We would like to thank Iwan Schie and Sebastian Wachsmann-Hogiu for their contributions in developing the DR-CARS technique. Tyler Weeks acknowledges support by the Lawrence Scholar Program from Lawrence Livermore National Laboratory. Thomas Huser is grateful for support from the American Heart Association through the Grant-in-Aid program. This work was also supported in part by funding from the National Science Foundation. The Center for Biophotonics, an NSF Science and Technology Center, is managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. Support is also acknowledged from the UCD Clinical Translational Science Center under grant number UL1 RR024146 from the National Center for Research Resources (NCRR).

1. Generation of CARS and DR-CARS Signals
In order to generate CARS and DR-CARS signals simultaneously, three tunable and synchronized short pulsed laser sources are required.
<ol>
 <li>To obtain three synchronized pulses we start with a single 10 W laser (PicoTrain, HighQ laser, Inc.). This laser has a fixed wavelength of 1064 nm, a fixed pulse length of 7 ps, and a fixed repetition rate of 76 MHz.</li>
 <li>Using a series of half-wave plates and polarizing beam splitter cubes the beam is split into three parts. A half-wave plate combined with a polarizing beam splitter cube allows us to adjust the amount of power in each component of the beam without changing its direction. Typically, two beams are adjusted to ~ 4.5 W each, and the third beam contains the remaining 1 W.</li>
 <li>The two high power beams are then directed into two independent optical parametric oscillators (OPO's, Avante, APE GmbH, Berlin, Germany). OPO's use difference frequency generation to convert a higher energy photon into two lower energy photons with different wavelengths. By controlling the temperature of the crystal used to achieve this effect, the wavelengths of the resulting photons can be controlled to within 0.1 nm. By frequency-doubling these signals, a laser with a fixed wavelength of 1064 nm can now be transformed into a laser beam that can be tuned anywhere between 780 nm to 910 nm. By pumping two separate OPO's with the same 1064 nm source, we obtain two independently tunable laser sources that are automatically synchronized to our original pump laser.</li>
 <li>The third (lowest power) beam from the 1064 nm pump laser is directed around the OPO's by a combination of dichroic mirrors so that all three beams (2 from the OPOs, 1 from the pump laser) can be recombined later. In order to efficiently produce coherent Raman signal photons the recombined pulses must be overlapped both temporally and spatially. Because OPO's contain ring cavities, allowing each laser pulse to pass through the crystal multiple times, the beams sent through the OPO's travel an extra distance resulting in a considerable delay relative to the original pump beam. This distance must be compensated for with the third beam by introducing extra mirrors that force this beam to travel the same distance as the other two before they are recombined using dichroic mirrors.</li>
 <li>In order to provide fine adjustment of the length of the path each beam travels adjustable delay stages, using prism retroreflectors, are introduced into each beam path.</li>
 <li>Another set of half-wave plates and polarizing beam splitter cubes are added to each beam to allow us to adjust the power of each beam independently without affecting the input of the OPO's.</li>
 <li>Dichroic mirrors are used to first combine the beams from the two OPO's and then with the 1064 nm beam. Care must be taken to ensure that the beams are precisely overlapped (i.e. they are collinear and have similar divergence). This can be checked by comparing the overlap of the beams close to (within a few centimeters) and far from (~ 1 m) the dichroic mirror. </li>
 <li>The advantage of using pulsed lasers is that each pulse can have a high peak energy at the sample, allowing for efficient generation of coherent Raman signals, while still maintaining a low average intensity. A high average intensity can damage the sample. Therefore in order to further control the average intensity without sacrificing peak energy, the three combined beams are sent through an electro-optic modulator (Pockel's cell, ConOptics) functioning as a pulse picker, which allows us to adjust the repetition rate of the pulses that arrive at our sample and therefore the average intensity.</li>
 <li>The combined laser beams are then coupled into an inverted microscope with a high NA objective. The objective is typically a 60 X water objective lens with a numerical aperture (NA) of 1.2 that has been corrected for chromatic aberration across the range of tunability of our OPO's and the 1064 nm beam. The tight focusing generated by the high NA objective lens allows for the most efficient generation of coherent Raman signals on the micron scale.</li>
 <li>The combined beams are expanded in order to overfill the back-port of the microscope objective. By overfilling the back of the microscope objective we can achieve the best focusing conditions and therefore the best spatial resolution in our microscope system.</li>
 <li>The objective lens in our microscope is mounted on an XYZ piezo stage which allows us to obtain images by raster-scanning the beams across the sample, similar to commercial beam-scanning confocal microscopes.</li>
</ol>
2. The Use of Three Short Pulsed Lasers
The use of three short pulsed lasers results in the production of several CARS signals, from the various combinations of two lasers, as well as 3-Color CARS and DR-FWM signals from the combination of all three lasers.
<ol>
 <li>The wavelengths of the signals can lie spectrally close to each other. When the signals are close together it can be difficult to separate them for analysis using fixed wavelength bandpass filters and dichroic mirrors. For this reason our signals are passed into an imaging spectrometer (SpectraPro 2300i, Acton Research) that also serves as an efficient monochromator to spatially separate signals at different wavelengths.</li>
 <li>An electronically actuated flip mirror within the spectrometer in the lowered position sends the signal to a back-illuminated deep-depletion charge-coupled device (CCD) camera which provides spectroscopic information across the entire signal range and allows us to identify and optimize the various coherent Raman signals.</li>
 <li>To select the signal with which we wish to image we simply rotate the grating within the spectrometer, using the vendor-supplied control and data acquisition software (WinSpec, Princeton Instruments), to center the peak of interest on the CCD camera and then change the position of the flip mirror to redirect this signal to a second exit port to which a single-photon counting avalanche photo-diode (APD) has been attached.</li>
 <li>The objective lens is then raster-scanned and the signals recorded on the APD are used to generate an image by displaying the photon count-rate for each pixel via data acquisition software (SymPhoTime, Picoquant GmbH, Berlin, Germany).</li>
 <li>We repeat this imaging procedure for each desired coherent Raman signal which allows us to compare the signals during post-processing.</li>
</ol>
3. Sample Preparation
In order to obtain clear, reproducible images some care must be taken in preparing the sample.
<ol>
 <li>Samples are typically prepared on ~150 micron thick glass coverslips. These coverslips are thin enough to allow for high resolution imaging with the 1.2 NA objectives that are typically used.</li>
 <li>Optical trapping of dielectric objects can occur when the laser light is diffracted through small transparent objects. The use of tightly focused, high peak power, short pulsed laser beams can then drag small cells or bacteria along, resulting in blurred or smeared images. In order to avoid this it may be necessary to immobilize the sample on the surface of the glass coverslip by first applying a thin layer of poly-L-lysine by spin-coating.</li>
 <li>For cells in culture glass bottom culture dishes can be utilized that allow for imaging without the need to detach the cells from their cell culture growth dishes.</li>
 <li>For demonstration in this contribution we first deposit a formaldehyde-fixed C. elegans nematode worm on a glass cover slip.</li>
 <li>Then add a 20 &mu;L droplet of 5 M deuterated glucose solution to the worm. The deuterated glucose solution provides a unique and strong Raman background signature.</li>
</ol>
4. Sample Analysis
In order to properly take advantage of the doubly-resonant enhancement effect the Raman spectra of both Raman-resonant substances must be known.
<ol>
 <li>Once the sample is prepared on the coverslip it is analyzed using a confocal Raman microscope to obtain the relevant spontaneous Raman spectrum and identify suitable peaks.</li>
 <li>To perform DR-CARS we identify the spectral locations of the CH stretch mode, typically associated with lipids, and the CD stretch mode, associated with the deuterated glucose, to be 2845 cm-1 and 2121 cm-1 respectively.</li>
 <li>Coherent Raman scattering is achieved when the frequency difference between two lasers matches the frequency of a molecular vibration. By tuning one OPO to 817 nm it will probe the 2845 cm-1 CH mode when combined with the 1064 nm laser beam and by tuning the other OPO to 868 nm it will probe the 2121 cm-1 peak when combined with the 1064 nm beam.</li>
 <li>By raster-scanning the sample on the CARS microscope we can now observe three coherent Raman signals of interest. A CARS signal probing the CH stretching vibration, a CARS signal probing the CD stretch mode, and a DR-CARS signal probing both.</li>
 <li>We select each peak as described in Section 2.3 and take an image as described above.</li>
</ol>
5. Image Processing
Extracting additional information based on these three images now requires some fairly simple image processing.
<ol>
 <li>First the images must be normalized. In theory normalization can be achieved by accounting for the intensity of each laser involved in each signal generation process. In practice, however this does not always work, mainly due to the non-uniform spectral response of dichroic mirrors, detectors, and gratings.</li>
 <li>A practical method for normalization relies on the fact that in regions where pure lipids dominate the signal, the CD resonance should not contribute to any of the signal. Likewise, in regions of pure glucose solution the CH resonance should not contribute to any of the signal. With this in mind, we normalize the DR-CARS image and the CARS image obtained on the CD resonance of the deuterated glucose solution to a region well outside the C. elegans worm that should exhibit no CH resonance. </li>
 <li>Then we identify a region within the worm in the CH-resonant CARS image that is rich in lipids and normalize it to the corresponding region within the normalized DR-CARS image. For this method to work properly we assume that deep within this region no deuterated glucose is present. This is a safe assumption since the lipids are hydrophobic and will not mix with the solution.</li>
 <li>Now, by subtracting the normalized CH-resonant CARS image from the normalized DR-CARS image we are left with just the amplified CD-resonant signal.</li>
 <li>Similarly by subtracting the normalized CD-resonant CARS image from the normalized DR-FWM image we are left with just the amplified CH-resonant signal.</li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/2085/2085fig1.jpg" alt="Figure 1" /> 
 Figure 1: Diagram of DR-CARS microscopy system as described above. 
<img src="/files/ftp_upload/2085/2085fig2.jpg" alt="Figure 2" /> 
 Figure 2: White light image of a C. elegans worm in deuterated glucose solution prepared on a glass coverslip and ready for imaging. 
<img src="/files/ftp_upload/2085/2085fig3.jpg" alt="Figure 3" /> 
 Figure 3: Raman spectrum of modified oleic acid (an unsaturated fatty acid) that includes an alkyne modification (a carbon triple-bond carbon group). The strong CH resonances at 2845 cm-1 and the alkyne resonance at 2100 cm-1 are both well isolated from the fingerprint region (the region of densely packed peaks), making them ideal markers for coherent Raman imaging.
<img src="/files/ftp_upload/2085/2085fig4.jpg" alt="Figure 4" /> 
 Figure 4: Typical spectrum of coherent Raman signals generated when three short-pulsed lasers are overlapped within the sample. The arrows point to the process(es) responsible for each signal as represented by energy diagrams. In the diagrams shown here dashed arrows indicate photons from the laser and the OPO's and solid arrows indicate the resulting signal. The solid horizontal lines indicate the energy of the Raman vibration and give a visual representation that, in DR-CARS, mixing the same 3 input photons simultaneously probes two different Raman vibrations.
<img src="/files/ftp_upload/2085/2085fig5.jpg" alt="Figure 5" /> 
 Figure 5: Typical results from imaging C. elegans worms using DR-CARS and CARS. The top row used the three signals indicated in Figure 4 to image a worm in a solution of deuterated glucose. In the second row the images were appropriately normalized and in the third row the difference images were produced by subtracting each of the CARS images from the DR-CARS image.

<ol>
	<li>Cheng, J. X., Volkmer, A., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+and+experimental+characterization+of+coherent+anti-Stokes+Raman+scattering+microscopy.">Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy.</a> J Opt Soc Am B. 19 (9), (2002).</li><li>Tolles, W. M., Nibler, J. W., McDonald, J. R., Harvey, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+the+Theory+and+Application+of+Coherent+Anti-Stokes+Raman+Spectroscopy+(CARS).">A Review of the Theory and Application of Coherent Anti-Stokes Raman Spectroscopy (CARS).</a> Appl Spectrosc. , (1977).</li><li>Weeks, T., Schie, I. W., Wachsmann-Hogiu, S., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+generation+and+Raman-resonant+imaging+by+non-degenerate+four-wave+mixing+under+tight+focusing+conditions.">Signal generation and Raman-resonant imaging by non-degenerate four-wave mixing under tight focusing conditions.</a> J Biophoton. 3 (3), 169-175 (2010).</li><li>Weeks, T., Wachsmann-Hogiu, S., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+Microscopy+based+on+Doubly-Resonant+Four-Wave+Mixing+(DR-FWM).">Raman Microscopy based on Doubly-Resonant Four-Wave Mixing (DR-FWM).</a> Opt Express. 17 (19), 17044-17051 (2009).</li><li>Schie, I. W., Weeks, T., McNerney, G. P., Fore, S., Sampson, J. K., Wachsmann-Hogiu, S., Rutledge, J. C., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+forward+and+epi-CARS+microscopy+with+a+single+detector+by+time-correlated+single+photon+counting.">Simultaneous forward and epi-CARS microscopy with a single detector by time-correlated single photon counting.</a> Optics Express. 16 (3), 2168-2175 (2008).</li></ol>

No conflicts of interest declared.

Raman spectroscopy and Raman-based imaging are powerful emerging tools in the bio sciences. Currently, this is particularly true for the in vivo and in vitro study of cellular metabolism and metabolic disorders in processing and storage of lipids. Most bio-macromolecules contain a large number of similar, mostly carbon-based molecular bonds, so that the Raman spectra obtained from cells and organisms are typically a convolution of contributions from lipids, proteins, nucleic acids, sugars, etc. Lipids are relatively easy to isolate from these complex spectra, because of their tendency to form dense droplets or bilayers and because they contain extended chains with a large number of aliphatic CH bonds. Our ability to isolate specific proteins, amino acids, RNA, or DNA within the complex cellular environment is, however, very limited. This is particularly true if these molecules of interest are only present at &mu;M concentrations and below. Here, the ability to probe weak Raman resonances utilizing our newly introduced DR-CARS difference imaging technique provides a potentially powerful approach for their chemical microanalysis and imaging. Admittedly, the most complicated portion of this protocol is the alignment and synchronization of the laser system. When starting from scratch, the synchronization of the pulses, i.e. ensuring that the pulses are overlapped in time despite the different paths they take can be facilitated by the use of a pulse autocorrelator. Once spatial and temporal overlap is achieved, CARS and DR-CARS signals should be readily detectable. However, the first alignment is often crude, resulting in weak signals. The best practice for aligning this system well is to initially generate weak signals and then to improve the signal strength by gently tweaking the mirrors along each path and adjusting the temporal overlap using the delay stages. Although the spectrometer/monochromator acts as a very efficient baffle for room light the cleanest results can be achieved by operating the system with the room lights turned off and curtains or lens tubing to minimize background introduced by the various other light sources (e.g computer monitors, indicator lights, LEDs, etc.). Our particular setup utilizes single-photon counting avalanche photo-diode (APD) detectors and time-correlated single photon counting (TCSPC) hardware for detection 5. This enables us to detect extremely weak signals with relatively low noise but many groups have found photo-multiplier tubes (PMT's) with variable gain advantageous when making similar measurements. The advantage of PMT's is that they offer variable gain and have a much larger detection area which can simplify alignment of the detector. Additionally, our setup utilizes piezo stages to translate the objective in order to achieve beam scanning. The advantage of this is that we have the ability to return to any spot within the previously scanned image with a high degree of accuracy and take additional measurements including spontaneous Raman spectra. Other groups have been successful utilizing scanning mirror assemblies, or even entire confocal scanning units such as the Olympus FluoView system, which offers much faster imaging but is limited in its ability to precisely return to arbitrary locations within an image. Tuning the lasers to match the Raman resonance is also a critical step that may require some optimization. Although the Raman peaks may be known the maximum spectral peak intensity obtained from DR-CARS and CARS does not necessarily correspond to the maximum of the spontaneous Raman peak. This is due to the intrinsic interference of signals generated by four-wave mixing leading to a non-resonant background signal and CARS, which distorts CARS spectra relative to spontaneous Raman spectra. The spectral location of the peak of the CARS signal can be calculated, but a more practical approach is to tune the OPOs in several, small spectral steps across the expected location of the Raman resonance. This process should yield a clear maximum. In fact, for the greatest sensitivity from DR-FWM both resonances must be tuned to this maximum. One last potential problem of the DR-CARS approach has to also be discussed, i.e. the DR-CARS signal will depend on a homogeneous distribution of the Raman-active amplifying molecule. For most biological objects, this could well be the broad OH resonance from water, which is abundant and almost omnipresent. Water is, however, excluded from hydrophobic regions with a cell, such as lipid droplets, leading a distortion of the signals obtained when utilizing the water resonance to amplify lipid modes. In our example, we have used a solution of deuterated glucose to generate an easily detectable and abundant signal for our biological sample. Similarly, deuterated water or deuterated biological buffers, such as d-HEPES could be used. In our example, the lipid droplets within the C. elegans worm were small enough to always contain both, the deuterated glucose solution and lipids within the focused laser spot of our system. This, however, is not generally true. A particular example would be adipocytes, which generate rather large lipid droplets within their cytoplasm. This means, any experiment conducted with the DR-CARS technique requires careful preparation and control experiments to verify the results.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>60X water immersion objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>UPLSAPO 60XW</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX-71SIF-3</td>
<td>&nbsp;</td>
<tr>
<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>ConOptics</td>
<td>350-160</td>
<td>&nbsp;</td>
<tr>
<td>Picotrain pump Laser</td>
<td>&nbsp;</td>
<td>HighQ</td>
<td>IC-1064-10000</td>
<td>&nbsp;</td>
<tr>
<td>Optical Parametric Oscillator</td>
<td>&nbsp;</td>
<td>APE</td>
<td>Levante IR</td>
<td>&nbsp;</td>
<tr>
<td>1.5 Glass cover slips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-102 25cm-1</td>
<td>&nbsp;</td>
<tr>
<td>Half-wave plates</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>AHWP05M-980</td>
<td>&nbsp;</td>
<tr>
<td>Polarizing Beam Splitter Cubes</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>PBS052 or PBS053</td>
<td>&nbsp;</td>
<tr>
<td>Spectrometer/Monochromator</td>
<td>&nbsp;</td>
<td>PI Acton</td>
<td>Spectra Pro 2300i</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera</td>
<td>&nbsp;</td>
<td>PI Acton</td>
<td>PIXIS: 100B</td>
<td>&nbsp;</td>
<tr>
<td>Avalanche Photo Diode</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>SPCM-AGR-14-12691</td>
<td>&nbsp;</td>
<tr>
<td>XYZ Piezo Stage</td>
<td>&nbsp;</td>
<td>Physik Instruments</td>
<td>P 733-2CL P 721.CDQ</td>
<td>This is a combination of an XY stage and a Z objective holder</td>
</tr>
<tr>
<td>Dichroic Mirrors</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>Ff01-720/SP-25 LPD01-633RS-25</td>
<td>These specific dichroics are not critical, any set with the appropriate transmission/reflection characteristics will be sufficient.</td>
</tr>
<tr>
<td>Dichroic Mirror</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>Z830rdc</td>
<td>To combine the different near-infrared laser beams</td>
</tr>
<tr>
<td>TCSPC board</td>
<td>&nbsp;</td>
<td>PicoQuant</td>
<td>Timeharp 200</td>
<td>&nbsp;</td>
<tr>
<td>Symphotime Imaging Software</td>
<td>&nbsp;</td>
<td>PicoQuant</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Matlab</td>
<td>&nbsp;</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

differential imaging of biological structures with doubly-resonant coherent anti-stokes raman scattering (cars)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Watch this Scientific Journal Video about Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS at JoVE.com

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

differential-imaging-biological-structures-with-doubly-resonant

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13.6K Views.  University of California, Davis. A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Video: Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...