two-photon in vivo imaging of the mouse retina

Two-Photon In Vivo Imaging of the Mouse Retina

Apply lubricant eyedrops to both eyes of the mouse. Rotate the main arm of the head holder until the pupil of one eye is oriented in line with the light path, and place a number 1.5 coverslip into the compact filter holder. After securing the holder to the microscope stage, lower the coverslip until it contacts the lubricant i-gel without touching the cornea. And adjust the stage in the xy dimension and the objective z position until the widefield excitation light fully covers the cornea.Use the eyepiece to continue to adjust the z position, until the fluorescent cells or structures in the retina come into focus, increasing the epifluorescence illuminator as necessary to resolve individual cells or structures of interest. Fine-tune alignment of the retina with the imaging light path. Adjust the head angle until only expansion or contraction of the out-of-focus light occurs when changing the focal plane, minimizing xy distortions.Then turn off the epifluorescence illuminator and close the illuminator shutter. For two-photon imaging, follow all institutional laser safety protocols. Turn off and cover all ambient light and switch the excitation light path to the laser and the emission light path to the PMTs.In the image acquisition software, set the frame size to 512 by 512 and the frame average to 3. Set the z-stepping to start at the top of the stack and progress downward, minimizing the two-photon laser activation of the photoreceptors. Turn on and enable the PMTs, and adjust the voltage to 680 volts.Enable the imaging and emission shutters. Begin a live image preview of the target tissue starting at a 1% laser power, and auto-adjust the display brightness to visualize the cells or structures of interest. If the target tissue is dim or unclear, increase the laser power percentage until structures become visible without surpassing 45 milliwatts.Maneuver the microscope stage in the xy direction to center on a desired imaging area, and navigate to the z plane, with the structures of interest in focus. For a chronic time-lapse experiment, open a previously obtained image to use as a reference for the cells of interest, taking care that the angle of imaging in the current image is similar to that of the previous images. Then navigate to the upper and lowermost z planes of interest to set the z limits of the imaging stack and acquire the image.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;1. Preparation of mouse for image acquisition<ol><li>Anesthetizing mouse for imaging NOTE:&#160;To mitigate exposure to isoflurane, ensure proper waste gas scavenging. Make sure the exhaust port of the anesthesia induction chamber is connected to a passive scavenging gas filter canister and the outlet of the mouse anesthesia nosepiece is connected to an active scavenging gas filter canister with a vacuum provided by a gas evacuation apparatus. Follow the manufacturer's guidelines for monitoring the filter canister weight for replacement.<ol style='list-style-type:decimal;'><li>Anesthetize the mouse using the ketamine/xylazine cocktail. Alternatively, induce anesthesia via isoflurane inhalation if also using isoflurane for maintenance of anesthesia (imaging session &gt; 30 min). Set the small animal anesthesia device to fill the induction chamber with 5% isoflurane mixed with room air at a flow rate of 0.5 L/min. Place the mouse in the induction chamber, and allow 15 s for the mouse to become anesthetized.</li><li>Switch the isoflurane vaporizer to 0% and direct the anesthesia flow to the nosepiece. Perform a 5-second "O2 flush" of the induction chamber. Take the mouse out of the induction chamber, and secure it in the head holder, attaching the nosepiece.</li><li>Use a mixture of 1% isoflurane with room air at a flow rate of 0.5 L/min for maintenance of anesthesia. Monitor respiratory rate at 5-minute intervals throughout anesthesia, adjusting the isoflurane percentage to maintain a respiratory rate of ~60 breaths/min. &#8203;NOTE:&#160;These settings (% isoflurane, flow rate) can also be used for the maintenance of anesthesia induced by the ketamine/xylazine cocktail.</li></ol></li><li>Pupil dilation<ol style='list-style-type:decimal;'><li>Prepare a solution of 1% w/v atropine and 2.5% w/v phenylephrine hydrochloride in water. Store the dilator solution at room temperature protected from light.</li><li>Using a disposable pipette eye dropper, apply a drop of the dilator solution to each eye that will be imaged. Turn off the room lights, and wait 5 min for the pupil to dilate. When the pupil is dilated, blot away the dilator solution with lint-free tissue. NOTE: Ensure dilator solution does not enter the nostrils.</li><li>Apply a large drop of lubricant eye gel to each eye that will be imaged. If both eyes are to be imaged, apply a small piece of plastic cling film over the eye gel on the non-imaging eye to prevent dehydration. If only imaging one eye, apply lubricant eye ointment to the eye that will not be imaged.</li></ol></li><li>Positioning mouse for imaging<ol style='list-style-type:decimal;'><li>To secure the mouse in the imaging head holder, rotate the main arm of the head holder until the earpiece bar is tilted at an angle of 60&#176; or more below horizontal. Secure the lower ear canal pin in the inward extended position and the upper ear canal pin in the withdrawn position.</li><li>With the mouse facing the bite bar, mount one ear onto the extended lower pin, inserting the pin into the ear canal. Loosen the screw securing the upper ear canal pin, and extend the pin into the other ear canal. Tighten the screw to secure the head.</li><li>Slide the bite bar toward the head of the mouse. Gently elevate the head of the mouse, then lower the maxillary incisors of the mouse into the hole of the bite bar. Retract the bite bar with gentle force to secure the head of the mouse, and secure the bite bar position by tightening the screw.</li><li>If using isoflurane, slide the nosepiece through its slot onto the bite bar prior to securing the incisors of the mouse. Secure and tighten the bite bar position as in step 1.3.3. Tighten the nosepiece using the two screws on its upper face until it fits snugly on the nose of the mouse, but does not constrict the snout.</li><li>Transfer the mouse, in the head holder, to the microscope stage beneath the objective. Rotate the main arm of the head holder until the pupil of the eye is oriented straight up, in line with the light path (Figure 1).</li><li>Place a #1.5 coverslip in the compact filter holder and attach the holder to the microscope stage. Lower the coverslip toward the eye, contacting the lubricant eye gel, so that it lies horizontally immediately above the cornea (Figure 1). Ensure that the coverslip does not touch the cornea. NOTE:&#160;On the microscope, ensure the excitation light path is set to epifluorescence and the emission light path is set to the eyepiece. Turn on the epifluorescence illuminator at its lowest power and open the illuminator shutter. Choose the epifluorescence illuminator wavelength and fluorescence filter corresponding to the fluorophore being imaged.</li><li>Maneuver the stage in the X-Y dimensions and objective in the Z-position using the stage control and motorized focus drive until the widefield excitation light fully covers the cornea. Looking through the eyepiece, continue to adjust the Z-position of the stage until the fluorescent cells or structures in the retina come into focus. Increase epifluorescence illuminator power if the sample signal is insufficiently bright to resolve individual cells or structures of interest through the eyepiece. NOTE: If having trouble locating the retina, the iris is a high-contrast landmark to anchor on and then focus down toward the retina. This step will also allow verification that the pupil is maximally dilated.</li><li>Obtain an imaging area on the axis with the mouse lens. Adjust the angle of the mouse using the various degrees of freedom on the head holder until only expansion or contraction of out-of-focus light occurs when adjusting the focal plane. Turn off the epifluorescence illuminator and close the illuminator shutter. NOTE: Significant X-Y parallax of out-of-focus light while scrolling through the Z-direction focal planes indicates that the retina is not on the axis with respect to the imaging light path.</li></ol></li></ol>2. Two-photon image acquisition<ol><li>Image setup and acquisition parameters &#8203;NOTE: Turn off room lights, and cover stray light sources in the room. Ensure the epifluorescence illuminator is off, and the illuminator shutter is closed.<ol style='list-style-type:decimal;'><li>Switch the excitation light path to the laser and the emission light path to the photomultiplier tubes (PMTs).</li><li>In the image acquisition software, set a frame size of 512 x 512 and a frame average of 3. Set the steps per slice to -8 &#181;m. Designate z-stepping to start at the top of the stack and progress downward, minimizing two-photon laser activation of photoreceptors. &#8203;NOTE:&#160;The step size can be reduced to increase Z-resolution at a cost of increased imaging time, but 8 &#181;m steps are sufficient to resolve cell somata in this imaging configuration.</li><li>Turn on and enable the PMTs. Adjust the PMT voltage to 680 V. Enable the excitation shutter. &#8203;NOTE:&#160;Powered PMTs are susceptible to light damage. Ensure that the epifluorescence illuminator is off, the microscope enclosure curtain is drawn, and the room lights are off.</li><li>Begin a live image preview of the target tissue, starting with 1% laser power. Auto-adjust the display brightness to visualize the cells or structures of interest. Auto-adjust the scan phase. If the target tissue is dim or unclear, increase the laser power percentage until structures become visible without surpassing the limit set in step 2.4.4, which corresponds to 45 mW. &#8203;NOTE:&#160;For a 16-bit image, a display value of ~1000 when auto-adjusting brightness in a Z-plane containing structures of interest indicates sufficient brightness of the sample.</li><li>Maneuver the microscope stage in the X-Y direction to center on a desired imaging area, then navigate to the Z-plane with the structures of interest in focus. &#8203;NOTE: Imaging adjacent to the optic nerve head allows it to serve as an unambiguous landmark for chronic imaging experiments.</li><li>If this is a chronic time-lapse experiment, open a previous image on the acquisition computer and use it as a reference to find the same area of interest. Ensure that the angle of imaging is similar to that of previous images to acquire the same set of cells with minimal parallax. NOTE: To image the same cells as in previous time points (Figure 2), further adjustment of the mouse head position is likely required.</li><li>Set the Z-limits of the imaging stack by navigating to the uppermost and lowermost Z-planes of interest, and acquire the image. Upon completion of image acquisition, disable the PMTs and the emission shutter. Switch the emission light path back to the eyepiece, and the excitation light path to epifluorescence illumination. Remove the mouse from the microscope stage.</li></ol></li></ol>

<figure class='table' style='width:1112pt;'><table class='ck-table-resized'><colgroup><col style='width:30.76%;'><col style='width:17.36%;'><col style='width:20.68%;'><col style='width:31.2%;'></colgroup><tbody><tr><td style='height:15.5pt;'>#1.5 coverslip</td><td>ThermoFisher</td><td>152440</td><td>Richard-Allan #1.5 24 mm x 40 mm</td></tr><tr><td style='height:15.5pt;'>50 mL glass syringe</td><td>Hamilton</td><td>80950</td><td>22G cemented needle</td></tr><tr><td style='height:15.5pt;'>Anesthesia Air Pump</td><td>RWD Life Science</td><td>R510-30</td><td>&#160;</td></tr><tr><td style='height:15.5pt;'>Atropine</td><td>Sigma</td><td>A0132</td><td>For pupil dilator solution</td></tr><tr><td style='height:15.5pt;'>Basic Small Animal Anesthesia Device</td><td>RWD Life Science</td><td>R500IE</td><td>&#160;</td></tr><tr><td style='height:15.5pt;'>Borosilicate glass capillary</td><td>Sutter</td><td>B150-86-10</td><td>Outside diameter 1.50 mm, inside diameter 0.86 mm, length 10 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oxytetracycline/polymyxin B antibiotic ophthalmic ointment</td><td>Zoetisus</td><td>NA</td><td>For use after intravitreal injection</td></tr><tr><td style='height:15.75pt;'>ThermoRack cooling system</td><td>Solid State Cooling Systems</td><td>ThermoRack 401</td><td>Set to 20 &#176;C</td></tr><tr><td style='height:15.75pt;'>Ultrafast Ti:Sapphire laser</td><td>Spectra-Physics</td><td>Mai Tai DeepSee</td><td>&#160;</td></tr><tr><td style='height:15.75pt;'>Vgat-Cre transgenic mice (Slc32a1tm2(cre)Lowl/J)</td><td>The Jackson Laboratory</td><td>16962</td><td>&#160;</td></tr><tr><td style='height:15.75pt;'>VGlut2-Cre transgenic mice (Slc17a6tm2(cre)Lowl/J)</td><td>The Jackson Laboratory</td><td>16963</td><td>&#160;</td></tr><tr><td style='height:15.75pt;'>VivoScope for In Vivo Imaging</td><td>Scientifica</td><td>S-MPVS-1200-00P</td><td>&#160;</td></tr><tr><td style='height:15.75pt;'>White petrolatum-mineral oil lubricant eye ointment</td><td>Stye</td><td>NA</td><td>For use after 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<a href='https://app.jove.com/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.

<img src='https://cloudfront.jove.com/files/ftp_upload/23422/23422_figure_1_editor_23422.jpg' alt='23422_figure_1.jpg' />Figure 1: Positioning mice for in vivo imaging. To position the mouse with the pupil on the axis with the light path, the anesthetized mouse is first restrained in a head holder, the head is rotated and angled, a large drop of lubricant eye gel is placed on the eye, and the mouse is placed on the stage. A coverslip is mounted in the coverslip holde...

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 ...

Take an anesthetized transgenic mouse with its head secured at an angle on a holder for in vivo imaging of the mouse retina.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;The retina contains retinal ganglion cells, or RGCs, expressing a fluorophore-tagged protein.Apply eye lubricant and mount a coverslip over the eye.Under a microscope, uniformly illuminate the retina to excite the fluorescent proteins in the RGCs.Using the emitted fluorescence, obtain a widefield view of the focal plane RGCs and out-of-focus RGCs.Switch to two-photon imaging mode, focusing low-energy near-infrared light into the RGC layer.At the focal point, the combined energy from two photons excites the fluorophore, which then releases energy as fluorescence.&#160;As the excitation is confined to a single point, fluorescence from other focal planes is minimized.Capture images at multiple focal planes along the z-axis to image the entire RGC layer.

11 조회수. Two-Photon In Vivo Imaging of the Mouse Retina

비디오: Two-Photon In Vivo Imaging of the Mouse Retina - 실험

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

두뇌 조각에서 신경 모 수석에서 2 광자 칼슘 이미지

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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JoVE Journal

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In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo에서 신생아 쥐에 있는 대뇌 피 질의 뉴런의 두 광자 영상

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

FRET 기반 센서 ATeam1.03YEMK를 사용하여 마우스 뇌의 오르가노티픽 조직 슬라이스에서 세포 내 ATP 이미징

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Two-Photon In Vivo Imaging of the Mouse Retina

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Two-Photon In Vivo Imaging of the Mouse Retina