This work was supported by grants from the Research to Prevent Blindness Foundation (Career Development Award to P.R.W. and an unrestricted grant to the Department of Ophthalmology and Visual Sciences at Washington University School of Medicine in St. Louis), National Glaucoma Research (a program of BrightFocus Foundation), and the McDonnell Center for Cellular and Molecular Neurobiology. Z.W. is supported by an Institutional National Research Service Award T32 EY013360. This work was also supported by the Hope Center Viral Vectors Core at Washington University School of Medicine.

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The authors have nothing to disclose.

The two-photon imaging procedure described herein enables longitudinal in vivo imaging of the mouse retina. Repeatable images of the same region of retina can be obtained for a continuous period of up to 6 or more h under isoflurane. The mouse can also be imaged on different days using cellular and vascular landmarks to locate the same imaging area (Figure 3). The use of a clear gel immersion combined with cover glass for this purpose has previously been applied to a range of procedures, including visualization of retina for subretinal injection, laser-induced retinal injury models, and fundus imaging20,21,22.
The anatomy of the eye presents unique challenges to in vivo imaging, as the high optical power of the mouse cornea and lens impedes direct imaging though the pupil without correction. Several other in vivo imaging methods rely on the use of a plano-concave contact lens for correction of the anterior optics of the mouse eye7,17,18,19.&#160;With only optical correction at the cornea, the high optical power of the mouse lens results in an inevitable amount of parallax, particularly of structures in the peripheral scan field, manifesting as stretching and translational movement in the X-Y dimension at different Z-planes. To minimize image parallax-related distortion in X and Y dimensions, it is crucial that the mouse eye is oriented such that the tangent plane to the retina at the imaging area is perpendicular to the microscope light path. The setup described here is conducive to precise manipulation of the angle of the eye to achieve this alignment. An adjustable mouse head holder that permits rotation along two axes allows for easy manual adjustments of the angle of the eye as the experimenter scrolls through the Z-dimension to minimize parallax. This tilting also circumvents the field stop effect of the pupil to permit imaging greater areas of the retina. The restraint of the head holder also greatly reduces motion artifacts caused by respiration.
Care must be taken to maintain clarity of the mouse eye, as image quality will deteriorate with opacification during continuous imaging. Frequent reapplication of lubricant gel during imaging, and ointment application after each imaging session help to prevent the eye from drying and developing opacities. Some corneal opacities will spontaneously resolve after 24-48 h. The use of clear gel and cover glass as described in this protocol provides for similar image quality and aberration correction as a contact lens7, while allowing for easier adjustments of the eye angle without the need to realign the cover glass. Additionally, the gel provides continual hydration to the eye, making it possible to perform acute imaging sessions of up to several hours. Finally, since the cover glass does not contact the cornea, it causes minimal irritation to the eye that may reduce optical clarity for repeat imaging sessions.
A limitation of this approach is the fact that optical aberrations are not entirely corrected. While this severely diminishes axial resolution due to the heavy parallax, quantitative measurements of the soma can be obtained in single-image planes. It should be noted that as fluorescence signal intensity of retinal neurons is dependent on sample alignment with this method, excitation and emission ratiometric based sensors are more appropriate for experiments comparing samples chronically across different imaging sessions. An approach to correct optical aberrations at the system level is adaptive optics, which allows for subcellular resolution in the retina8,9,14,21. However, adaptive optics requires highly specialized equipment and extensive expertise to implement.
Alternative approaches to two-photon in vivo&#160;retinal imaging are confocal microscopy or ophthalmoscopy6. The approach presented here should be readily translatable to widefield or&#160;confocal microscopy. Single photon imaging is perhaps more robust and poses less risk of damaging the retina due to high energy of the two-photon laser necessary to achieve efficient two-photon effect through the cornea and lens of the eye. To avoid two-photon laser damage, the threshold for maximal laser power should be empirically determined by examining wholemount retinas after completion of imaging experiments and immunostaining for cell types in the layers imaged. In the system presented here, RGCs were labelled with the pan-RGC marker, Rbpms, and densities were normal up to 45 mW imaging power, whereas 55 mW caused a significant loss of RGCs (not shown).
A drawback of single-photon imaging is the fact that this approach will very heavily stimulate the native visual circuits of the retina compared to two-photon imaging23. Previous experiments using retinal wholemounts or eyecup preparations have shown that two-photon laser scanning elicits circuit activation that is largely transient24. Here, imaging of RGC activity with the Ca2+ sensor Twitch2b shows that the onset of laser scanning induces Ca2+ elevations, which return to baseline over the course of 5-20 s in most RGCs (Figure 8). Given that the laser power in this protocol is in the range of previous experiments reporting in vivo&#160;retinal light response8, the currently described method is likely amenable to recordings of circuit activity in the retina.&#160;Such considerations are important for experiments that may be influenced by circuit activity.
This protocol demonstrates in vivo imaging of two types of retinal neurons, RGCs and amacrine cells. Similar labeling of other major cell types can be achieved, including horizontal cells (Cx57-Cre25), bipolar cells (Chx10-Cre26; mGluR6-GFP27), cone photoreceptors (S- or M-opsin-Cre28), rod photoreceptors (Nrl-Cre29), M&#252;ller glia (Foxg1-Cre26), and pericytes (NG2-DsRed9). Transgenic mice are also available to label discrete subsets of RGCs (e.g., KCNG4-Cre for &#945;RGCs30; OPN4-Cre for ipRGCs31; JAM-B-CreER for J-RGCs32) and amacrine cells (e.g., ChAT-Cre for starburst amacrine cells26 and neuropeptide promoter drivers for various amacrine cell subtypes 3,34). Viral vectors can be used to target specific cell populations in lieu of transgenic mice. Intravitreal injections of AAV2 with a ubiquitous CAG promoter element almost exclusively label RGCs, amacrine cells and horizontal cells25. Pairing the modified AAV2.7m8-Y444F capsid with an engineered mGluR6 promoter construct allows for broad labeling of ON bipolar cells35. Subretinal injections of AAV lead to an enrichment of photoreceptors, with serotype AAV2/5 having the highest transduction efficiency36. Shh10, a modified AAV6 capsid protein, paired with glial fibrillary acidic protein promoter elements has been demonstrated specific for M&#252;ller glia37.
The ability to observe cells in the central nervous system with a completely non-invasive approach can be used to study both basic properties of neural circuits8, as well as mechanisms of neurodegeneration3,4,5,6,38. Many blinding diseases target cellular populations in the retina, and in vivo imaging approaches in mice have been used to study optic nerve injury1,3,4, macular degeneration13, stroke5, glaucoma2,6, and uveitis7. Furthermore, many central nervous system neurodegenerative conditions manifest in the retina including Alzheimer&#39;s disease39, multiple sclerosis40, and Parkinson&#39;s disease41. Therefore, this readily accessible technique for in vivo imaging of the retina can be applied as a tool to study a broad set of neurodegenerative conditions.

In vivo visualization of the central nervous system generally requires invasive procedures like skull thinning and installation of glass windows or optical relay lenses. The retina is the only structure in the nervous system that can be directly observed without the need for invasive preparation as it natively receives light from the environment. The ease of optical access to the retina makes it an attractive model system for studying the central nervous system.
Live fluorescent imaging of the retina in mice has been used to track RGC death in models of glaucoma1,2, optic nerve injury1,3,4, and stroke5, as well as changes in microglial activation6,7,8 and vasculature9 in degenerative conditions. Intrinsic signals can also be used to visualize photoreceptors10,11,12 and retinal pigment epithelial cells13. Many approaches to in vivo imaging of the retina use either highly specialized devices specifically designed for ophthalmological purposes6 or highly modified optical systems to correct for the native aberrations of the cornea and lens8,9,11,12,13,14.
The present protocol demonstrates an approach to in vivo imaging of fluorescent signals in the retina at cellular resolution, utilizing a basic method of partially correcting for the anterior optics of the mouse eye. This strategy requires very minor adaptations to multiphoton microscope setups that are commonly used for in vivo imaging of the brain. As this approach is straightforward to set up, and the mice are under little stress, it is conducive to perform time-lapse experiments over both acute and chronic durations. Additionally, genetic and organic dye-based procedures that label individual retinal components, including RGCs, amacrine cells, microglia, and vasculature, are compatible with this imaging technique and enable in vivo observation of cell types and structures critical for retinal function. These tools can be adapted to label most other neuronal cell types as well as glial and vascular components of the retina.

<table><tbody><tr><td>#1.5 coverslip</td><td>ThermoFisher</td><td>152440</td><td>Richard-Allan #1.5 24 mm x 40 mm</td></tr><tr><td>50 mL glass syringe</td><td>Hamilton</td><td>80950</td><td>22G cemented needle</td></tr><tr><td>Adeno-associated virus (AAV2)</td><td>Hope Center Viral Core</td><td>NA</td><td></td></tr><tr><td>Anesthesia Air Pump</td><td>RWD Life Science</td><td>R510-30</td><td></td></tr><tr><td>Atropine</td><td>Sigma</td><td>A0132</td><td>For pupil dilator solution</td></tr><tr><td>Basic Small Animal Anesthesia Device</td><td>RWD Life Science</td><td>R500IE</td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>Sutter</td><td>B150-86-10</td><td>Outside diameter 1.50 mm, inside diameter 0.86 mm, length 10 cm</td></tr><tr><td>CFP/YFP filter cube</td><td>Chroma</td><td>custom</td><td>480/40, 505 long pass, 535/30</td></tr><tr><td>ChromoFlex - Two channel PMT detection unit</td><td>Scientifica</td><td>S-MPLG-1002</td><td></td></tr><tr><td>Circulating heating pump</td><td>Braintree Scientific</td><td>tp-700</td><td>Set to 37 &amp;deg;C</td></tr><tr><td>Cling film</td><td>VWR</td><td>10713-916</td><td></td></tr><tr><td>Compact Filter Holder</td><td>ThorLabs</td><td>DH1</td><td>Holds coverslip over mouse eye</td></tr><tr><td>Cx3cr1-GFP transgenic mice (B6.129P2(Cg)-Cx3cr1tm1Litt/J)</td><td>The Jackson Laboratory</td><td>005582</td><td></td></tr><tr><td>DAQ controller chassis</td><td>National Instruments</td><td>PXIe-1073</td><td></td></tr><tr><td>Data acquisition device</td><td>National Instruments</td><td>BNC-2090A</td><td></td></tr><tr><td>Evans Blue dye</td><td>Fisher Scientific</td><td>AAA1677409</td><td></td></tr><tr><td>FPGA module with digitizer</td><td>National Instruments</td><td>NI-5734</td><td></td></tr><tr><td>Gas Evacuation Apparatus</td><td>RWD Life Science</td><td>R546W</td><td></td></tr><tr><td>GenTeal Severe lubricant eye gel&amp;nbsp;&amp;nbsp;</td><td>Alcon</td><td>(from local pharmacy)</td><td>For use during imaging</td></tr><tr><td>GFP/Red filter cube</td><td>Chroma</td><td>custom</td><td>535/30, 560 long pass, 605/70</td></tr><tr><td>Heating pad</td><td>McKesson Medical and Surgical</td><td>190147</td><td></td></tr><tr><td>HyperScope Launch Optics for use with Pockels Cell</td><td>Scientifica</td><td>S-MP-101080</td><td></td></tr><tr><td>HyperScope Main module</td><td>Scientifica</td><td>S-MP-100466</td><td></td></tr><tr><td>HyperScope Scan Path</td><td>Scientifica</td><td>S-MP-100406</td><td></td></tr><tr><td>HyperScope X galvo Module</td><td>Scientifica</td><td>MP-100443</td><td></td></tr><tr><td>ImageJ Fiji software</td><td></td><td>Freeware</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>NDC 14043-704-06</td><td></td></tr><tr><td>Isoflurane gas filter cannister (active scavenging)</td><td>RWD Life Science</td><td>R510-31</td><td></td></tr><tr><td>Isoflurane gas filter cannister (passive scavenging)</td><td>RWD Life Science</td><td>R510-31S</td><td></td></tr><tr><td>ketamine HCl (100 mg/mL)</td><td>Vedco</td><td>NDC 50989-161-06</td><td></td></tr><tr><td>M32 to M26 adapter</td><td>ThorLabs</td><td>M32M26S</td><td></td></tr><tr><td>MaiTai GUI software</td><td>Spectra-Physics</td><td>NA</td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks</td><td>NA</td><td>R2015b</td></tr><tr><td>meloxicam (5 mg/mL)</td><td>Boehringer Ingelheim</td><td>NDC 0010-6013-01</td><td>Analgesic</td></tr><tr><td>Micorscope Objective</td><td>Edmund Optics</td><td>46-404</td><td>Mitutoyo WE715042319</td></tr><tr><td>micropipette puller</td><td>Sutter</td><td>Flaming/Brown Model P-97</td><td></td></tr><tr><td>Mineral oil</td><td>Fisher</td><td>BP2629-1</td><td></td></tr><tr><td>Mini bulldog hemostatic clamp</td><td>Fine Science Tools</td><td>18053-28</td><td></td></tr><tr><td>Miniature EVA Tubing 0.02&quot; ID, 0.06&quot; OD</td><td>McMaster Carr</td><td>1883T1</td><td></td></tr><tr><td>Miniature EVA Tubing 0.05&quot; ID, 0.09&quot; OD</td><td>McMaster Carr</td><td>1883T4</td><td></td></tr><tr><td>Mouse head holder</td><td>Narishige</td><td>SGM-4</td><td></td></tr><tr><td>No. 5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Optic Posts 1/2&quot;</td><td>ThorLabs</td><td>TR3-P5</td><td></td></tr><tr><td>Optical power meter kit</td><td>ThorLabs</td><td>PM100D</td><td></td></tr><tr><td>pE-300 Ultra LLG Deivery</td><td>Scientifica</td><td>COO-LED3ULLGs</td><td></td></tr><tr><td>Phenylephrine hydrochloride</td><td>Sigma</td><td>P6126</td><td>For pupil dilator solution</td></tr><tr><td>Pockels cell</td><td>Conoptics</td><td>350-80-02</td><td></td></tr><tr><td>Pockels cell amplifier</td><td>Conoptics</td><td>Model 302RM</td><td></td></tr><tr><td>Proparacaine hydrochloride</td><td>Sigma</td><td>1571001</td><td>For eye immobilization</td></tr><tr><td>Red &amp;amp; Far Red short pass filter Cube</td><td>Chroma</td><td>custom</td><td>560 short pass</td></tr><tr><td>Rotating 1/2&quot; post clamp</td><td>ThorLabs</td><td>SWC</td><td></td></tr><tr><td>ScanImage package</td><td>Vidrio Technologies</td><td>Freeware</td><td>Image acquisition software; Version 5.4.0 (2018); requires MATLAB</td></tr><tr><td>sodium chloride solution, sterile (0.9%)</td><td>Fresenius Kabi</td><td>NDC 63323-186-01</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>S9 E</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Oxford</td><td>Benchmate C8</td><td></td></tr><tr><td>Terramycin oxytetracycline/polymyxin B antibiotic ophthalmic ointment</td><td>Zoetisus</td><td>NA</td><td>For use after intravitreal injection</td></tr><tr><td>ThermoRack cooling system</td><td>Solid State Cooling Systems</td><td>ThermoRack 401</td><td>Set to 20 &amp;deg;C</td></tr><tr><td>Ultrafast Ti:Sapphire laser</td><td>Spectra-Physics</td><td>Mai Tai DeepSee</td><td></td></tr><tr><td>Vgat-Cre transgenic mice (Slc32a1tm2(cre)Lowl/J)</td><td>The Jackson Laboratory</td><td>016962</td><td></td></tr><tr><td>VGlut2-Cre transgenic mice (Slc17a6tm2(cre)Lowl/J)</td><td>The Jackson Laboratory</td><td>016963</td><td></td></tr><tr><td>VivoScope for In Vivo Imaging</td><td>Scientifica</td><td>S-MPVS-1200-00P</td><td></td></tr><tr><td>White petrolatum-mineral oil lubricant eye ointment</td><td>Stye</td><td>NA</td><td>For use after imaging</td></tr><tr><td>xylazine HCl (20 mg/mL)</td><td>Akorn</td><td>NDC 59399-110-20</td><td></td></tr></tbody></table>

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.

Various transgenic, viral vector, or inorganic dye-labeling approaches can be used to specifically visualize several retinal cells types and structures in vivo using a simple adaptation of a basic multiphoton microscope. To visualize RGCs and amacrine cells, VGlut2-Cre and VGat-Cre transgenic mice, respectively, were given an intravitreal injection of a Cre-dependent AAV expression construct encoding Twitch2b, a cytoplasmic fluorescence resonance energy transfer (FRET)-based Ca2+ sensor that contains cyan and yellow fluorescent proteins (CFP and YFP, respectively) and the Ca2+ binding domain of troponin15. In VGlut2-Cre mice, RGC somas are clearly discernable, and fascicles of axons are often apparent (Figure 3).
It should be noted that the trajectory of axons and the negative image of the vasculature makes it very straightforward to identify the optic nerve head in VGlut2-Cre mice, which is useful as a landmark in chronic imaging experiments (Figure 3). Although amacrine cells appear less bright than RGCs, possibly due to their smaller soma size and/or less efficient AAV transduction, their somas are still readily apparent in the inner nuclear layer. In contrast to RGCs, amacrine cell neurites are more often observed in the inner plexiform layers (Figure 4). Retinal microglia can be imaged in the Cx3cr1-GFP transgenic mouse line6. Microglia associate with the vasculature, making it possible to find the same region in time-lapse imaging experiments.
This approach can be used to track the dynamics of fine microglia processes, a procedure that has better spatial resolution in single-plane images, or if maximum-intensity projections are prepared focusing on individual cells (Figure 5). The poor axial resolution caused by optical aberration through the mouse lens precludes examination of fine details in the z-dimension. To determine whether this imaging technique can observe degenerative changes in cellular ultrastructure, 1 &#181;L of 50 mM N-methyl-D-aspartate (NMDA) was injected into the vitreous to induce excitotoxic lesion. One day after injection, the microglia demonstrated short processes or amoeboid morphology (Figure 5) in accordance with previous reports16. It should be noted that cells in the Cx3cr1-GFP transgenic line exhibited more uniform and complete fluorescent protein expression across the cellular cohort than in experiments with AAV-mediated delivery of fluorescent protein expression cassettes. The benefits of varied and sparse versus complete and uniform labeling should be considered when designing experiments.
To label retinal vasculature as previously described8, mice were injected intraperitoneally with 200 &#181;L of Evans blue dye (20 mg/mL in sterile saline) 30-60 min before imaging. This led to strong labeling of blood vessels emanating from the optic nerve head (Figure 6). Surprisingly, the fluorescent signal of a single injection persisted for at least seven days. Two distinct methods were used to estimate the dimensions of the in vivo images. First, the same retinal regions were imaged in vivo and after fixation in flattened retinal wholemounts using confocal microscopy (Figure 7). Random cell pairs were selected from four different in vivo&#160;samples, and the true distance between cell pairs was measured in confocal scans and matched with in vivo pixel distance to obtain an average pixel size of 0.99 &#181;m with 1x digital magnification. Using similar methods correlating in vivo images with confocal wholemount scans has revealed that a single head position allows for imaging over a roughly 650 mm2 patch of retina.
Repositioning of the head holder along one torsional axis can allow access to a linear region of the retina 2.2 mm in length (not shown). Further, 1 or 2 &#181;m diameter fluorescent microspheres were injected into the eyes of mice and their diameter was measured as full-width half-maximum of line scans from in vivo images with 10x digital zoom. This gave a slightly larger pixel size estimate, but with more variance (Figure 7). Overall, confocal imaging of wholemount samples after completion of in vivo experiments is the most consistent method to assign scale to individual images, as variance in corneal and lens properties may alter image scale from sample to sample.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61970/61970fig1v2.jpg" /> 
Figure 1: Light path schematic. The basic components of the two-photon microscope used in this protocol consist of a Pockels Cell to modulate laser power, a lens pair to reduce the laser beam diameter to match the back aperture of the microscope objective, and a pair of galvo scan mirrors for beam steering. A pair of steering mirrors is present before each major optical component. The focus is controlled by a motor that drives the objective mount. The emission light path can be customized for different fluorophores by changing out dichroic and barrier filters. A general setup for cyan/yellow/red imaging is displayed in which a short pass dichroic mirror directs red light to the first PMT, and a long pass dichroic mirror paired with appropriate band pass filters is used to separate cyan and yellow emissions. Abbreviation: PMT = photomultiplier tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61970/61970fig2v2.jpg" /> 
Figure 2: Positioning mice for in vivo imaging. To position the mouse with the pupil on axis with the light path, the anesthetized mouse is first restrained in a head holder, the head is rotated and angled, a large drop of lubricant eye gel is placed on the eye, and the mouse is placed on the stage. A coverslip is mounted in the coverslip holder perpendicular to the light path and lowered down towards the eye. The coverslip should not contact the cornea or mouse head (left), which will be evident if the coverslip is deflected. However, the coverslip should also be close enough to avoid waisting of the droplet (right), because this will have a demagnifying effect on the sample. After applying the gel immersion and securing the coverslip, the stage should be moved in place directly under the microscope objective. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig2v2large.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/61970/61970fig3v3.jpg" /> 
Figure 3: Imaging retinal ganglion cells. For image display, maximum intensity projections with the z-planes containing cells of interest are created, and resultant images are median-filtered to remove PMT shot noise. Two examples of retinal ganglion cells labelled by injecting AAV-EF1&#945;-FLEX-Twitch2b into VGlut2-Cre transgenic mice are shown, specifically the CFP signal. Images were acquired at sessions four days apart, and vascular landmarks were used to return to the same region near the optic nerve head. The optic nerve head is oriented towards the bottom of the image. Although both samples show some variance in orientation (regions with decreased intensity are indicated with arrows), most cells are present at both time points. Scale bar = approximately 50 &#181;m. Abbreviations: PMT = photomultiplier tube; AAV = adeno-associated virus; EF1&#945; = elongation factor-1alpha; FLEX = flip-excision; VGlut2 = vesicular glutamate transporter 2; CFP = cyan fluorescent protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61970/61970fig5v2.jpg" /> 
Figure 4: Imaging amacrine cells. Amacrine cells were labeled by injecting AAV-EF1&#945;-FLEX-Twitch2b into VGat-Cre transgenic mice. The CFP signal of Twitch 2b is specifically shown. Small maximum-intensity projections focused on the depths of the inner nuclear layer indicate amacrine cell somas, while focusing on the inner plexiform resolves amacrine cell neurites (arrow). The optic nerve head is oriented towards the right of the image. Scale bar = approximately 50 &#181;m. Abbreviations: AAV = adeno-associated virus; EF1&#945; = elongation factor-1alpha; FLEX = flip-excision; VGat = vesicular gamma aminobutyric acid transporter; CFP = cyan fluorescent protein; INL = inner nuclear layer; IPL = inner plexiform layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61970/61970fig4v2.jpg" /> 
Figure 5: Imaging microglia. The transgenic mouse line Cx3cr1-GFP was used to label microglia. A maximum-intensity projection of the full scan volume shows many microglia, some with fine process detail that can be resolved. Note that cells towards the lower left of the field have less distortion in the maximum projection than those towards the upper right due to parallax in this region. Maximum-intensity projections containing only the cell of interest significantly reduces this parallax (center, boxed in corresponding colors). Furthermore, this imaging strategy can document the dynamics of fine microglia process remodeling (lower panels). Comparatively, many microglia can be seen with short processes or amoeboid morphology one day after an excitotoxic lesion by intravitreal injection of 50 mM NMDA (right). Scale bar = approximately 50 &#181;m. Abbreviations: GFP = green fluorescent protein; Cx3cr1 = Cx3 chemokine receptor 1; NMDA = N-methyl-D-aspartate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61970/61970fig6v2.jpg" /> 
Figure 6: Labelling vascular landmarks. Mice were injected with 200 &#181;L of 20 mg/mL Evans blue intraperitoneally 30-60 min prior to the first imaging session. Full thickness maximum-intensity projections demonstrate lasting fluorescence in the retinal vasculature that persisted for at least seven days. Scale bar = approximately 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61970/61970fig7v2.jpg" /> 
Figure 7: Image dimensions. Retinal ganglion cells labelled by injecting AAV-EF1&#945;-FLEX-Twitch2b into VGlut2-Cre transgenic mice were imaged in vivo, and the same region was then imaged by confocal laser scanning microscopy after fixation and wholemount preparation of the retina. Yellow fluorescent protein channel is shown for both. Colored arrow pairs indicate the same cell in both preparations (upper panels). Single-plane image of 2 &#181;m diameter fluorescent microspheres injected intravitreally and imaged in vivo&#160;(lower left panel). Microspheres did not settle and thus were in constant motion making measurement of axial resolution impossible. Pixel sizes calculated from full-width half-maximum measurements of in vivo fluorescent microspheres or correlative confocal measurements taken from 2-4 retinas per group (lower right). Scale bar = 50 &#181;m. Abbreviations: AAV = adeno-associated virus; EF1&#945; = elongation factor-1alpha; FLEX = flip-excision; VGlut2 = vesicular glutamate transporter 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61970/61970fig8v2.jpg" /> 
Figure 8: Calcium activity induced by two-photon scanning. Retinal ganglion cells labelled by injecting AAV-EF1&#945;-FLEX-Twitch2b into VGlut2-Cre transgenic mice, YFP is pseudocolored magenta and CFP green, imaged in a single plane as a time series at 4.22 Hz. All RGCs had a similar starting YFP/CFP ratio. Most responded with an increase in FRET ratio (excluding the orange cell), and one maintained a high YFP/CFP ratio throughout the time series (yellow cell). YFP/CFP ratios were normalized to the first frame average, and colored circles match with colored traces. Asterisks indicate time points with representative images displayed on left. Scale bar = 20 &#181;m. Abbreviations: AAV = adeno-associated virus; EF1&#945; = elongation factor-1alpha; FLEX = flip-excision; VGlut2 = vesicular glutamate transporter 2; YFP = yellow fluorescent protein; CFP = cyan fluorescent protein; RGCs = retinal ganglion cells; FRET = fluorescence resonance energy transfer.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61970/61970fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina at JoVE.com

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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 ...

transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina

Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.2K Views.  Washington University School of Medicine. 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.

Video: Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Cancer Research

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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 ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Bioengineering

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 ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Medicine

 Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature 

Source:<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/53582/quantification-of-cerebral-vascular-architecture-using-two-photon-microscopy-in-a-mouse-model-of-hiv-induced-neuroinflammation'><span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:10pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Nishimura, C.&#160;et al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016).</a> &#160;This video demonstrates the in vivo visualization of cortical capillaries in the mouse brain using two-photon microscopy. It outlines the steps for injecting a fluorescent dye into an anesthetized mouse with a thin-skull cortical window, preparing the setup for imaging, and acquiring detailed images of brain microvascular structures for analysis.

Source:&#160;Nishimura, C.&#160;et al. Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurovascular & Blood Flow Imaging

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Live Imaging & In Vivo Tracking

Two-Photon Microscopy for Imaging Neuronal Morphology  in a Mouse Pup

Source:&#160;<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/58340/in-vivo-two-photon-imaging-of-cortical-neurons-in-neonatal-mice'>Mizuno, H.,&#160;et al.&#160;In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice.&#160;J. Vis. Exp. (2018)</a>This video showcases two-photon microscopy being used to image neurons in the brain of an anesthetized transgenic mouse pup. The two-photon laser allows for deeper imaging with high spatial resolution and minimal light scattering, enabling the detailed capture of neuronal morphology, including dendritic projections.

Source:&#160;Mizuno, H.,&#160;et al.&#160;In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice.&#160;J. Vis. Exp. (2018)This video showcases ...

Synaptic & Cellular Imaging

Visualizing Protein Kinase A Activity in a Mouse Using Two-Photon Fluorescence Lifetime Imaging Microscopy 

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/59526/visualizing-protein-kinase-a-activity-in-head-fixed-behaving-mice-using-in-vivo-two-photon-fluorescence-lifetime-imaging-microscopy'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Jongbloets, B. C.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy. J. Vis. Exp. (2019)</a> &#160;This video demonstrates a two-photon fluorescence lifetime imaging microscopy procedure for visualizing protein kinase A activity in head-fixed, behaving mice during enforced locomotion.

Source:&#160;Jongbloets, B. C.,&#160;et al. Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Summary

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Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature

Two-Photon In Vivo Imaging of the Mouse Retina

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Visualizing Protein Kinase A Activity in a Mouse Using Two-Photon Fluorescence Lifetime Imaging Microscopy