This study was supported by NIH R35 GM133435 to D.F.

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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=Video-rate+molecular+imaging+in+vivo+with+stimulated+Raman+scattering.">Video-rate molecular imaging in vivo with stimulated Raman scattering.</a> Science. 330 (6009), 1368-1370 (2010).</li><li>Hill, A. H., Hill, A. 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S., et al., Frostig, R. 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=Principles,+design,+and+construction+of+a+two-photon+laser-scanning+microscope+for+in+vitro+and+in+vivo+brain+imaging.">Principles, design, and construction of a two-photon laser-scanning microscope for in vitro and in vivo brain imaging.</a> In Vivo Optical Imaging of Brain. , 113-171 (2002).</li><li>Francis, A., Berry, K., Chen, Y., Figueroa, B., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+pathology+by+spectrally+sliced+femtosecond+stimulated+Raman+scattering+(SRS)+microscopy.">Label-free pathology by spectrally sliced femtosecond stimulated Raman scattering (SRS) microscopy.</a> PLoS One. 12 (5), 0178750 (2017).</li><li>Figueroa, B., 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=Broadband+hyperspectral+stimulated+Raman+scattering+microscopy+with+a+parabolic+fiber+amplifier+source.">Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source.</a> Biomedical Optics Express. 9 (12), 6116-6131 (2018).</li><li>Kong, L., 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=Multicolor+stimulated+Raman+scattering+microscopy+with+a+rapidly+tunable+optical+parametric+oscillator.">Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator.</a> Optics Letters. 38, 145-147 (2013).</li><li>He, R., 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=Dual-phase+stimulated+Raman+scattering+microscopy+for+real-time+two-color+imaging.">Dual-phase stimulated Raman scattering microscopy for real-time two-color imaging.</a> Optica. 4 (1), 44-47 (2017).</li><li>Pence, I. J., Kuzma, B. A., Brinkmann, M., Hellwig, T., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-window+sparse+spectral+sampling+stimulated+Raman+scattering+microscopy.">Multi-window sparse spectral sampling stimulated Raman scattering microscopy.</a> Biomedical Optics Express. 12 (10), 6095-6114 (2021).</li><li>Wei, M., 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=Volumetric+chemical+imaging+by+clearing-enhanced+stimulated+Raman+scattering+microscopy.">Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (14), 6608-6617 (2019).</li><li>Wright, A. J., 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=Adaptive+optics+for+enhanced+signal+in+CARS+microscopy.">Adaptive optics for enhanced signal in CARS microscopy.</a> Optics Express. 15 (26), 18209-18219 (2007).</li></ol>

The authors declare that there are no conflicts of interest.

The two-color SRS imaging scheme presented in this protocol hinges on the proper implementation of one-color SRS imaging. In one-color SRS imaging, the critical steps are spatial alignment, temporal alignment, modulation depth, and phase shift. Spatially combining the two beams is accomplished by a dichroic mirror. Several steering mirrors are used for fine adjustment when sending the beams to the dichroic mirror. Once the beams are combined with the dichroic mirror, spatial alignment can be confirmed by picking off the ...

Traditional tissue diagnostics rely on staining protocols followed by examination under an optical microscope. One common staining method used by pathologists is H&#38;E staining: hematoxylin stains cell nuclei a purplish blue, and eosin stains the extracellular matrix and cytoplasm pink. This simple staining remains the gold standard in pathology for many tissue diagnoses tasks, particularly cancer diagnosis. However, H&#38;E histopathology, particularly the frozen sectioning technique used in an intraoperative setting, still has limitations. The staining procedure is a laborious process involving tissue embedding, sectioning, fixation, and staining1. The typical turnaround time is 20 min or longer. Performing H&#38;E during frozen sectioning can sometimes become more challenging when multiple sections are processed at once due to the need to evaluate cellular features or growth patterns in 3D for margin assessment. Moreover, intraoperative histological techniques require skilled technicians and clinicians. Limitation in the number of board-certified pathologists in many hospitals is a constraint for intraoperative consultation in many cases. Such limitations may be alleviated with the fast development interests in digital pathology and artificial intelligence-based diagnosis2. However, the H&#38;E staining results are variable, depending on the experience of the technician, which presents additional challenges for computer-based diagnosis2.
These challenges can potentially be addressed with label-free optical imaging techniques. One such technique is SRS microscopy. SRS uses synchronized pulsed lasers&#8212;pump and Stokes&#8212;to excite molecular vibrations with high efficiency3. Recent reports have demonstrated that SRS imaging of proteins and lipids can generate H&#38;E-equivalent images (also known as stimulated Raman histology or SRH) with intact fresh tissue, which bypasses the need for any tissue processing, significantly shortens the time needed for diagnosis, and has been adapted intraoperatively4. Moreover, SRS imaging can provide 3D images, which offers additional information for diagnosis when 2D images are insufficient5. SRH is unbiased and generates digital images that are readily available for computer-based diagnosis. It quickly emerges as a possible solution for intraoperative cancer diagnosis and tumor margin analysis, especially in brain cancer6,7,8. More recently, SRS imaging of chemical changes of tissue has also been suggested to provide useful diagnostic information that can further help clinicians stratify different cancer types or stages9.
Despite its tremendous potential in tissue diagnosis applications, SRS imaging is mostly limited to academic laboratories specialized in optics due to the complexity associated with the imaging platform, which includes ultrafast lasers, the laser scanning microscope, and sophisticated detection electronics. This protocol provides a detailed workflow to demonstrate the use of a common femtosecond laser source for real-time, two-color SRS imaging and the generation of pseudo-H&#38;E images from mouse brain tissue. The protocol will cover the following procedures:
Alignment and chirp optimization 
Most SRS imaging schemes use either picosecond or femtosecond lasers as the excitation source. With femtosecond lasers, the bandwidth of the laser is much larger than the Raman linewidth. To overcome this limitation, a spectral focusing approach is used to chirp the femtosecond lasers to a picosecond timescale to achieve narrow spectral resolution10. Optimal spectral resolution is only achieved when the temporal chirp (also known as the group delay dispersion or just dispersion) is properly matched for the pump and the Stokes lasers. The alignment procedure and the steps needed to optimize the dispersion of the laser beams using highly dispersive glass rods are demonstrated here.
Frequency calibration 
An advantage of spectral focusing SRS is that the Raman excitation can be quickly tuned by changing the time delay between the pump and the Stokes lasers. Such tuning affords fast imaging and reliable spectral acquisition compared to tuning laser wavelengths. However, the linear relationship between excitation frequency and time delay requires external calibration. Organic solvents with known Raman peaks are used to calibrate the Raman frequency for spectral focusing SRS.
Real-time, two-color imaging 
It is important to increase the imaging speed in tissue diagnosis applications to shorten the time needed for analyzing large tissue specimens. Simultaneous two-color SRS imaging of lipids and proteins obviates the need to tune the laser or time delay, which increases the imaging speed by more than two-fold. This is achieved by using a novel orthogonal modulation technique and dual-channel demodulation with a lock-in amplifier11. This paper describes the protocol for orthogonal modulation and dual-channel image acquisition.
Epi-mode SRS imaging 
The majority of SRS imaging shown to date is performed in transmission mode. Epi-mode imaging detects backscattered photons from tissue12. For pathology applications, surgical specimens can be quite large. For transmission mode imaging, tissue sectioning is often necessary, which undesirably requires extra time. In contrast, epi-mode imaging can work with intact surgical specimens. Because the same objective is used to collect backscattered light, there is also no need for aligning a high numerical-aperture condenser required for transmission imaging. Epi-mode is also the only option when tissue sectioning is difficult, such as with bone. Previously we have demonstrated that for brain tissue, epi-mode imaging offers superior imaging quality for tissue thickness &#62; 2 mm13. This protocol uses a polarizing beam splitter (PBS) to collect scattered photons depolarized by tissue. It is possible to collect more photons with an annular detector at the expense of the complexity of customized detector assembly12. The PBS approach is simpler to implement (similar to fluorescence), with the standard photodiode already being used for transmission mode detection.
Pseudo-H&#38;E image generation 
Once two-color SRS images are collected, they can be recolored to simulate H&#38;E staining. This paper demonstrates the procedure for converting lipid and protein SRS images to pseudo-H&#38;E SRS images for pathology applications. The experimental protocol details critical steps needed to generate high-quality SRS images. The procedure shown here is not only applicable to tissue diagnosis but also can be adapted for many other hyperspectral SRS imaging applications such as drug imaging and metabolic imaging14,15.
General system requirements 
The laser system for this protocol must be able to output 2 synchronized femtosecond laser beams. Systems ideally feature an Optical Parametric Oscillator (OPO) for broad wavelength tuning of one of the laser beams. The setup in this protocol uses a commercial laser system Insight DS+ that outputs two lasers (one fixed beam at 1,040 nm and one OPO-based tunable beam, ranging from 680 to 1,300 nm) with a repetition rate of 80 MHz. Laser scanning microscopes, either from major microscope manufacturers or home-built, can be used for SRS imaging. The utilized microscope is an upright laser scanning microscope built on top of a commercial upright microscope frame. A pair of 5 mm galvo mirrors are used to scan the laser beam. For users choosing to adopt a homebuilt laser scanning microscope, refer to a previously published protocol for the construction of a laser scanning microscope16.

<table><tbody><tr><td>100 mm Achromatic Lens</td><td>THORLABS</td><td>AC254-100-B</td><td>Broadband, 650 - 1,050 nm, achromatic lens focal length, 100 mm</td></tr><tr><td>20 MHz bandpass filter</td><td>Minicircuits</td><td>BBP-21.4+</td><td>Lumped LC Band Pass Filter, 19.2 - 23.6 MHz, 50 &amp;Omega;</td></tr><tr><td>200 mm Achromatic Lens</td><td>THORLABS</td><td>AC254-200-B</td><td>Broadband, 650 - 1,050 nm, achromatic lens focal length, 200 mm</td></tr><tr><td>Achromatic Half Waveplate</td><td>Union Optic</td><td>WPA2210-650-1100-M25.4</td><td>Broadband half waveplate</td></tr><tr><td>Achromatic Quarter Waveplate</td><td>Union Optic</td><td>WPA4210-650-1100-M25.4</td><td>Broadband quarter waveplate</td></tr><tr><td>Beam Sampler</td><td>THORLABS</td><td>BSN11</td><td>10:90 Plate Beamsplitter</td></tr><tr><td>Dichroic Mirror</td><td>THORLABS</td><td>DMSP1000</td><td>Other dichroics with a center wavelength around 1,000 nm can be used.</td></tr><tr><td>DMSO (Dimethyl sulfoxide)</td><td>Sigma Aldrich</td><td>472301</td><td>Solvent for calibration of Raman shift. Other solvents with known Raman peaks can be used.</td></tr><tr><td>Electrooptic Amplitude Modulator</td><td>THORLABS</td><td>EO-AM-NR-C1</td><td>Two EOMs are needed for orthogonal modulation and dual-channel imaging. Resonant version is recommended so lower driving voltage can be used.</td></tr><tr><td>False H&amp;amp;E Staining Script</td><td>Matlab</td><td></td><td>https://github.com/TheFuGroup/HE_Staining</td></tr><tr><td>Fanout Buffer</td><td>PRL-414B</td><td>Pulse Research Lab</td><td>1:4 TTL/CMOS Fanout Buffer and Line Driver, for generating the EOM driving frequency and the reference to the lock-in</td></tr><tr><td>Fast Photodiode</td><td>THORLABS</td><td>DET10A2</td><td>Si Detector, 1 ns Rise Time</td></tr><tr><td>Frequency Divider</td><td>PRL-220A</td><td>Pulse Research Lab</td><td>TTL Freq. Divider (f/2, f/4, f/8, f/16), for generating 20MHz from the laser output.</td></tr><tr><td>Highly Dispersive Glass Rods</td><td>Union Optic</td><td>CYLROD01</td><td>High dispersion H-ZF52A Rod lens 120 mm, SF11 Rod lens 100 mm</td></tr><tr><td>Insight DS+</td><td>Newport</td><td></td><td>Laser system capable of outputting two synchronzied pulsed lasers (one fixed beam at 1, 040 nm and one tunable beam, ranging from 680-1,300 nm) with a repetition rate of 80 MHz.&amp;nbsp;</td></tr><tr><td>Lock-in Amplifier</td><td>Liquid Instruments</td><td>Moku Lab</td><td>Lock-in amplifier to extract SRS signal from the photodiode. A Zurich Instrument HF2LI or similar instrument can be used as well.</td></tr><tr><td>Mirrors</td><td>THORLABS</td><td>BB05-E03-10</td><td>Broadband Dielectric Mirror, 750 - 1,100 nm. Silver mirrors can also be used.</td></tr><tr><td>Motorized Delay Stage</td><td>Zaber</td><td>X-DMQ12P-DE52</td><td>Delay stage for fine control of the temporal overlap of the pump and the Stokes lasers. Any other motorized stage should work.</td></tr><tr><td>Oil Immersion Condensor</td><td>Nikon</td><td>CSC1003</td><td>1.4 NA. Other condensers with NA&gt;1.2 can be used.</td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TDS7054</td><td>Any other oscilloscope with 400 MHz bandwdith or higher should work.</td></tr><tr><td>Phase Shifter</td><td>SigaTek</td><td>SF50A2</td><td>For shifting the phase of the modulation frequency</td></tr><tr><td>Photodiode</td><td>Hamamatsu Corp</td><td>S3994-01</td><td>Silicon PIN diode with large area (10 x 10 cm&lt;sup&gt;2&lt;/sup&gt;). Other diodes with large area and low capacitance can be used.</td></tr><tr><td>Polarizing Beam Splitter</td><td>Union Optic</td><td>PBS9025-620-1000</td><td>Broadband polarizing beamsplitter</td></tr><tr><td>Refactive Index Database</td><td></td><td></td><td>refractiveindex.info</td></tr><tr><td>Retro-reflector</td><td>Edmund Optics</td><td>34-408</td><td>BBAR Right Angle Prism. Other prisms or retroreflector can be used.</td></tr><tr><td>RF Power Amplifier</td><td>Minicircuits</td><td>ZHL-1-2W+</td><td>Gain Block, 5 - 500 MHz, 50 &amp;Omega;</td></tr><tr><td>Scan Mirrors</td><td>Cambridge Technologies</td><td>6215H</td><td>We used a 5mm mirror set with silver coating</td></tr><tr><td>ScanImage</td><td>Vidrio</td><td>ScanImage Basic</td><td>Laser scanning microscope control software</td></tr><tr><td>Shortpass Filter</td><td>THORLABS</td><td>FESH1000</td><td>25.0 mm Premium Shortpass Filter, Cut-Off Wavelength: 1,000 nm. For efficient suppression of the Stokes, two filters may be necessary.</td></tr><tr><td>Upright Microscope</td><td>Nikon</td><td>Eclipse FN1</td><td>Any other microscope frame can be used. If a laser scanning microscope is available, it can be used directly. Otherwise, a galvo scanner and scan lens needed to be added to the microscope.</td></tr><tr><td>Water Immersion Objective</td><td>Olympus</td><td>XLPLN25XWMP2</td><td>The multiphoton 25X Objective has a NA of 1.05. Other similar objectives can be used.</td></tr></tbody></table>

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.

Optimizing spectral resolution: 
Dispersion through a material is affected by the dispersive medium (length and material) and wavelength. Changing the dispersion rod length affects the spectral resolution and the signal size. It is a give-and-take relationship that can be weighed differently depending on the application. The rods stretch out the beam pulse from being wide in frequency and narrow in time to being narrow in frequency and broad in time. Figure 7 shows the ...

Watch this Scientific Journal Video about Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis at JoVE.com

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

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 ...

real-time-two-color-stimulated-raman-scattering-imaging-mouse-brain

Research

JoVE Journal

Bioengineering

3.0K Views.  University of Washington. 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.

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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Medicine

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Neuroscience

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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.

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) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

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

Summary

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Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Highly-Multiplexed Tissue Imaging with Raman Dyes

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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