This work was supported by a grant of the Swiss National Science Foundation (SNSF; #320030_156019). The authors received nonfinancial support from Heidelberg Engineering GmBH, Germany.

In all procedures, BALB/c adult male and female mice who express gfp under the promoter of Cx3cr1 were used24. Mice were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and all procedures were approved from the Swiss government according to the Federal Swiss Regulations on Animal Welfare. Mice were anesthetized by a subcutaneous injection of medetomidine hydrochloride (0.75 mg/kg) and ketamine (45 mg/kg). Proper anesthesia was confirmed by monitoring the respiratory rate and the animal&#39;s reflex against a tail pinch. At the end of the experiments, mice were euthanized with CO2 inhalation.
NOTE: Perform each imaging session as quickly as possible (maximum 20 min), since cataract formation following anesthesia may hamper retinal visualization30.
1. System Configuration
<ol>
	<li>Turn on the computer.</li>
	<li>Turn on the power supply. The message &#34;Start acquisition module&#34; will appear on the device`s control panel screen.</li>
	<li>Double click on the software`s desktop shortcut to operate the software.</li>
	<li>On the database view, click the &#34;add patient&#34; icon.</li>
	<li>In the pop-up window, insert all necessary information (last name, first name, title, date of birth, sex, and patient-ID) and click &#34;ok&#34;. On the pop window set the corneal curvature to 2 mm and click &#34;ok&#34;.</li>
	<li>Position a 78D standard ophthalmic non-contact slit lamp lens in front of the standard 30 &#176; optic and fasten it safely with tape.</li>
	<li>Make sure that the filter lever on the left of the device is positioned on the indication &#34;A&#34; to allow both IR+OCT and AF imaging (Figure 1).</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Use a medetomidine hydrochloride dose of 0.75 mg/kg and a ketamine dose of 45 mg/kg in phosphate-buffered saline (PBS, pH 7.4) to prepare anesthesia solution. Thus, for a 20 g mouse, mix 15 &#181;L of medetomidine hydrochloride with 18 &#181;L of ketamine to a final volume of 50 &#181;L in PBS. Store the solution at 4 &#176;C for up to one week.</li>
	<li>Grasp the mouse by the scruff and apply one drop of tropicamide 0.5% + phenylephrine hydrochloride 2.5% on each eye to achieve pupil dilation.</li>
	<li>Keep the mouse restrained, and with an insulin syringe attached to a 30G needle, inject 50 &#181;L of the anesthesia solution subcutaneously. Put the mouse back in its cage that is placed above a heating pad. Wait 3-5 min until the mouse is fully sedated. To assess anesthetic depth, pinch the tail of the mouse. Check the corneal reflex by gently touching the mouse cornea with a cotton swab. If no positive reflex is observed, continue with the imaging. 
	NOTE: It is important to monitor respiratory rate during anesthesia. If the mouse is not fully sedated, the respiratory rate will increase upon a painful stimulus.</li>
	<li>Hydrate the mouse cornea with an application of 2% hydroxypropylmethylcellulose drops on each eye.</li>
</ol>
3. SD-OCT
<ol>
	<li>Place a warming patch of approximately 32 &#176;C on the custom-made platform attached on the device`s chin rest (Figure 1).</li>
	<li>To image the right eye, place the mouse on the left part of the platform (Figure 1). Ensure that the right orbit of the mouse faces the lens and its body lies prone on the left part of the platform.</li>
	<li>Apply a drop of hydroxypropylmethylcellulose on the right eye and carefully position a +4 diopter rigid gas permeable contact lens on it (spherical power: -25.00 to +25.00 diopters). Store the contact lens in balanced salt solution (BSS). To grab the lens, use plastic or silicon forceps to avoid damage.</li>
	<li>Press the yellow box on the right corner of the control panel display to start the acquisition module (green box on Figure 2A). The yellow box will turn green and the control panel menu will appear on the screen (Figure 2A).</li>
	<li>For acquisition of B-scans, select the option IR+OCT on the control panel (Figure 2A). Selected settings are highlighted in blue on the control panel.</li>
	<li>Select &#34;Retina&#34; under &#34;Application and Structure&#34; in the software and move the lens towards the mouse eye using the micromanipulator on the device (Figure 1). Before focusing onto the retina, check that the indication &#34;OD&#34; is selected on the left bottom part of the screen. The software automatically identifies the left (OS) and the right (OD) eye based on the position of the objective. 
	NOTE: If the objective is positioned on the middle of the platform, an error message will appear on the bottom of the screen. In that case, re-position the mouse slightly until the software recognizes the correct eye.</li>
	<li>With the focus knob, focus on the retina until the big vessels are clearly seen in the fundus image on the left of the computer screen. Move the camera by turning the micromanipulator left or right, to move the camera in that direction, or clockwise or counterclockwise, to move the camera up or down, respectively. 
	NOTE: Reposition the mouse on the platform or move the lens upwards or downwards to achieve the preferred localization of the optic nerve head in the infrared image.</li>
	<li>Turn the sensitivity knob counterclockwise to reduce or clockwise to increase the brightness of the fundus image. When optimal focus is achieved, an SD-OCT B-scan will appear on the right of the screen. 
	NOTE: If the B-scan cannot be visualized by adjusting the focus, press the combination Ctrl+Alt+Shift+O on the keyboard. In the pop-up window, adjust the value of the reference arm until the B-scan appears on the screen.</li>
	<li>On the bottom right of the screen, choose the single scan from the pattern menu (single line in Figure 2B).</li>
	<li>Turn the micromanipulator of the device (Figure 1) left, right, forward, or backward (to turn the camera in that direction) to ensure that the B-scan is located between the top and bottom corners of the SD-OCT scan window.</li>
	<li>Set the Automatic Real-Time (ART) value to at least 9 to obtain high quality images. 
	NOTE: ART improves image quality by averaging several consecutive scans. The higher the &#34;ART&#34; value, the higher the signal-to-noise ratio and thus the image quality. However, by increasing the &#34;ART&#34; value, the acquiring time is also increased.</li>
	<li>Press the &#34;acquire&#34; button (Figure 2) on the control panel screen and acquire the images.</li>
	<li>Re-position the mouse to image the left eye and repeat steps 3.2-3.12.</li>
	<li>Remove the contact lens with plastic/silicon forceps and place it into BSS.</li>
	<li>Hydrate the mouse cornea with a fresh drop of hydroxypropylmethylcellulose and remove the excess with a tissue paper.</li>
	<li>Remove the standard 30 &#176; optic by rotating it counterclockwise.</li>
	<li>Mount the 55 &#176; lens and repeat steps 3.4-3.12 for each eye.</li>
</ol>
4. Auto Fluorescence Imaging
<ol>
	<li>Without moving the mouse on the platform, select IR on the control panel.</li>
	<li>With the focus knob, focus on the big retinal vessels.</li>
	<li>On the control panel select AF.</li>
	<li>Turn the sensitivity knob counterclockwise to reduce or clockwise to increase the brightness of the image.</li>
	<li>Press the sensitivity knob and set the &#34;ART&#34; value to 67 or more to obtain high quality images.</li>
	<li>When the set &#34;ART&#34; value has been reached, press &#34;acquire&#34; on the control panel to acquire the image. Press the sensitivity knob again to stop averaging. Adjust the focus, to visualize different retinal layers.</li>
	<li>Remove the 55&#176;&#160; lens and mount the wide field 102&#176;&#160; lens. Follow steps 4.1&#8211;4.6 for both eyes of each mouse and for every mouse.</li>
	<li>Click &#8220;Save images&#8221; on the left upper border of the window and then click &#8220;exit&#8221;. To save the images in the computer, select the mouse from the list on the right part of the screen by double clicking on the mouse name. Double click on each image separately and then click on the floppy disc icon. Save the image as .tif, .bmp, .jpg, or .png at the desired folder. To exit the software press &#8220;File&#8221; and &#8220;Exit&#8221;.</li>
</ol>
5. Anesthesia Reversal
<ol>
	<li>Hydrate the mouse eyes with one drop of hydroxypropylmethylcellulose and return the mouse on a heating pad.</li>
	<li>Prepare atipamezole solution in a dose of 2.25 mg/kg to reverse the sedative effects of medetomidine hydrochloride. For a 20 g mouse, add 9 &#181;L of atipamezol in a final volume of 150 &#181;L PBS. Store the solution at 4 &#176;C for up to one week.</li>
	<li>min after anesthesia injection (step 2.2), inject 150 &#181;L of atipamezole solution subcutaneously.</li>
	<li>Monitor the mouse until recovery from anesthesia. When the mouse is fully recovered (5-10 min after atipamezole injection) put it back in its cage. Recovery is indicated by the ability of the animal to turn its body when laid on one side, regaining of walking activity, and reacting in response to environmental stimulation.</li>
</ol>
6. Manual Retinal Thickness Measurement from SD-OCT Images
<ol>
	<li>Open the OCT scan by double clicking on the name of the animal.</li>
	<li>Open the B-scan obtained with the 30 &#176; or 55 &#176; lens.</li>
	<li>Choose &#34;thickness profile&#34;.</li>
	<li>Press the &#34;Edit Layer Segmentations&#34; icon (Figure 2B). The software automatically identifies the inner limiting and base membrane.</li>
	<li>If necessary, manually correct the position of the inner limiting and base membrane. To do so, choose the layer to be modified from the left side of the screen and then the red circle option (Figure 2B). Move the circle with the mouse button pressed to modify the line.</li>
	<li>Modify the line to position the corresponding layer correctly. Click &#34;Save and close&#34; to exit the window. 
	NOTE: Due to reflectivity of the choroid, the software may identify the base membrane incorrectly. Thus, it is preferable to define it manually before proceeding to retinal thickness measurements.</li>
	<li>Choose &#34;Retina&#34; under the &#34;Layer&#34; option. A diagram of retinal thickness will appear on the bottom of the screen.</li>
	<li>Click on a different position either on the diagram or on the B-scan to view the retinal thickness (indicated in &#181;m) for the selected position.</li>
	<li>Measure the retinal thickness in the desired distance from the optic nerve head and copy the values in a spreadsheet.</li>
	<li>Repeat steps 5.1-5.9 for each mouse for both the 30 &#176; and the 55 &#176; lens.</li>
	<li>Perform statistical analysis with desired software.</li>
</ol>

<ol>
	<li>Madeira, M. H., Boia, R., Santos, P. F., Ambrosio, A. F., Santiago, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia-mediated+neuroinflammation+to+retinal+degenerative+diseases.">Contribution of microglia-mediated neuroinflammation to retinal degenerative diseases.</a> Mediators Inflamm. , 673090 (2015).</li><li>Ng, T. F., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+migration+of+retinal+microglia+into+the+subretinal+space.">Light-induced migration of retinal microglia into the subretinal space.</a> Invest Ophthalmol Vis Sci. 42 (13), 3301-3310 (2001).</li><li>Langmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+activation+in+retinal+degeneration.">Microglia activation in retinal degeneration.</a> J Leukoc Biol. 81 (6), 1345-1351 (2007).</li><li>Joly, S., 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=Cooperative+phagocytes:+resident+microglia+and+bone+marrow+immigrants+remove+dead+photoreceptors+in+retinal+lesions.">Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.</a> Am J Pathol. 174 (6), 2310-2323 (2009).</li><li>Arroba, A. I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+IGF-I+neuroprotection+in+the+rd10+mouse+model+of+retinitis+pigmentosa.">Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52 (12), 9124-9130 (2011).</li><li>Zhang, C., Lam, T. T., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+populations+of+microglia/macrophages+in+the+retina+and+their+activation+after+retinal+ischemia+and+reperfusion+injury.">Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury.</a> Exp Eye Res. 81 (6), 700-709 (2005).</li><li>Geng, Y., 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=Optical+properties+of+the+mouse+eye.">Optical properties of the mouse eye.</a> Biomed Opt Express. 2 (4), 717-738 (2011).</li><li>Lozano, D. C., Twa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rat+schematic+eye+from+in+vivo+biometry+and+the+correction+of+lateral+magnification+in+SD-OCT+imaging.">Development of a rat schematic eye from in vivo biometry and the correction of lateral magnification in SD-OCT imaging.</a> Invest Ophthalmol Vis Sci. 54 (9), 6446-6455 (2013).</li><li>Vaz-Pereira, S., 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=Optical+Coherence+Tomography+Features+Of+Active+And+Inactive+Retinal+Neovascularization+In+Proliferative+Diabetic+Retinopathy.">Optical Coherence Tomography Features Of Active And Inactive Retinal Neovascularization In Proliferative Diabetic Retinopathy.</a> Retina. 36 (6), 1132-1142 (2016).</li><li>Kokona, D., Haner, N. U., Ebneter, A., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+macrophage+dynamics+with+optical+coherence+tomography+in+anterior+ischemic+optic+neuropathy.">Imaging of macrophage dynamics with optical coherence tomography in anterior ischemic optic neuropathy.</a> Exp Eye Res. , (2016).</li><li>Makiyama, Y., 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=Macular+cone+abnormalities+in+retinitis+pigmentosa+with+preserved+central+vision+using+adaptive+optics+scanning+laser+ophthalmoscopy.">Macular cone abnormalities in retinitis pigmentosa with preserved central vision using adaptive optics scanning laser ophthalmoscopy.</a> PLoS One. 8 (11), e79447 (2013).</li><li>Paques, 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=High+resolution+fundus+imaging+by+confocal+scanning+laser+ophthalmoscopy+in+the+mouse.">High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse.</a> Vision Res. 46 (8-9), 1336-1345 (2006).</li><li>Joshi, 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=Spontaneously+occurring+fundus+findings+observed+using+confocal+scanning+laser+ophthalmoscopy+in+wild+type+Sprague+Dawley+rats.">Spontaneously occurring fundus findings observed using confocal scanning laser ophthalmoscopy in wild type Sprague Dawley rats.</a> Regul Toxicol Pharmacol. 77, 160-166 (2016).</li><li>Muraoka, Y., 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=Real-time+imaging+of+rabbit+retina+with+retinal+degeneration+by+using+spectral-domain+optical+coherence+tomography.">Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography.</a> PLoS One. 7 (4), e36135 (2012).</li><li>Fischer, M. 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=Noninvasive,+in+vivo+assessment+of+mouse+retinal+structure+using+optical+coherence+tomography.">Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.</a> PLoS One. 4 (10), e7507 (2009).</li><li>Bell, B. A., 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=Retinal+vasculature+of+adult+zebrafish:+in+vivo+imaging+using+confocal+scanning+laser+ophthalmoscopy.">Retinal vasculature of adult zebrafish: in vivo imaging using confocal scanning laser ophthalmoscopy.</a> Exp Eye Res. 129, 107-118 (2014).</li><li>Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-domain+optical+coherence+tomography+as+a+noninvasive+method+to+assess+damaged+and+regenerating+adult+zebrafish+retinas.">Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas.</a> Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).</li><li>Huber, 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=Spectral+domain+optical+coherence+tomography+in+mouse+models+of+retinal+degeneration.">Spectral domain optical coherence tomography in mouse models of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 50 (12), 5888-5895 (2009).</li><li>Dysli, C., Enzmann, V., Sznitman, R., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Mouse+Retinal+Layers+Using+Automated+Segmentation+of+Spectral+Domain+Optical+Coherence+Tomography+Images.">Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.</a> Transl Vis Sci Technol. 4 (4), 9 (2015).</li><li>Sim, D. A., 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=A+simple+method+for+in+vivo+labelling+of+infiltrating+leukocytes+in+the+mouse+retina+using+indocyanine+green+dye.">A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.</a> Dis Model Mech. 8 (11), 1479-1487 (2015).</li><li>Bosco, A., Romero, C. O., Ambati, B. K., Vetter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+retinal+microglial+activation+during+neurodegeneration:+confocal+ophthalmoscopic+imaging+and+cell+morphometry+in+mouse+glaucoma.">In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma.</a> J Vis Exp. (99), e52731 (2015).</li><li>Acton, J. H., Cubbidge, R. P., King, H., Galsworthy, P., Gibson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+detection+in+retro-mode+imaging+by+a+scanning+laser+ophthalmoscope.">Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope.</a> Acta Ophthalmol. 89 (5), e404-e411 (2011).</li><li>Greenstein, V. C., 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=Structural+and+functional+changes+associated+with+normal+and+abnormal+fundus+autofluorescence+in+patients+with+retinitis+pigmentosa.">Structural and functional changes associated with normal and abnormal fundus autofluorescence in patients with retinitis pigmentosa.</a> Retina. 32 (2), 349-357 (2012).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Wang, X., 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=Requirement+for+Microglia+for+the+Maintenance+of+Synaptic+Function+and+Integrity+in+the+Mature+Retina.">Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.</a> J Neurosci. 36 (9), 2827-2842 (2016).</li><li>Ebneter, A., Casson, R. J., Wood, J. P., Chidlow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+the+visual+pathway+in+experimental+glaucoma:+spatiotemporal+characterization+and+correlation+with+axonal+injury.">Microglial activation in the visual pathway in experimental glaucoma: spatiotemporal characterization and correlation with axonal injury.</a> Invest Ophthalmol Vis Sci. 51 (12), 6448-6460 (2010).</li><li>Ebneter, A., Kokona, D., Schneider, N., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Activation+and+Recruitment+of+Circulating+Macrophages+During+Ischemic+Experimental+Branch+Retinal+Vein+Occlusion.">Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.</a> Invest Ophthalmol Vis Sci. 58 (2), 944-953 (2017).</li><li>Lin, Y. L., Potter-Baker, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, C., 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. A., 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. A., 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=A+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Effect+of+Oral+CSF-1R+Kinase+Inhibitor+on+Retinal+Microglia+Revealed+by+In+Vivo+Scanning+Laser+Ophthalmoscopy.">Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy.</a> Transl Vis Sci Technol. 6 (2), 10 (2017).</li><li>Gabriele, M. 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, S., 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. H., 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

The authors have nothing to disclose.

The present article demonstrates a protocol for the acquisition of retinal B-scans and imaging of gfp positive microglia distribution in the mouse retina in the same imaging session. SD-OCT and SLO are increasingly used in animal models of retinal disease to provide information of retinal alterations over time10,14,17,18,21. With this protocol, Cx3cr1...

In experimental ophthalmology, examination of retinal pathology is usually evaluated using histological techniques. However, histology requires animal euthanization and may cause alteration to the actual properties of the tissue. SD-OCT and SLO are routinely used in clinical ophthalmology for diagnostic purposes and for the monitoring of several retinal diseases such as diabetic macular edema9, anterior ischemic optic neuropathy10, or retinitis pigmentosa11. SD-OCT and SLO are non-invasive techniques that generate high-resolution images of the retina, which are visualized through the dilated pupil without further intervention. SD-OCT provides information of retinal structure and retinal thickness by collecting backscattering data to create cross-sectional images of the retina, while SLO collects fluorescence data to produce stereoscopic high-contrast images of the retina. Nowadays, both techniques are increasingly used in experimental ophthalmology using small rodents12,13,14,15 (or even zebrafish16,17) and can provide both qualitative and quantitative information12,17,18,19,20,21.
Accumulation of endogenous fluorophores like lipofuscins or the formation of drusen in the retina can be visualized by SLO as auto fluorescent signal. This feature makes SLO a valuable technique for diagnosis and monitoring of retinal diseases such as age-related macular degeneration or retinitis pigmentosa22,23. In experimental ophthalmology, auto fluorescence imaging (AF) can be used for the detection of specific cell types in reporter mouse lines. For example, mice heterozygous for the expression of gfp under the promoter of Cx3cr124 are advantageous for in vivo visualization of microglial cells in the normal retina and for the investigation of microglia/macrophage dynamics in retinal disease21. Microglia are the resident macrophages of the retina, which play a crucial role on tissue homeostasis and tissue repair upon injury1,25,26. Microglia activation in the retina has been reported in retinal injury, ischemia, and degeneration, suggesting a role of these cells in retinal disease2,3,4,5,6.
The aim of the present protocol is to describe a relatively simple method for retinal imaging and measurement of retinal thickness using SD-OCT, and for visualization of gfp positive microglia cells in the Cx3cr1gfp/gfp mouse retina using SLO (Heidelberg Spectralis HRA+OCT system). This protocol can be utilized for imaging and thickness measurements of healthy or diseased retinas in various mouse lines. Additionally, morphometric analyses can be performed for the identification and quantification of microglia numbers and microglia activation in the retina using SLO21. Microglia cells are associated with degenerative diseases in the central nervous system (CNS), including the retina27,28,29. Thus, by combining the two methods used in the present protocol, correlation of microglia distribution and retinal degeneration can be made, which can facilitate monitoring disease severity or the effectiveness of therapeutic approaches in vivo.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

Using the protocol presented here, SD-OCT scans and SLO images were obtained from Cx3cr1gfp/gfp mice in the same imaging session. Figure 3 includes representative SD-OCT single scans obtained with a 30 &#176; or a 55 &#176; lens (Figure 3A) and representative SLO images obtained with a 55 &#176; or a 102 &#176; lens, where gfp positive microglia cells are visualized. Higher reflectivity of the choroid is obser...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

in-vivo-imaging-cx3cr1gfpgfp-reporter-mice-with-spectral-domain

Research

JoVE Journal

Medicine

10.5K Views.  Bern University Hospital and University of Bern. This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Video: In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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 ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...