in vivo visible light optical coherence tomography (oct) mouse retinal imaging

마우스 Retina OCT Fibergram Alignment with Confocal Images

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.

저자 스포트라이트: 녹내장 진단 및 치료 개선을 위한 In Vivo 및 Ex Vivo 망막 영상의 발전 

Align of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina(가시광선 광간섭 단층 촬영 파이버그램과 동일한 마우스 망막의 컨포칼 이미지 정렬)

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 In Vivo Visible Light Optical Coherence Tomography OCT Mouse Retinal Imaging at JoVE.com

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

In Vivo 가시광선 광간섭 단층촬영(OCT) 마우스 망막 이미징

마취된 마우스를 동물 홀더에 놓고 두 개의 벨크로 스트랩으로 고정하는 것으로 시작합니다. 컴퓨터를 켜고 소프트웨어를 열어 레이저를 활성화하십시오. 레이저가 쥐의 눈 중앙에 오도록 동물 홀더를 조정합니다.얼굴 미리보기를 통해 후방 부분, 표재성 혈관신경총의 시야, B 스캔 및 시야 내 망막 단면을 시각화합니다. 가시광선 광간섭 단층촬영(optical coherence tomography) 또는 광학 초점을 약간 조정한 후 vis-OCT 부피를 획득합니다. 시야의 네 모서리 각각에 시신경 머리를 정렬하여 서로 다른 망막 영역을 덮습니다.강도 기반 임계값 방법을 사용하여 망막 표면을 감지하고 부피에서 vis-OCT 섬유 그램을 생성합니다. 처음 16마이크로미터 깊이를 선택하여 망막 신경 섬유층(RNFL)을 자릅니다. 다음으로, 축 방향을 따라 평균 강도 투영을 계산하여 망막 신경절 세포 축삭 다발과 주변 혈관 구조로 구성된 섬유 그램 이미지를 생성합니다.그래픽 편집기를 사용하여 혈관을 정렬하여 4개의 이미지를 후처리 상태로 몽타주합니다. 복합 vis-OCT fiber gram은 망막 신경절 축삭돌기에 대해 TUJ1 면역염색된 평판 장착 망막의 해당 컨포칼 이미지와 비교됩니다. 혈관은 공초점 이미지에서 ICAM II 표지된 혈관과 일치시킬 수 있는 구별 가능한 분지 구조를 나타냅니다.생체 외 공초점 현미경과 in vivo vis-OCT를 나란히 비교한 결과, 동일한 망막 신경절 엑손 번들 네트워크와 주변 망막 혈관 구조가 밝혀졌습니다.

in-vivo-visible-light-optical-coherence-tomography-oct-mouse-retinal

Research

JoVE Journal

Neuroscience

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

179 조회수.  University of Virginia. 마취된 마우스를 동물 홀더에 놓고 두 개의 벨크로 스트랩으로 고정하는 것으로 시작합니다. 컴퓨터를 켜고 소프트웨어를 열어 레이저를 활성화하십시오. 레이저가 쥐의 눈 중앙에 오도록 동물 홀더를 조정합니다.얼굴 미리보기를 통해 후방 부분, 표재성 혈관신경총의 시야, B 스캔 및 시야 내 망막 단면을 시각화합니다. 가시광선 광간섭 단층촬영(optical coherence tomography...

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

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 ...

연구

신경과학

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.

Ex Vivo Confocal Imaging of Mouse Retina

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.

생쥐 망막의 Ex Vivo 컨포칼 이미징

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.