Name: a custom multiphoton microscopy platform for live imaging of mouse cornea and conjunctiva
Uploaded: 2020-05-17T14:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva at JoVE.com

We thank the grant support from Ministry of Science and Technology, Taiwan (106-2627-M-002-034, 107-2314-B-182A-089, 108-2628-B-002-023, 108-2628-B-002-023), National Taiwan University Hospital (NTUH108-T17) and Chang Gung Memorial Hospital, Taiwan (CMRPG3G1621, CMRPG3G1622, CMRPG3G1623).

All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University and Chang Gung Memorial Hospital.
1. Multiphoton microscopy setup
<ol>
	<li>Build a system based on an upright microscope with water immersion 20x 1.00 NA objective (Figure 1A).</li>
	<li>Use Ti: Sapphire laser (with tunable wavelength) as the excitation source. Set the laser output wavelength at 880 nm for EGFP and 940 nm for tdTomato (Figure 1A).</li>
	<li>Include two dichroic mirrors (495 nm and 580 nm) for the separation of SHG/EGFP and EGFP/tdTomato (Figure 1A). Spectrally separate the SHG signals, EGFP and tdTomato by bandpass filters 434/17nm, 510/84nm and 585/40nm (Figure 1A).</li>
	<li>In order to optimize the image quality and to avoid photobleaching and tissue damage, set the laser power to be about 35 mW for imaging cornea and 50 mW for limbus. Measure the laser power before the laser passes the optical system. The exact laser power on samples is about 8-9 mW. The complete microscopic design is shown in Figure 1A. 
	NOTE: The upper limit of laser power is set to 70 mW to avoid photo-bleaching and tissue damage.</li>
</ol>
2. Animal preparation for live imaging
<ol>
	<li>Use 8-12-week-old mice for the experiment. Intramuscularly inject 50-80 mg/kg of tiletamine HCl and zolazepam HCl for general anesthesia. Check for the lack of response to withdrawal reflex by pinching a toe. Sufficient anesthetization is important to allow stable breathing rate monitoring. 
	NOTE: Mice at the age of 8 weeks or above are recommended because their eyeballs are matured.</li>
	<li>Place the mouse under anesthesia on a heated stage and insert the temperature monitoring probe into the anus. 
	CAUTION: The probe must be inserted fully into the anal cavity without exposure to the air, to avoid overheating of the heater and induction of heatstroke.</li>
</ol>
3. Eye holding for live imaging of ocular surface
<ol>
	<li>For live imaging of the ocular surface, use the custom designed stereotaxic mouse holder consisting of two parts: a head holder to stabilize the head and an eye holder to retract the eyelids and expose the entire ocular surface (Figure 1B-D).</li>
	<li>Insert ear bars into the external auditory meatus and maintain the three-point fixation of the head holder (Figure 1B,D).</li>
	<li>Topically apply a solution of 0.4% oxybuprocaine hydrochloride in saline and leave it for 3 min to anesthetize the ocular surface.</li>
	<li>Ensure the eyeball is protruded by proper manual eyelid retraction. Otherwise, ischemia and bleeding of the eyeball can occur.</li>
	<li>Carefully place a loop of the polyethylene tube of eye holder along the eyelid margin to expose the ocular surface. Stabilize the eyeball with the eye holder composed of a No. 5 Dumont forceps with its tips covered with the loop of polyethylene tube (Figure 1C,D).</li>
	<li>Screw forceps using a knob in distal forceps of eye holder to keep the eyeball stable (Figure 1D).</li>
	<li>Apply an eye gel with the refractive index of 1.338 on the corneal surface as an immersion medium to maintain the moisture of the ocular surface every hour. In addition, regular application of the eye gel every hour avoid clouding in cornea during imaging.</li>
	<li>Rotate the eyeball with the holder that locked on the stepper-motorized stage for imaging across the entire corneal surface from the central cornea to the peripheral region (Figure 1C,D). 
	CAUTION: Both excess and insufficient amounts of eye gel can impact the quality of images during imaging. Therefore, supplementing eye gel every hour to keep the surface moist regularly is important for imaging.</li>
</ol>
4. Z-serial image acquisition
NOTE: Set the first and last slide in every stack to reduce the dropping motion artifacts.
<ol>
	<li>Before taking the images, image the targeting field with a mercury light source.</li>
	<li>Click the symbol of the microscope software to turn on the software.</li>
	<li>Select proper PMT gain and digital gain to visualize the cellular structure in the ocular surface.</li>
	<li>Set the first slide and the last slide to acquire a stack.</li>
	<li>Enter numerical values for image resolution and z-step, e.g., 512 x 512 and 1 &#956;m as z-step.</li>
	<li>Click on the Start button to collect z-serial images.</li>
	<li>Acquire live images twice in the same area, first at 880 nm excitation for SHG/EGFP signals collection and second at 940 nm excitation for EGFP/tdTomato signals collection. 
	NOTE: The combination of two stacks provides 3 channels images. The image resolution and scan format size were 512 x 512 pixels and 157 &#956;m x157 &#956;m, respectively.</li>
</ol>
5. Image processing and 3D reconstruction
<ol>
	<li>Load the z-serial images into Fiji software18.</li>
	<li>Select the plugin Median 3D filter in Fiji to reduce background noises.</li>
	<li>Select the Package Unsharp Mask filter in Fiji to sharpen the images.</li>
	<li>Click &#8220;Auto&#8221; in brightness/contrast to automatically optimize the quality of images.</li>
	<li>Save the images as image sequences to be able to export the z-serial images.</li>
	<li>Load z-serial images into commercial software (e.g., Avizo lite) for 3D reconstruction using volume rendering.</li>
	<li>In all MPM images, present EGFP, tdTomato, and SHG signals in pseudo-green, red, and cyan color respectively.</li>
	<li>Capture 3D structure pictures by the snapshot.</li>
</ol>

<ol>
	<li>DelMonte, D. W., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+cornea.">Anatomy and physiology of the cornea.</a> Journal of Cataract &amp; Refractive Surgery. 37 (3), 588-598 (2011).</li><li>Van Buskirk, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+limbus.">The anatomy of the limbus.</a> Eye (London). 3, 101-108 (1989).</li><li>Hodges, R. R., Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tear+film+mucins:+front+line+defenders+of+the+ocular+surface;+comparison+with+airway+and+gastrointestinal+tract+mucins.">Tear film mucins: front line defenders of the ocular surface; comparison with airway and gastrointestinal tract mucins.</a> Experimental Eye Research. 117, 62-78 (2013).</li><li>Tan, H. 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=Multiphoton+fluorescence+and+second+harmonic+generation+imaging+of+the+structural+alterations+in+keratoconus+ex+vivo.">Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo.</a> Investigative Ophthalmology &amp; Visual Science. 47 (12), 5251-5259 (2006).</li><li>Konig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+in+life+sciences.">Multiphoton microscopy in life sciences.</a> Journal of Microscopy. 200, 83-104 (2000).</li><li>Rompolas, P., 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=Spatiotemporal+coordination+of+stem+cell+commitment+during+epidermal+homeostasis.">Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.</a> Science. 352 (6292), 1471-1474 (2016).</li><li>Park, 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=Tissue-scale+coordination+of+cellular+behaviour+promotes+epidermal+wound+repair+in+live+mice.">Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice.</a> Nature Cell Biology. 19 (2), 155-163 (2017).</li><li>Xin, T., Gonzalez, D., Rompolas, P., Greco, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+fate+determination+ensures+robust+differentiation+in+the+hair+follicle.">Flexible fate determination ensures robust differentiation in the hair follicle.</a> Nature Cell Biology. 20 (12), 1361-1369 (2018).</li><li>Wu, Y. F., 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=Intravital+multiphoton+microscopic+imaging+platform+for+ocular+surface+imaging.">Intravital multiphoton microscopic imaging platform for ocular surface imaging.</a> Experimental Eye Research. 182, 194-201 (2019).</li><li>Wu, Y. F., Tan, H. Y., Lin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Intravital+Imaging+of+the+Cornea,+Skin,+and+Hair+Follicle+by+Multiphoton+Microscope.">Long-Term Intravital Imaging of the Cornea, Skin, and Hair Follicle by Multiphoton Microscope.</a> Methods in Molecular Biology. , (2019).</li><li>Lo, W., 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=Fast+Fourier+transform-based+analysis+of+second-harmonic+generation+image+in+keratoconic+cornea.">Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3501-3507 (2012).</li><li>Tan, H. 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=Multiphoton+fluorescence+and+second+harmonic+generation+microscopy+for+imaging+infectious+keratitis.">Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis.</a> Journal of Biomedical Optics. 12 (2), 024013 (2007).</li><li>Jester, J. V., 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=Four-dimensional+multiphoton+confocal+microscopy:+the+new+frontier+in+cellular+imaging.">Four-dimensional multiphoton confocal microscopy: the new frontier in cellular imaging.</a> Oculur Surface. 2 (1), 10-20 (2004).</li><li>Morishige, N., 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=Second-harmonic+imaging+microscopy+of+normal+human+and+keratoconus+cornea.">Second-harmonic imaging microscopy of normal human and keratoconus cornea.</a> Investigative Ophthalmology &amp; Visual Science. 48 (3), 1087-1094 (2007).</li><li>Hao, 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=In+vivo+non-linear+optical+(NLO)+imaging+in+live+rabbit+eyes+using+the+Heidelberg+Two-Photon+Laser+Ophthalmoscope.">In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope.</a> Experimental Eye Research. 91 (2), 308-314 (2010).</li><li>Chen, Y. T., 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=R26R-GR:+a+Cre-activable+dual+fluorescent+protein+reporter+mouse.">R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.</a> PLoS One. 7 (9), 46171 (2012).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Masihzadeh, O., Lei, T. C., Ammar, D. A., Kahook, M. Y., Gibson, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiphoton+microscope+platform+for+imaging+the+mouse+eye.">A multiphoton microscope platform for imaging the mouse eye.</a> Molecular Vision. 18, 1840-1848 (2012).</li><li>Zhang, 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=Two-photon+imaging+of+the+cornea+visualized+in+the+living+mouse+using+vital+dyes.">Two-photon imaging of the cornea visualized in the living mouse using vital dyes.</a> Investigative Ophthalmology &amp; Visual Science. 54 (10), 6526-6536 (2013).</li><li>Lee, J. 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=Comparison+of+confocal+microscopy+and+two-photon+microscopy+in+mouse+cornea+in+vivo.">Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.</a> Experimental Eye Research. 132, 101-108 (2015).</li><li>Ehmke, T., 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=In+vivo+nonlinear+imaging+of+corneal+structures+with+special+focus+on+BALB/c+and+streptozotocin-diabetic+Thy1-YFP+mice.">In vivo nonlinear imaging of corneal structures with special focus on BALB/c and streptozotocin-diabetic Thy1-YFP mice.</a> Experimental Eye Research. 146, 137-144 (2016).</li><li>Webster, M. T., Manor, U., Lippincott-Schwartz, J., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+Reveals+Ghost+Fibers+as+Architectural+Units+Guiding+Myogenic+Progenitors+during+Regeneration.">Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration.</a> Cell Stem Cell. 18 (2), 243-252 (2016).</li><li>Mesa, K. 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=Homeostatic+Epidermal+Stem+Cell+Self-Renewal+Is+Driven+by+Local+Differentiation.">Homeostatic Epidermal Stem Cell Self-Renewal Is Driven by Local Differentiation.</a> Cell Stem Cell. 23 (5), 677-686 (2018).</li><li>Goh, C. 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=Real-time+imaging+of+dendritic+cell+responses+to+sterile+tissue+injury.">Real-time imaging of dendritic cell responses to sterile tissue injury.</a> Journal of Investigative Dermatology. 135 (4), 1181-1184 (2015).</li><li>Kissenpfennig, 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=Dynamics+and+function+of+Langerhans+cells+in+vivo:+dermal+dendritic+cells+colonize+lymph+node+areas+distinct+from+slower+migrating+Langerhans+cells.">Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.</a> Immunity. 22 (5), 643-654 (2005).</li><li>Chow, Z., Mueller, S. N., Deane, J. A., Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+regulatory+T+cells+display+distinct+migratory+behavior+that+is+modulated+during+adaptive+and+innate+inflammation.">Dermal regulatory T cells display distinct migratory behavior that is modulated during adaptive and innate inflammation.</a> Journal of Immunology. 191 (6), 3049-3056 (2013).</li><li>Dudeck, 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=Mast+cells+are+key+promoters+of+contact+allergy+that+mediate+the+adjuvant+effects+of+haptens.">Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.</a> Immunity. 34 (6), 973-984 (2011).</li></ol>

The authors declare that they have no competing financial interests.

This custom-built MPM imaging platform with a control software was used for intravital imaging of mouse epithelial organs, including skin10, hair follicle10 and ocular surface9,10 (Figure 1A). The custom-built system was used for its flexibility in changing the optical components for various experiments, since the beginning of our project. This imaging methodology is versatile for commercial MPM products. This protocol describes a detailed method for intravital imaging of the mouse ocular surface by MPM imaging platform. Using a stereotaxic mouse holder (Figure 1B), we were able to visualize different regions across the entire ocular surface. The accurate positioning capability makes it possible to monitor temporal cell dynamic changes in a specified region.
Although several studies of murine corneal MPM imaging have been reported in vivo19,20,21,22, there are several technical limitations which one needs to overcome to achieve continuous imaging over a long period e.g., holding the eyeball in a stable position, decreasing motion artifacts during imaging and acquiring the images for a long period without photobleaching. Compared to the plastic eyecup designed for corneal imaging in vivo with relatively limited access to the eye surface20, our eye holder not only keeps the eyeball fully exposed but also makes the entire ocular surface accessible to the objective lens (Figure 1C).
Although vital dyes or probes are routinely used as fluorescent reporters to label cells20,21,22, the invasiveness of needle injection may disrupt the natural cell dynamics of organs during the physiological homeostatic state or tissue regeneration process. R26R-GR transgenic mice strain expresses stable and bright green fluorescent protein in the cell nucleus and has been used for the visualization of the cell dynamics of muscle progenitors in ghost fibers23. mT-mG reporter transgenic mouse line was used to analyze the differentiation of epidermal cells inhomeostasis24. Compared with a single reporter fluorescent transgenic mouse line, this dual fluorescent transgenic mouse strain reduced the time of image acquisition to half by simultaneously providing the cell structure information of both cell nuclei and cell membranes. By rotating the mouse holder, flat nuclei of both endothelial and blood cells were visualized within the vascular lumen using dual fluorescent transgenic mouse strain (Figure 6). Therefore, this transgenic mouse line can be used to investigate the cell dynamics of capillaries in the future, which is the key to the pathological corneal neovascularization. In addition, this in vivo imaging platform can also be used to explore immune responses in corneal pathologies by fluorescent labeling of different immune cells, such as neutrophils25, langerhans cells26, regulatory T cells (Tregs)27 and mast cells28.
In conclusion, we demonstrated a powerful intravital MPM imaging platform for mouse ocular surface study. The combination of live MPM stereotaxic stage and special fluorescent transgenic mice enables real-time visualization of distinct cellular and extracellular structures in the ocular surface, including cornea, limbus and conjunctiva. This intravital imaging platform can help to explore the cell dynamics of the ocular surface and, with further development, can be potentially used for ophthalmic drug screening in the future.

The ocular surface structures, including the cornea and conjunctiva, protect other deeper ocular tissues from external disturbances. The cornea, the transparent front part of the eye, functions both as a refractive lens for directing light into the eye and as a protective barrier. Corneal epithelium is the outermost layer of the cornea and consists of distinct layers of superficial cells, wing cells and basal cells. Corneal stroma is composed of sophisticatedly packed collagenous lamellae embedded with keratocytes. Corneal endothelium, a single layer of flat hexagonal cells, has an important role in maintaining the transparency of cornea by keeping corneal stroma in a relatively dehydrated state through its pumping functions1. Limbus forms the border between the cornea and the conjunctiva, and is the reservoir of corneal epithelial stem cells2. The highly vascularized conjunctiva helps to lubricate the eyes by producing mucus and tears3.
Cell dynamics of the corneal surface structures are conventionally studied by either histological analysis or in vitro cell culture, which might not adequately simulate the in vivo cell dynamics. A non-invasive live imaging approach can, therefore, bridge such the gap. Due to its advantages, which include high resolution, minimal photodamage and deeper imaging depth, MPM has become a powerful modality in diverse areas of biological research4,5,6,7,8. For corneal imaging, MPM provides cellular information from intrinsic autofluorescence derived from the intracellular NAD(P)H. Second harmonic generation (SHG) signals derived from the non-centrosymmetric type I collagen fibers under femtosecond laser scanning provides collagenous stromal structures without additional staining procedures9. Previously, we and other groups have exploited MPM for imaging of animal and human corneas9,10,11,12,13,14,15.
Transgenic mouse lines exhibiting fluorescent proteins in specific cell populations have been widely used for various studies in cell biology, including development, tissue homeostasis, tissue regeneration, and carcinogenesis. We used transgenic mouse strains labeled with fluorescent proteins for in vivo imaging of corneas9,10, hair follicles10 and epidermis10 by MPM. The dual fluorescent mouse strain with cell membrane labeled with tdTomato and cell nucleus tagged with EGFP is bred from two mouse strains: R26R-GR (B6;129-Gt (ROSA)26Sortm1Ytchn/J, #021847)16 and mT-mG (Gt(ROSA26)ACTB-tdTomato-EGFP, #007676)17. R26R-GR transgenic mouse line contains a dual fluorescent protein reporter constructs, including an H2B-EGFP fusion gene and mCherry-GPI anchor signal fusion gene, inserted into the Gt (ROSA)26Sor locus. The mT-mG transgenic strain is a cell membrane-targeted tdTomato and EGFP fluorescent Cre-reporter mice. Prior to Cre recombination, cell membrane protein with tdTomato fluorescence expression is widely present in various cells. This transgenic mouse strain enables us to visualize nuclei-EGFP and membrane with tdTomato without Cre excitation. Two females (R26R-GR+/+) and one male (mT-mG+/+) transgenic mouse were bred together to produce sufficient mice for experiments. Their offspring with R26R-GR+/-;mT-mG+/- genotype, a dual fluorescent mice strain, were used in this study. Compared with one fluorescent reporter mouse line as previously described9,10, this dual fluorescent reporter mouse strain provides us with a 50% reduced acquirement of imaging time.
In this work, we describe a detailed technical protocol for in vivo imaging of the ocular surface in a step-by-step manner using our imaging platform and dual fluorescent transgenic mice.

<table><tbody><tr><td>AVIZO Lite software</td><td>Thermo Fisher Scientific</td><td></td><td>Version: 2019.3.0</td></tr><tr><td>Bandpass filters</td><td>Semrock</td><td>FF01-434/17&lt;br/&gt; FF01-500/24&lt;br/&gt; FF01-585/40</td><td></td></tr><tr><td>Dichroic mirrors</td><td>Semrock</td><td>FF495-Di01-25x36&lt;br /&gt;FF580-Di01-25x36</td><td></td></tr><tr><td>Galvano</td><td>Thorlabs</td><td>GVS002</td><td></td></tr><tr><td>Jade BIO control software</td><td>SouthPort Corporation</td><td>Jade BIO</td><td></td></tr><tr><td>Oxybuprocaine hydrochloride</td><td>Sigma</td><td>O0270000</td><td></td></tr><tr><td>PMT</td><td>Hamamatsu</td><td>H7422A-40</td><td></td></tr><tr><td>Polyesthylene Tube</td><td>BECTON DICKINSON</td><td>427401</td><td></td></tr><tr><td>Stereotaxic mouse holder</td><td>Step Technology Co.,Ltd</td><td>000111</td><td></td></tr><tr><td>Ti: Sapphire laser</td><td>Spectra-Physics</td><td>Mai-Tai DeepSee</td><td></td></tr><tr><td>Upright microscopy</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>Vidisic Gel</td><td>Dr. Gerhard Mann Chem-pharm. Fabrik GmbH</td><td>D13581</td><td></td></tr><tr><td>Zoletil</td><td>Virbac</td><td>VR-2831</td><td></td></tr></tbody></table>

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.

Using this live imaging platform, the mouse ocular surface can be visualized at cellular levels. To visualize individual single cells in the ocular surface, we employed the dual fluorescent transgenic mice with EGFP expressed in the nucleus and tdTomato expressed in the cell membrane. The collagen-rich corneal stroma was highlighted by SHG signals.
In corneal epithelium, superficial cells, wing cells and basal cells (Figure 2) were visualized. In the dual fluorescent transgenic mice, we were able to map single cells from the basal layer to the superficial layers in both corneal and limbal epithelium (Figure 2). The hexagonal superficial cells were observed (white arrowhead in Figure 2). Both nuclear size and internuclear spacing from the basal layer toward the outer layers increased in corneal epithelium (Figure 2). The cytoplasmic signals of tdTomato fluorescence indicated that the membrane protein-rich intracellular vesicular system, including the Golgi apparatus, endoplasmic reticulum, were scattered in the wing cells (Figure 2).
Within the collagenous stroma, the stellate-shaped keratocytes were outlined by the membrane-targeting tdTomato fluorescence in dual fluorescent transgenic mice (yellow arrowhead in Figure 3 and Figure 4). The keratocytes embedded in the collagen stroma were more loosely spaced than the endothelial cells. In addition, thin branching nerves in corneal stroma were also visualized by membrane-targeting tdTomato signals (white arrowhead in Figure 3). Monolayer of corneal endothelial cells showed a relatively homogenous hexagonal shape connected into a honeycombed pattern (white arrowhead in Figure 4). The limbal epithelium consisted of 1&#8211;2 layers of epithelial cells (Figure 5). The dual fluorescent reporter transgenic mouse strain also enabled us to image the capillaries within conjunctiva (Figure 6A). 3D architecture of capillaries was reconstructed by outlining vascular endothelium (Figure 6B,6C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60944/60944fig01.jpg" /> 
Figure 1: The setup of MPM and rotating mouse holder. 
(A) The setup of the MPM. (B) The design of the mouse holder. The mouse holder consists of a rotating head holder and an eye holder. (C) The design of the mouse eye holder. The eye holder retracts eyelids by a plastic loop to expose the cornea and conjunctiva. The head holder and eye holder are screwed together (B) on the stage to allow precise and controllable rotation and imaging of ocular surface. (D) Photograph of live mouse for corneal imaging with eye holder. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig01large.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/60944/60944fig02.jpg" /> 
Figure 2: Live imaging of corneal epithelium in dual fluorescent transgenic mice. 
Corneal epithelium was imaged layer-by-layer, to include superficial cells, wing cells, and basal cells. Superficial cells are marked with a white arrowhead. Scale bar = 50 &#956;m. (Z = depth from the top surface of epithelium (&#181;m)). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig02large.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/60944/60944fig03.jpg" /> 
Figure 3: Live imaging of the corneal stroma. 
Within the corneal stroma, the keratocytes (yellow arrowhead) and the nerve fibers (white arrowhead) were embedded in the collagenous stroma (in pseudo-blue color). Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig03large.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/60944/60944fig04.jpg" /> 
Figure 4: Live imaging of corneal endothelium in central cornea. 
Monolayer of hexagonal corneal endothelial cells (white arrowhead). Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig04large.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/60944/60944fig05.jpg" /> 
Figure 5: Live imaging of limbal epithelium. 
Live images of the limbal epithelium in dual fluorescent transgenic mice showed vacuolated nuclei. Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig05large.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/60944/60944fig06.jpg" /> 
Figure 6: Live imaging and the three-dimensional reconstruction of vascular network in conjunctiva. 
(A) Capillaries in conjunctival stroma were visualized. Scale bar = 50 &#956;m. (B) 3D reconstruction of capillary networks performed using a commercial software. Scale bar = 50 &#956;m. (C,D) Magnified 3D views of the image shown in panel B. Fluorescent vascular endothelial cells outlined the capillaries (white and yellow arrowheads). Scale bar for panel C = 6.26 &#956;m and for panel D = 10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60944/60944fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva at JoVE.com

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse 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. ...

a-custom-multiphoton-microscopy-platform-for-live-imaging-mouse

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.3K Views.  National Taiwan University. Dit protocol maakt gebruik van een multi-filter microscoop voor live beeldvorming van de gehele structuur van de muis oculaire oppervlak in de dubbele fluorescerende transgene muis model. De combinatie van een aangepaste multifoton microscopisch platform en dubbele fluorescerende transgene muizen maakt de real-time visualisatie van de en de structuur van het oculaire oppervlak. Om de multiphoton microscoop in te stellen, selecteert u de wateronderdompeling 20X, 1.00 NA doelstelling en stel de...

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

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Developmental Biology

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Immunology and Infection

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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.

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.

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

Medicine

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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

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

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

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

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 ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video