Supported by NIH R01 DC 17924, R01 DC 18401 (Klug), and NIH 1R15HD105231-01, T32DC012280, and FRAXA (McCullagh). The CARS imaging was performed in the Advanced Light Microscopy Core part of the NeuroTechnology Center at the University of Colorado Anschutz Medical Campus supported in part by NIH P30 NS048154 and NIH P30 DK116073.

All experiments complied with all applicable laws, National Institutes of Health guidelines, and were approved by the University of Colorado Anschutz Institutional Animal Care and Use Committee.
1. Animals
<ol>
	<li>Use C57BL/6J (stock #000664) mice (Mus musculus) obtained from The Jackson Laboratory or Mongolian gerbils (Meriones unguiculatus) originally obtained from Charles River.</li>
</ol>
2. Tissue preparation
<ol>
	<li>For transcardial perfusion, overdose rodent species of interest with pentobarbital (120 mg/kg body weight) and transcardially perfuse them with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.76 mM KH2PO4, 10 mM Na2HPO4) followed by 4% paraformaldehyde (PFA)24.
	<ol>
		<li>Specifically, open the abdomen and the rib cage using scissors and hold the rib cage in place with Kelly hemostatic forceps to expose the heart.</li>
		<li>Insert a 23 GA needle connected to a perfusion pump into the left ventricle and quickly cut the right atrium using fine scissors.</li>
		<li>Administer PBS through the perfusion pump and the needle in the heart for 10 min to clear the brain and body of blood.</li>
		<li>Switch the perfusion pump to 4% PFA for 10 min and check for rigidity of limbs and tail to confirm successful perfusion.</li>
	</ol>
	</li>
	<li>After perfusion, decapitate the animals and remove their brain from the skull. Keep the brains overnight in 4% PFA before transferring to PBS. Embed brainstems in 4% agarose (in PBS) and slice coronally using a vibratome at 200 &#181;m thickness.</li>
</ol>
3. Staining
<ol>
	<li>Stain free floating sections for Nissl, to visualize cell bodies (1:100), in antibody media (AB media: 0.1 M phosphate buffer (PB: 50 mM KH2PO4, 150 mM Na2HPO4), 150 mM NaCl, 3 mM Triton-X, 1% bovine serum albumin (BSA)) for 30 min at room temperature on a standard laboratory shaker25.
	<ol>
		<li>Protect sections from light using aluminum foil and/or a cover. 550 nm or below wavelengths are compatible with CARS imaging (Figure 1). 
		NOTE: While we do not expect that Triton-X or other reagents have an impact on CARS imaging of lipids, additional controls with specific antibody media may be warranted.</li>
	</ol>
	</li>
	<li>PAUSE POINT: Store free floating sections (while protected from light) in PBS until imaging. Once sectioned, image brain sections within 2 weeks.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64013/64013fig01.jpg" /> 
Figure 1: CARS imaging can be combined with immunofluorescent imaging. The graphs shows that CARS imaging occurs at 660/640 nm red signal spectrum26. This wavelength is sufficiently far removed from the green, blue, or UV range, allowing for combination of the CARS signal with immunofluorescence in these ranges. Specifically, the graph also indicates the excitation and emission for Nissl tagged with blue fluorophore, which was combined with CARS during the collection of representative results for this publication. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64013/64013fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Imaging
NOTE: The CARS laser set up contains a fiber laser that provides the 80 MHz clock, and an OPO (Optical Parametric Oscillator) laser with a tunable range of 770-990 nm with the Stokes beam fixed at 1031 nm, which are needed for collecting the CARS signal. There is one aperture for both beams.
<ol>
	<li>Before bringing samples to the microscope, turn on and warm up CARS laser for at least 1 h, align the CARS laser, and Koehler the condenser optics and the diaphragm of the microscope for forward CARS imaging. 
	NOTE: This step is critical for proper function of CARS microscopy.
	<ol>
		<li>For spatial alignment of the two laser beams (pump and stokes), access the two internal PSDs (position sensitive detectors) via the CARS laser GUI.</li>
		<li>Achieve temporal alignment by using the delay function in the CARS laser GUI, which can help overlap the pulses of the two lasers (pump and stokes) that have different dispersions because of their different wavelengths. Therefore, both the temporal and spatial overlapping of the pump and stokes beams are done with the user input through the GUI.</li>
		<li>Adjust the external periscope (last two mirrors of the setup) to center the spatially overlapped two lasers onto the scanning head mirrors of the microscope.</li>
		<li>For best forward CARS non-descanned detection, make sure the condenser is Koehler-ed (meaning the condenser is centered and focused onto the diaphragm to achieve uniform illumination)</li>
	</ol>
	</li>
	<li>For immunofluorescence confocal imaging and CARS imaging, fit the CARS laser, with both forward and epi CARS non-descanned detectors (NDDs), by incorporating a confocal microscope equipped with visible lasers for fluorescence imaging (Figure 2).</li>
	<li>Place sections in a culture dish with coverslip (for inverted microscopy), PBS to avoid the tissue drying out, and a glass weight to keep tissue near coverslip.</li>
	<li>Take z-stacks or single images with a 60X, 1.2 NA infrared corrected water objective, which serves for collection of the CARS signal in the epi direction and through a 0.55 NA condenser in the forward direction for imaging brain areas such as the medial nucleus of the trapezoid body (MNTB).
	<ol>
		<li>Take the CARS images at approximately 600 mW pump/probe and 300 mW Stokes, by using the CARS laser GUI. These laser power values are measured internally by the system. Both lasers&#39; powers at the sample location are less than 25 mW and safe for the tissue sample.</li>
		<li>Overlap the pump and Stokes beams spatially and temporally. Tune the OPO to 797.2 nm. This yields a CARS wavelength of 650 nm. Because of the higher energy level, the resulting return to the ground state is anti-Stokes (blue shifted) to the excitation.</li>
		<li>Capture the CARS signal in epi or forward non-descanned detectors using bandpass filters (640-680 nm) followed by sequential detection of the immunofluorescence label (in this instance fluorescently labeled Nissl). 
		NOTE: The Nissl neuronal soma marker is not caught in the CARS 640-680 nm bandpass filter, allowing the combination of fluorescence and CARS imaging in the images presented below.</li>
		<li>The CARS and fluorescence do not share PMTs. Use these settings for optimal lipid signal to selectively image myelination in the brain area. 
		CAUTION: Shield the user from the laser beam</li>
	</ol>
	</li>
	<li>Save the images as .oib files, which can be imported into an image analysis program for further quantification (Figure 3).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64013/64013fig02.jpg" /> 
Figure 2: CARS instrument diagram showing CARS lasers (red arrows) and non-descanned (NDD) epi and forward detection incorporated onto a laser scanning confocal. In forward NDD we acquire CARS for C-H bonds (dark red arrows) and SHG (second harmonic generatioin) at 515 nm (orange arrow). In epi NDD, we acquire CARS for C-H bonds (dark red arrows) and 2PE (two-photon emission) autofluorescence (light blue arrow). Sequentially, fluorescence confocal images can be acquired (green arrows for visible laser, blue arrows for confocal detection). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64013/64013fig02large.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/64013/64013fig03v2.jpg" /> 
Figure 3: CARS can illuminate myelin (magenta) in brain tissue (brainstem) while also imaging Nissl (cyan) or fluorescent markers. The two panels show representative results from a Mongolian gerbil (single image M. unguiculatus, Figure 3A,C,E) and mouse (z-stack max projection M. musculus, Figure 2B,D,F) brain, indicating that this technique can be used across species. Figure 3A,B showing Nissl in cyan, C,D show the CARS signal in magenta, E,F combine the Nissl and CARS signals with each panel for gerbil or mouse, respectively. Both sets of images show a section of the medial nucleus of the trapezoid body (MNTB) in the brain stem. Neurons in the MNTB receive inputs from heavily myelinated axons, which terminate in the calyx of held, a type of giant synapse27. Scale bar is 20 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/64013/64013fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

A growing body of literature emphasizes the role of myelin in brain function13,16,21,28. Moreover, we know that myelination thickness and myelination pattern can change in several neurological conditions such as multiple sclerosis (reviewed in29), aging (reviewed in30), autism20,31, and ma...

Action potentials are the basic unit of information in the brain, and action potential propagation through axons forms one pillar of information processing1,2,3. Neurons typically receive afferent inputs from multiple other neurons and integrate these inputs within a given narrow time window4,5. Therefore, the mechanisms of action potential propagation in axons have received a significant amount of attention from investigators.
When propagating through an axon, an action potential is regenerated repeatedly along the axon to ensure reliable propagation6. In most neurons of jawed vertebrates (gnathostomes) axons are surrounded by a sheath of myelin, which is a lipid-rich substance produced by nearby oligodendrocytes or Schwann cells, which are types of glial cells (reviewed in7,8). This myelin sheath electrically insulates the axon, reducing its capacitance and allowing action potential propagation efficiently, quickly, and with lower energy consumption. Myelin does not cover the axon uniformly, but it sheaths the axon in segments that have short gaps in between them, called the nodes of Ranvier (reviewed in9,10). Both myelination thickness, which controls the level of electrical insulation of an axon, and the spacing of the nodes of Ranvier, which control the frequency with which action potentials are regenerated along an axon, influence the speed of action potential propagation (reviewed in11).
There is a large body of literature suggesting that myelination thickness affects the speed of action potential propagation in axons12,13,14. Moreover, alterations in axon myelination can result in a number of CNS deficits15,16,17,18,19,20,21. It is therefore not surprising that the focus of many research efforts involves the measurement and characterization of axon myelination. Measurements of myelin thickness have most commonly been done with electron microscopy, a technique that requires a significant amount of tissue preparation and is challenging to use in combination with immunohistochemistry. However, there is also a faster and simpler technique to measure axon myelination that is based on Coherent Anti-Stokes Raman Spectroscopy (CARS). A CARS laser can be tuned to various frequencies and when tuned to frequencies that are suitable to excite lipids, myelin can be imaged without the need for any additional labels22. The lipid imaging can be combined with standard immunohistochemistry such that lipids can be imaged together with several fluorescent channels23. Imaging myelination with CARS is significantly faster than electron microscopy and has a resolution that is, albeit lower than EM, sufficient to detect even small differences in myelination in the same type of axons.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL disposable syringe with needle 27 GA x 0.5&quot;</td><td>Exel int</td><td>260040</td><td></td></tr><tr><td>Fatal +</td><td>Vortech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surgery:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors - 8mm Cutting Edge</td><td>Fine Science Tools</td><td>15024-10</td><td></td></tr><tr><td>Standard tweezers</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>&lt;strong&gt;Perfusion:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Fisher Chemical</td><td>SF994 (CS)</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14063-11</td><td></td></tr><tr><td>Kelly hemostats</td><td>Fine Science Tools</td><td>13019-14</td><td></td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Needle tip, 23 GA x 1&quot;</td><td>BD precision glide</td><td>305193</td><td></td></tr><tr><td>Phosphate buffered saline (PBS):</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobase</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>pump with variable flow or equivalent</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Chemical</td><td>s271-1</td><td></td></tr><tr><td>Sodiumphosphate dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>&lt;strong&gt;Dissection:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL vial with 4% PFA</td><td></td><td></td><td></td></tr><tr><td>Bochem Chemical Spoon 180mm</td><td>Bochem</td><td>230331000</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14063-11</td><td></td></tr><tr><td>Noyes Spring Scissors</td><td>Fine Science Tools</td><td>15011-12</td><td></td></tr><tr><td>Pair of fine (Graefe) tweezers</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Shallow glass or plastic tray, approximately 10&quot; x 10&quot;</td><td></td><td></td><td></td></tr><tr><td>Standard tweezers</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Surgical Scissors - Blunt</td><td>Fine Science Tools</td><td>14000-20</td><td></td></tr><tr><td>&lt;strong&gt;Slicing:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agar, plant</td><td>RPI</td><td>9002-18-0</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000s</td><td></td></tr><tr><td>well plate</td><td>Alkali Sci.</td><td>TPN1048-NT</td><td></td></tr><tr><td>&lt;strong&gt;Staining:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AB Media:</td><td></td><td></td><td>1n 1,000 mL of Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phosphate buffered (PB):</td><td></td><td></td><td></td></tr><tr><td>Potassium Phosphate Monobase</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Sodium Phosohate Dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma life science</td><td>A2153-100g</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>s271-1</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma - Aldrich</td><td>x100-500ml</td><td></td></tr><tr><td>Nissl 435/455</td><td>Invitrogen</td><td>N21479</td><td></td></tr><tr><td>&lt;strong&gt;CARS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>APE&amp;nbsp;picoemerald laser</td><td>Angewandte Physik &amp;amp; Elektronik&amp;nbsp;GmbH</td><td></td><td></td></tr><tr><td>bandpass filter (420-520 nm)</td><td>Chroma Technology</td><td>HQ470/100m-2P</td><td></td></tr><tr><td>bandpass filter (500-530 nm)</td><td>Chroma Technology</td><td>HQ515/30m-2P</td><td></td></tr><tr><td>bandpass filters (640-680 nm)</td><td>Chroma Technology</td><td>HQ660/40m-2P</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Cut Transfer pipet</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>dichroic longpass 565 nm</td><td>Chroma Technology</td><td>565dcxr</td><td></td></tr><tr><td>dichroic longpass 585 nm</td><td>Chroma Technology</td><td>585dcxr</td><td></td></tr><tr><td>dichroic shortpass 750 nm</td><td>Chroma Technology</td><td>T750spxrxt</td><td></td></tr><tr><td>glass bottom culture dish</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>glass weight (10 mm x 10 mm boro rod)</td><td>Allen Scientific Glass Inc</td><td></td><td></td></tr><tr><td>multiphoton shortpass emission filter 680 nm</td><td>Chroma Technology</td><td>ET680sp-2p8</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr></tbody></table>

coherent anti-stokes raman spectroscopy (cars) application for imaging myelination in brain slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

One of the biggest advantages of CARS microscopy over other techniques is the compatibility with fluorescent imaging23. Figure 1 shows the CARS spectra compared to Nissl tagged with immunofluorescent marker showing little/no overlap in spectra. Figure 2 illustrates the laser set up for CARS in combination with confocal microscopy. Figure 3 demonstrates two representative images, one as a single stack and one ...

Watch this Scientific Journal Video about Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices at JoVE.com

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

coherent-anti-stokes-raman-spectroscopy-cars-application-for-imaging

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

2.6K Views.  Oklahoma State University. Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Video: Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

Engineering

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Chemistry

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.

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.

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 ...

Biology

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Bioengineering

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 ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

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Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

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

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

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

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy