</ol>
	</li>
	<li>Set up the laser&#8217;s computer control to fire a single shot when electronically commanded. Write a LabVIEW or a similar computer language to perform the control. For automated single-shot pulses, an external trigger is required.
	<ol>
		<li>Connect a trigger TTL pulse to the External Trigger port on the back of the laser control module with a BNC cable. Enable the external trigger option on the laser control module. The TTL pulse will now trigger the laser to fire a single shot.</li>
	</ol>
	</li>
	<li>Set up the appropriate sensor apparatus. Set up the entrance of the spectrometer pointing to the impact point.
	<ol>
		<li>Use a lens to couple the light from filamentation impact point into a spectrometer. Make sure that the distance between the lens and filamentation is about the focal length.</li>
		<li>Connect the spectrometer with computer using USB cable. Use software to monitor the spectrum. Open the software and the spectrum, and then click the Run button.</li>
		<li>Use the mouse to zoom in the range that is recorded in the experiment. Optimize the spectrometer position after seeing the signal on the screen.</li>
		<li>For imaging measurements, replace the spectrometer with a photomultiplier tube or a CCD camera.</li>
	</ol>
	</li>
	<li>Write a program in LabVIEW or a similar computer language to perform a loop over the following steps: Fire a single shot from the laser; collect and save the resulting data; move the translation stage to the next coordinate point.</li>
</ol>

<ol>
	<li>Kocharocsky, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-swept+superrandiance+applied+to+the+stand-off+detection+of+trace+impurities+in+the+atmosphere.">Gain-swept superrandiance applied to the stand-off detection of trace impurities in the atmosphere.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (22), 7806-7811 (2005).</li><li>Hemmer\, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standoff+spectroscopy+via+remote+generation+of+a+backward-propagation+laser+beam.">Standoff spectroscopy via remote generation of a backward-propagation laser beam.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (8), 3130-3134 (2011).</li><li>Zuber, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mars+Observer+laser+altimeter+investigation.">The Mars Observer laser altimeter investigation.</a> Journal of Geophysical Research. 97, 7781 (1992).</li><li>Mejean, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+and+identification+of+biological+aerosols+using+a+femtosecond+terawatt+lidar+system.">Remote detection and identification of biological aerosols using a femtosecond terawatt lidar system.</a> Applied Physics B: Lasers and Optics. 78 (5), 535-537 (2004).</li><li>Boyd, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nonlinear Optics. , (2008).</li><li>Gattass, R. R., Mazur, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+micromachining+in+transparent+materials.">Femtosecond laser micromachining in transparent materials.</a> Nature Photonics. 2, 219-225 (2008).</li><li>Tognoni, E., Palleschi, V., Corsi, M., Christoforetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+micro-analysis+by+laser-induced+breakdown+spectroscopy:+a+review+of+the+experimental+approaches.">Quantitative micro-analysis by laser-induced breakdown spectroscopy: a review of the experimental approaches.</a> Spectrochimica Acta Part B: Atomic Spectroscopy. 57 (7), 1115-1130 (2002).</li><li>Beadie, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+FAST+CARS+anthrax+detector:+coherence+preparation+using+simultaneous+femtosecond+laser+pulses.">Toward a FAST CARS anthrax detector: coherence preparation using simultaneous femtosecond laser pulses.</a> Optics Communications. 244, 423-430 (2005).</li><li>Scully, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAST+CARS:+Engineering+of+a+laser+spectroscopic+technique+for+rapid+identification+of+bacterial+spores.">FAST CARS: Engineering of a laser spectroscopic technique for rapid identification of bacterial spores.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (17), 10994-11001 (2002).</li><li>Pestov, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+laser-pulse+configuration+for+coherent+Raman+spectroscopy.">Optimizing the laser-pulse configuration for coherent Raman spectroscopy.</a> Science. 316 (5822), 265-268 (2007).</li><li>Braun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-channeling+of+high-peak-power+femtosecond+laser+pulses+in+air.">Self-channeling of high-peak-power femtosecond laser pulses in air.</a> Optics Letters. 20 (1), 73-75 (1995).</li><li>Couairon, A., Mysyrowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+filamentation+in+transparent+media.">Femtosecond filamentation in transparent media.</a> Physics Reports. 441, 47-189 (2007).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+sub-diffraction+imaging+with+femtosecond+laser+filaments.">Remote sub-diffraction imaging with femtosecond laser filaments.</a> Optics Letters. 37 (8), 1343-1345 (2012).</li><li>Strycker, B. 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No conflicts of interest declared.

The method presented above is a laboratory protocol for the use of high-intensity laser light delivered at classically intractable distances. Of the numerous possible applications of such light &#8211; CARS, FIBS, THz radiation, photoacoustics, superradiance, etc. &#8211; many applications can deliver point information about surface material properties. Femtosecond laser filaments with sub-classical-diffraction-limited spot size allows use of these techniques while scanning the surface on a point-by-point basis. This protocol is an ideal test bed for the development of such techniques.
The most critical aspect of the protocol is to generate the laser filamentation. To generate the stable laser filamentation, the critical laser intensity is a few 1013 W/cm2 and the clamped intensity is around 1.4x1014 W/cm2 measured in experiment16. There is no laser filamentation when the intensity is either high or low. If the intensity is too high, the medium might be ionized strongly at the focal point and a laser induced break-down will happen. A bright spark instead of a laser filamentation will be observed. In that case, attenuate the power or use a lens with a longer focal length. Conversely, if the power is low (no plasma generation is observed), increase the power or use a lens with short focal length. Moreover, in either case, it is worthwhile to adjust the chirp to help to form a laser filamentation.
This scanning technique is generally better suited for laboratory use and proof-of-concept rather than field deployment since remote sensing in the field generally does not allow fine translation-stage control of the target under investigation. In those scenarios the same lab-developed laser techniques can be used, but the laser itself will have to be scanned through more traditional beam steering methods such as changing the orientation of the laser apparatus itself.
The protocol could be relatively easily extended to involve experiments with multiple filaments, filament bundles, pump-probe experiments, standoff spectroscopy, waveguide, or numerous other possibilities. In each case, one of the major experimental hurdles is the alignment of the intersecting focal spots, but with this protocol, this need only be done once. The optical elements are fixed in place and the sample itself is the only object required to move. This can be done very precisely with a translation stage. Further modification of this protocol to achieve further control over the location of the filament formation distance, including filament formation at hundreds of meters from the laser, is possible in principle by careful control of the output laser pulse. Multi-filamentation will also form a waveguide during the propagation, which could help to deliver a light in free space.
Remote sensing is a broad subject that spans disciplines such as physics, chemistry, engineering, environmental science, etc. In the supplementary material, we propose additional remote sensing schemes including stand-off spectroscopy and superradiance in addition to filamentation.

The spatial coherence and corresponding small divergence angle of laser beams have led to numerous applications in remote sensing, including chemically-sensitive measurements of the atmosphere1,2, range-finding3, and remote spectroscopy4. The same coherence properties allow very tight focusing of laser light that can deliver continuous focused intensities of billions of watts per square centimeter and pulsed intensities of 1013 watts per square centimeter over a period of a few femtoseconds. Such extreme intensities are useful for numerous applications including examining the nonlinear optical properties of matter5, precision optical micromachining6, materials characterization through laser-induced-breakdown spectroscopy7, stimulated Raman spectroscopy8,9,10, and trace chemical detection11.
However, the physical limitations of Gaussian beams set limits on the ability to apply these properties of extreme intensity and small divergence angle simultaneously. A laser beam focused to a small spot size will necessarily diverge with a greater angle. Classically, the beam divergence angle is given by , where &#955; is the wavelength and w0 is the radius of the beam waist. Since the divergence angle is set by the diameter of the laser beam and the focal length f of the focusing lens, , and tight focusing is not possible at distances of many meters as f becomes large compared to d.
Workers in the field of amplified femtosecond pulses noticed that this limitation on intensity vs. range was violated for high-intensity femtosecond pulses, with burn marks smaller than the diffraction limit appearing on targets at large distance from the originating laser12. This was found to be due to Kerr-effect self-focusing. The refractive index of the air is modified in proportion to the intensity of the laser field, and when the laser has a Gaussian intensity profile, the resulting refractive intensity profile becomes functionally a lens5. The beam self-focuses as it propagates, resulting in a narrow and intense filament of less than 100 &#181;m radius whose small size is maintained by a dynamic balance among classical diffraction, Kerr self-focusing, and defocusing due to plasma generation13.
With femtosecond laser filaments, intensities on the order of 1013 W/cm2 can be delivered to targets at distances of many meters with commercially-available femtosecond chirped-pulse amplifiers. Thus, many experiments which previously required tight focusing conditions and targets very close to a lens of high numerical aperture can now be done at distance more typical of remote sensing applications. However, intensities much higher than this threshold are not easily possible with filamentation, as the beam tends to break up into multiple filaments where each individual filament is near the critical power for self-focusing13.
Numerous applications are possible. We present a protocol primarily applicable to imaging and spectroscopy of remote targets using a femtosecond laser filament scanned over the target surface. The experimental setup is shown in Figure 1.

<table><tbody><tr><td>Femtosecond laser system</td><td>Coherent Co</td><td>Legend Elite System</td><td>1 kHz system, fs system pulse energy 4 mJ</td></tr><tr><td>IRIS</td><td>Thorlabs</td><td>id25</td><td>Mounted Standard Iris, &amp;Oslash;25.0 mm Max Aperture, TR3 Post</td></tr><tr><td>Lens</td><td>Thorlabs</td><td>LA1908-C</td><td>L=50 cm, Plano-Convex Lenses (AR Coating: 1050 - 1700 nm)</td></tr><tr><td>Mirrors</td><td>Thorlabs</td><td>PF10-03-P01</td><td>Plano metallic mirror</td></tr><tr><td>Photodetector</td><td>Hamamatsu</td><td>H12694</td><td>Thermoelectric cooled NIR-PMT unit</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>OCEAN_HDX_VIS_NIR</td><td>Spectrometer, high dynamic range, 350-950</td></tr><tr><td>Translation Stage</td><td>Thorlabs</td><td>PT3-Z8</td><td>25 mm (0.98&quot;) Three-Axis Motorized Translation Stage, 1/4&quot;-20 Taps</td></tr></tbody></table>

femtosecond laser filaments for use in sub-diffraction-limited imaging and remote sensing

Probing remote matter with laser light is a ubiquitous technique used in circumstances as diverse as laser-induced breakdown spectroscopy and barcode scanners. In classical optics, the intensity that can be brought to bear on a remote target is limited by the spot size of the laser at the distance of the target. This spot size has a lower bound determined by the diffraction limit of classical optics. However, amplified femtosecond laser pulses generate intensity sufficient to modify the refractive index of the ambient air and undergo self-focusing. This self-focusing effect leads to the generation of highly intense laser filaments which maintain their intensity and small sub-millimeter diameter size at distances well beyond the classical Rayleigh length. Such intensity provides the capability of remote scanning, imaging, sensing, and spectroscopy with enhanced spatial resolution. We describe a technique for generating filaments with a femtosecond regenerative chirped-pulse amplifier, and for using the resulting filament to conduct imaging and spectroscopic measurements at remote distances of at least several meters.

The resolution of the scanned images is limited optically only by the ~100 &#181;m. Therefore, the translation stage motion should be of this order of magnitude or smaller for maximum resolution. However, this level of resolution is not necessary for all measurements. This protocol has been used for both imaging14&#160;and spectroscopic15&#160;measurements. Figure 1 shows the experimental setup. The pulse is generated in an amplifier system. The pulse is 1 kHz, 50 fs, and centered at 800 nm. Figure 2 compares a scan of a small Texas A&#38;M logo target taken with a laser at the diffraction limit compared to a scan taken with a filament-forming beam. This experiment was performed using filaments in liquid water, but the results may be rescaled for air in remote sensing13. Figure 3 shows spatially-resolved filament-induced breakdown spectroscopy scans of metal objects of different composition buried approximately two millimeters below a layer of sand. The shapes and compositions of the metal objects are apparent. In general, filamentation provides a number of mechanisms for target effects. The initial pulse can provide information on the surface layer, while subsequent pulses can provide information on deeper portions of the material through ablation or mechanical removal of surface layers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58207/58207fig1.jpg" /> 
Figure 1. The experimental setup. The laser is 1 kHz, 50 fs, and centered at 800 nm. It is focused with a lens to reach the intensity (~1013 W/cm2) to form laser filaments. The object is under sand and put on a translation stage. The scattered light is collected with a spectrometer. <a href="https://www.jove.com/files/ftp_upload/58207/58207fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58207/58207fig2.jpg" /> 
Figure 2. Sub-diffraction-limited Imaging. Remote images generated by scanning a laser beam across a printed Texas A&#38;M logo at a distance of several meters. a) Logo imaged with non-filamented beam. b) Logo imaged with filamented beam. <a href="https://www.jove.com/files/ftp_upload/58207/58207fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58207/58207fig3.jpg" /> 
Figure 3. The chemical map. Spectrally and spatially resolved image of metal objects buried under sand. a) Objects above sand. b) Objects below 2.3 &#177; 0.3 mm of sand. c) Image with material composition color-coded to metal spectral features. Composite image of the buried objects with aluminum (Al), copper (Cu), and stainless steel (SS) corresponding to the red, green, and cyan color components, respectively <a href="https://www.jove.com/files/ftp_upload/58207/58207fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing at JoVE.com

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing

High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

Probing remote matter with laser light is a ubiquitous technique used in circumstances as diverse as laser-induced breakdown spectroscopy and barcode ...

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7.5K Views.  Texas A&M University. High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

Video: Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb explosion imaging setup in which a hitherto-impractical camera angle is realized. In our imaging technique, diatomic molecules are irradiated with a circularly polarized strong laser pulse. The ejected atomic ions are accelerated perpendicularly to the laser propagation. The ions lying in the laser polarization plane are selected through the use of a mechanical slit and imaged with a high-throughput, 2-dimensional detector installed parallel to the polarization plane. Because a circularly polarized (isotropic) Coulomb exploding pulse is used, the observed angular distribution of the ejected ions directly corresponds to the squared rotational wave function at the time of the pulse irradiation. To create a real-time movie of molecular rotation, the present imaging technique is combined with a femtosecond pump-probe optical setup in which the pump pulses create unidirectionally rotating molecular ensembles. Due to the high image throughput of our detection system, the pump-probe experimental condition can be easily optimized by monitoring a real-time snapshot. As a result, the quality of the observed movie is sufficiently high for visualizing the detailed wave nature of motion. We also note that the present technique can be implemented in existing standard ion imaging setups, offering a new camera angle or viewpoint for the molecular systems without the need for extensive modification.

We present a protocol for creating a real-time movie of a molecular rotational wave packet using a high-resolution Coulomb explosion imaging setup.

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb ...

Chemistry

Fabrication of 1-D Photonic Crystal Cavity on a Nanofiber Using Femtosecond Laser-induced Ablation

We present a protocol for fabricating 1-D Photonic Crystal (PhC) cavities on subwavelength-diameter tapered optical fibers, optical nanofibers, using femtosecond laser-induced ablation. We show that thousands of periodic nano-craters are fabricated on an optical nanofiber by irradiating with just a single femtosecond laser pulse. For a typical sample, periodic nano-craters with a period of 350 nm and with diameter gradually varying from 50 - 250 nm over a length of 1 mm are fabricated on a nanofiber with diameter around 450 - 550 nm. A key aspect of such a nanofabrication is that the nanofiber itself acts as a cylindrical lens and focuses the femtosecond laser beam on its shadow surface. Moreover, the single-shot fabrication makes it immune to mechanical instabilities and other fabrication imperfections. Such periodic nano-craters on nanofiber, act as a 1-D PhC and enable strong and broadband reflection while maintaining the high transmission out of the stopband. We also present a method to control the profile of the nano-crater array to fabricate apodized and defect-induced PhC cavities on the nanofiber. The strong confinement of the field, both transverse and longitudinal, in the nanofiber-based PhC cavities and the efficient integration to the fiber networks, may open new possibilities for nanophotonic applications and quantum information science.

We present a protocol for fabricating 1-D photonic crystal cavities on subwavelength diameter silica fibers (optical nanofibers) using femtosecond laser-induced ablation.

We present a protocol for fabricating 1-D Photonic Crystal (PhC) cavities on subwavelength-diameter tapered optical fibers, optical nanofibers, using ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

An Experimental Protocol for Femtosecond NIR/UV - XUV Pump-Probe Experiments with Free-Electron Lasers

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a free-electron laser. This includes methods to establish the spatial and temporal overlap between the optical and free-electron laser pulses during the experiment, as well as important aspects of the data analysis, such as corrections for arrival time jitter, which are necessary to obtain high-quality pump-probe data sets with the best possible temporal resolution. These methods are demonstrated for an exemplary experiment performed at the FLASH (Free-electron LASer Hamburg) free-electron laser in order to study ultrafast photochemistry in gas-phase molecules by means of velocity map ion imaging. However, most of the strategies are also applicable to similar pump-probe experiments using other targets or other experimental techniques.

This protocol describes the key steps for performing and analyzing pump-probe experiments combining a femtosecond optical laser with a free-electron laser in order to study ultrafast photochemical reactions in gas-phase molecules.

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Bioengineering

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped fiber laser is built completely using commercially available parts, including $8,000 in fiber optic and pump laser components, plus $4,800 in standard optical components and extra-cavity accessories. Researchers new to fiber optic device fabrication may also consider investing in basic fiber splicing and laser pulse characterization equipment (~$63,000). Important for optimal laser operation, methods to verify true versus apparent (partial or noise-like) mode-locked performance are presented. This system achieves 70 fs pulse duration with a center wavelength of approximately 1,070 nm and a pulse repetition rate of 31 MHz. This fiber laser exhibits the peak performance that may be obtained for an easily assembled fiber laser system, which makes this design ideal for research laboratories aiming to develop compact and portable fs laser technologies that enable new implementations of clinical multiphoton microscopy and fs surgery.

A method is presented to build a custom low-cost, mode-locked femtosecond fiber laser for potential applications in multiphoton microscopy, endoscopy, and photomedicine. This laser is built using commercially available parts and basic splicing techniques.

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Biology

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...