ex vivo confocal imaging of mouse retina

Konfokal Görüntülerle Fare Retina OCT Fibergram Hizalaması

Retinal ganglion cells (RGCs) play a critical role in visual information processing, receiving synaptic inputs through their dendritic trees in the inner plexiform layer (IPL) and transmitting the information via their axons in the retinal nerve fiber layer (RNFL) to the brain1,2,3,4. In diseased conditions such as glaucoma, early RGC degeneration may result in subtle changes in the RNFL, the ganglion cell layer (GCL), the IPL, and the optic nerve in both patients and rodent models5,6,7,8,9. Early detection of these morphological changes in RGCs is thus essential for timely intervention to prevent RGC and vision loss.
We have recently developed a new clinical-ready imaging technology called visible-light optical coherence tomography (vis-OCT) to satisfy the need for in vivo monitoring of RGC damage. Vis-OCT improved the axial resolution, reaching 1.3 &#181;m in the retina10, 11, allowing for the visualization of individual RGC axon bundles in the RNFL. Subsequently, vis-OCT fibergraphy (vis-OCTF) was established to track and quantify RGC damage at the single axon bundle level in mice11,12,13. However, ex vivo confocal imaging of the same retina as the gold standard is often necessary to validate the in vivo findings. Therefore, this study will demonstrate how to align in vivo images acquired by vis-OCTF with ex vivo confocal images of the same mouse retina. The protocol aims to validate the in vivo findings by ex vivo confocal imaging and establish a foundation for examining the molecular and cellular changes underlying RGC damage in diseased conditions.

Yazar Spotlight: Gelişmiş Glokom Tanı ve Tedavisi için In Vivo ve Ex Vivo Retinal Görüntülemedeki Gelişmeler 

Görünür Işık Optik Koherens Tomografi Fibergramlarının Aynı Fare Retinasının Konfokal Görüntüleri ile Hizalanması

We present a protocol for aligning in vivo VIS-OCT images with ex vivo immunostained retinal nerve fibers to validate our novel imaging technology. VIS-OCT enables visualization of retinal nerve fibers in vivo, and this process confirms the accuracy of our findings. Our protocol combines in vivo and ex vivo imaging to provide a real time visualization of retina structure and confirm findings.The process is applicable to mouse models of eye diseases, provided the anterior eye portion allows for optical imaging. This includes diabetic retinopathy and retinal ischemia models. These studies lay the foundation for establishing an objective evaluation of neuro damage in humans, which can significantly improve glaucoma diagnosis and treatment in the future.

Watch this Scientific Journal Video about Ex Vivo Confocal Imaging of Mouse Retina at JoVE.com

Ex Vivo Confocal Imaging of Mouse Retina  

Fare Retinasının Ex Vivo Konfokal Görüntülemesi

Ötenazi yapılmış bir farenin gözlerini enüklee edin, oryantasyon amacıyla temporal tarafı işaretleyin. Ön kamarayı çıkarın. Ardından lensi ve vitreusu çıkarın ve göz kaplarını 30 dakika boyunca paraformaldehit içine yerleştirin.Göz bardaklarını PBS ile 30 dakika yıkayın. Çözeltiyi üç kez değiştirmek. Daha sonra %0,5 Triton X-100 ile 30 dakika yıkayınız.Göz kaplarını oda sıcaklığında iki saat boyunca bloke edici tamponda inkübe edin. İmmün boyamadan sonra, retinaları bir mikroskop kullanarak göz kapaklarından izole edin. Retinaları dört yaprağa bölün ve retina ganglion hücre tabakası yukarı bakacak şekilde düz bir şekilde monte edin.Yönlendirme için zamansal taraftaki işaretin takılı kaldığından emin olun. Retinaları montaj ortamı ile örtün. Kapak fişlerini yerleştirin.Ve slaytları oje kullanarak kapatın. Konfokal mikroskobu açın ve Konum modu altında, göz merceğini kullanarak ilgilendiğiniz alanı bulun. Alım moduna geçin ve tüm bilgi katmanları için Z-Stack dilimleriyle birlikte tüm retinayı kaplayacak döşemeler ayarlayın.Toplam hacmi elde etmek için tüm retina boyunca en az 25 karo görüntüleyin. İki boyutlu yüz konfokal mikroskopi görüntüleri oluşturmak için Z-Stack dilimlerini yansıtın. Kompozit vis-OCT fibergramı, RGC aksonları için TuJ1 ile immün boyanmış düz monte retinanın karşılık gelen konfokal görüntüsü ile karşılaştırıldı.Kan damarları, konfokal görüntüde ICAM-2 işaretli kan damarları ile eşleştirilebilen ayırt edilebilir dallanma yapıları sergiler. Ex vivo konfokal mikroskopi ve in vivo vis-OKT arasındaki yan yana karşılaştırma, aynı RGC akson demeti ağlarını ve çevresindeki retinal vaskülatürü ortaya çıkardı.

Research

JoVE Journal

Neuroscience

Ex Vivo Confocal Imaging of Mouse Retina

268 Görüntüleme.  University of Virginia. Ötenazi yapılmış bir farenin gözlerini enüklee edin, oryantasyon amacıyla temporal tarafı işaretleyin. Ön kamarayı çıkarın. Ardından lensi ve vitreusu çıkarın ve göz kaplarını 30 dakika boyunca paraformaldehit içine yerleştirin.Göz bardaklarını PBS ile 30 dakika yıkayın. Çözeltiyi üç kez değiştirmek. Daha sonra %0,5 Triton X-100 ile 30 dakika yıkayınız.Göz kaplarını oda sıcaklığında iki saat boyunca bloke edici tamponda inkübe edin. ?...

Video: Ex Vivo Confocal Imaging of Mouse Retina  

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Nörobilim

In Vivo Visible Light Optical Coherence Tomography (OCT) Mouse Retinal Imaging

Start by positioning the anesthetized mouse on the animal holder and securing it with two Velcro straps. Turn on the computer and open the software to activate the laser. Adjust the animal holder to center the laser in the mouse's eye.Visualize the posterior part through an on-face preview, the field of view of the superficial vascular plexus, a B scan, and the retina cross-section within the field of view. Acquire a visible light optical coherence tomography, or vis-OCT volume after making minor adjustments to the optical focus. Align the optical nerve head in each of the four corners of the field of view to cover different retinal areas.Use an intensity-based threshold method to detect the retina surface, and generate vis-OCT fiber grams from the volume. Crop the retinal nerve fiber layer, or RNFL, by selecting the first 16 micrometers depth. Next, calculate the mean intensity projection along the axial direction to generate the fiber gram image composed of retinal ganglion cell axon bundles and surrounding vasculature.Align the blood vessels using a graphics editor to montage the four images post-processing. The composite vis-OCT fiber gram is compared with the corresponding confocal image of flat-mounted retina immunostained with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM II labeled blood vessels on the confocal image.Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC exon bundle networks and surrounding retinal vasculature.

In Vivo Görünür Işık Optik Koherens Tomografi (OCT) Fare Retinal Görüntüleme

Enucleate the eyes of a euthanized mouse, marking the temporal side for orientation purposes. Remove the anterior chamber. Then remove the lens and vitreous and place eye cups in paraformaldehyde for 30 minutes.Wash eye cups with PBS for 30 minutes. Replacing the solution three times. Then, wash with 0.5%Triton X-100 for 30 minutes.Incubate eye cups in the blocking buffer for two hours at room temperature. After immunostaining, isolate the retinas from the eye cups using a microscope. Divide the retinas into four leaves and flat mount them with the retinal ganglion cell layer facing up.Ensure the mark on the temporal side remains attached for orientation. Cover the retinas with mounting medium. Place cover slips.And seal the slides using nail polish. Turn on the confocal microscope and under Locate mode, find the area of interest using the eye piece. Switch to Acquisition mode and set up tiles to cover the entire retina, along with Z-Stack slices for all layers of information.Image at least 25 tiles across the whole retina to achieve a total volume. Project the Z-Stack slices to generate two-dimensional on-face confocal microscopy images. The composite vis-OCT fibergram is compared with the corresponding confocal image of flat mounted retina immunostained with TuJ1 for RGC axons.Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM-2 labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature.

Alignment of In Vivo and Ex Vivo Mouse Retinal Images

Start by creating a composite image using the four obtained images from each mouse. Utilize a graphics editor to align all the blood vessels, employ the optic nerve head and blood vessel patterns as landmarks for aligning the composite fiber gram overlap the composite images and the confocal images of the same retina stained with ICAM2, to achieve in vivo and ex vivo alignment. The composite vis-oct fiber gram is compared with the corresponding confocal image of flat mounted retina.Immuno stain with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM2 labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-oct revealed identical RGC Axon bundle networks and surrounding retinal vasculature.