We thank Drs. Yichen Huang, Chunming Guo and Zhenfang Zhou for their technical support. This research was funded by NIH/NIDDK (1R01DK091645-01A1, XL) and American Heart Association (AHA, 13GRNT16950006, XL).

모든 동물의 사용은 보스턴 아동 병원의 기관 동물 관리 및 사용위원회에 의해 승인됩니다. 1. 샘플 준비 <ol><li> 시간 제한 짝짓기 <ol><li> 같은 케이지에서 함께 C57BL6 야생 형 남성과 여성을 배치합니다. </li><li> 상대 플러그의 형성 초기에 다음날 아침에 확인합니다. 결합 플러그 (일명, 질 또는 교미 플러그) 차단 질을 강화 된 젤라틴 증착이다. </li><li> 배아 일 0.5 (E0.5)와 같은 플러그 날짜를 설정합니다. </li></ol></li><li> 마우스 배아의 분리 <ol><li> CO 2 질식에 의해 임신 한 여성을 안락사. </li><li> 흡수 패드 부정사 쥐를 넣고 70 % 에탄올로 복부를 스프레이. </li><li> 피부를 들어 올려 수술 가위를 사용하여 꼬리 복부에서 초기 5mm 절개를합니다. 잘라 완전히 내부 장기를 노출 복부 피부를 제거 </li><li> 버지니아 근처 잘라GINA는 전체 자궁을 제거합니다. </li><li> 자궁 경적을 따라 주입 사이트간에 잘라. 각 세그먼트 또는 수태 안에 하나의 배아를해야한다. </li><li> 1 배 얼음처럼 차가운 인산 완충 식염수 (PBS)에서 자궁 세그먼트를 유지합니다. </li></ol></li><li> 배아 해부 <ol><li> 입체경 아래에 얼음처럼 차가운 PBS와 별도의 접시에 각각의 수태를 놓습니다. </li><li> 순차적으로 미세 해부 집게를 사용하여 자궁 조직, 태반 후 태아의 세포막을 제거합니다. </li><li> 1 배 얼음 차가운 PBS 큰 전송 피펫을 사용하여 함께 50 ML 튜브에 해부 배아를 전송합니다. 조직 손상을 최소화하기 위해 집게와 직접 배를 따기 마십시오. </li></ol></li><li> 정착 <ol><li> 정착액의 10 샘플 볼륨보다 24 시간 동안 4 ° C에서 10 % 중성 완충 포르말린 용액의 해부 배아를 수정합니다. </li></ol></li><li> 전처리 <ol><li> 어음 솔루션을 준비 : 4M 우레아, 증류수 10 % 글리세롤 4 % 소듐 도데 실 설페이트 (SDS). </li><li> 어음 솔루션과 정착액을 교체합니다. </li><li> 이주 - 조심스럽게 1 시간 동안 실온에서 로커에 시료를 회전한다. 두 번째와 세 번째 날에 한 번 클리어 솔루션을 교환한다. 시편 천천히 명확하고 반투명하게된다. </li><li> 10 % 포르말린으로 지우기 솔루션을 교체하고 2 일 동안 로커에 둡니다. </li></ol></li><li> 탈수 <ol><li> 5 분마다 1X PBS로 3 회 전처리 샘플을 씻으십시오. </li><li> 부드러운 로커에 실온에서 상승 에탄올 시리즈 샘플 탈수. E15.5 태아를 들어, 50 %, 60 %, 70 %, 80 %, 85 %, 90 %, 95 % 및 100 % 에탄올, 2 시간의 각 단계를 포함하는 상승 에탄올 시리즈를 사용한다. 이전 배아, 배양 시간을 증가시킬 필요가있다. </li></ol></li><li> 더럽히는 것 <ol><li> 에오신 Y 품목 소금과 0.02 0.1375 g을 추가 염색 용액을 제조아 크리 딘 오렌지 헤미 (아연 클로라이드) 100 % 에탄올 50ml로 소금 75g. 2 시간 동안 교반한다. 필터 종이 필터. 실온에서 저장하고 알루미늄 호일로 덮어 빛을 피하십시오. </li><li> 1 시간 동안 염색 용액에 시료를 전송합니다. </li><li> 신선한 염색 솔루션 및 얼룩 하룻밤로 교체하십시오. </li></ol></li><li> 침투 <ol><li> 침투 솔루션을 준비 : JB-4 삽입 키트의 해결 방법 100 ㎖, 촉매 C 1.25 g, 에오신 Y 품목 소금의 0.275 g 및 아 크리 딘 오렌지 헤미 (염화 아연) 염 0.055 g을 결합한다. 2 시간 동안 얼음 버킷 (200 RPM) 혼합 교반 한 후 여과지로 걸러. </li><li> 4 ° C에서 침투 솔루션을 저장하고 알루미늄 호일로 덮어 빛을 피하십시오. </li><li> (1 : 1) 염색 솔루션 : 솔루션을 침투하기 위해 배아를 전송, 4 ° C에서 로커에 적어도 3 시간 동안 배양한다. </li><li> 솔루션을 침투하기 위해 배아 전송하고 3 시간 동안 배양추운 방에 로커에 대한 연구. </li><li> 한 번 매일 침투 솔루션을 대체, 3 일 이상 부드러운 로커에 추운 방에 둡니다. </li></ol></li></ol> 2. 퍼가기 <ol><li> 얼음에 해결 B 및 침투 솔루션을 유지합니다. </li><li> 바로 삽입하기 전에 (용액 B 및 침투 솔루션 1:25) 임베딩 솔루션을합니다. </li><li> 포함 금형 동양 시편은, 부드럽게 삽입 금형에 차가운 임베딩 솔루션을 붓는다. 다시 방향 필요한 경우 핀. </li><li> 퍼가기 솔루션이 응고되어 있는지 여부를 조사하기 위해 핀을 사용합니다. 시편 블록을 밀어하고, 필요한 경우 트림. </li><li> 교차 방향을 얻기 위하여 별도의 금형을 사용하여 시험편 블록의 방향. </li><li> 매립 금형의 상단에 블록 홀더를 넣습니다. 금형 및 블록 홀더 솔루션을 포함하기로 침수 될 때까지 조심스럽게 금형에 임베딩 솔루션을 붓는다. </li><li> 보조 용기에 시료를 유지하고 맡겨흄 후드에서 4 시간 - 3 얼음. </li><li> 4 °의 C에서 응고 표본을 저장합니다. </li><li> 매입 형을 벗겨. 블록의 시험편의 위치를 ​​검사하도록 경사 조명하여 실체 현미경 블록을 넣는다. 시편 주변 블록의 얼굴에, 마크 그리드 선. </li><li> 참조로 표시된 눈금 선을 이용하여 물질을 매립 접근 양을 제거 할 수있는 블록을 낸다. </li></ol> 3. 이미지 인식 <ol><li> 마이크로톰에 시편 블록을 고정하고 신선한 잘라 표면을 구하십시오. 설정 섹션 두께 (예를 들어, e9.5-10.5, 1.5 μm의, e11.5-12.5, 2.0 μm의, e13.5-15.5, 2.5 μm의, E16.5-이전, 3 μm의). 마이크로톰의 홈 위치에서 시료 / 블록을 유지합니다. </li><li> 수평 시편의 블록 표면에 직면 스테레오 줌 현미경을 탑재합니다. </li><li> 컴퓨터와 현미경 켜고 이미지 수집 소프트웨어를 시작합니다. </li><li> 시각화 및 블록면에 광학 초점이미징 소프트웨어의 &#39;라이브 뷰&#39;모드에서. </li><li> 블록 얼굴이 뷰 파인더의 중심에 있도록 필요한 경우 현미경 마이크로톰을 맞 춥니 다. </li><li> 전체 블록 얼굴이 뷰 파인더 안에 들어 오도록 줌을 조정합니다. </li><li> 녹색 형광 단백질 (GFP) 필터 (여자 470 ± 20 nm의, 다이크로 익 495 nm의, 방출 525 ± 25 nm의)를 선택하고 필요한 경우 집중할. </li><li> 수동 측정하고 노출 시간을 설정 (예를 들면, 80-400 미니 초). </li><li> 각 신선한 잘라 후 블록 얼굴의 이미지를 획득하고 가능하면 자동으로 이미지를 저장합니다. </li><li> 해상도, 단면 두께 및 줌을 포함하여 기록 중요한 매개 변수. </li><li> 전체 표본, 수출을 마무리하고 필요한 경우 JPEG 형식으로 모든 이미지 파일을 변환 한 후. </li><li> 이미징 처리 소프트웨어를 사용하여 모든 원본 이미지를 반전하고, 밝기 및 대비를 조정합니다. </li><li> 별도의 폴더에있는 모든 처리 된 이미지를 저장합니다. </li></ol> 4. 3D심상 <ol><li> 지침에 따라 3 차원 시각화 소프트웨어에 대한 모든 처리 된 이미지를 업로드합니다. </li><li> 입력 해상도와 부분 두께는 체적 데이터 (즉, 복셀 크기)의 모든 이미지를 변환 및 3D 파일을 저장합니다. </li><li> 자동 모드를 사용하여 모든 이미지를 맞 춥니 다. 필요한 경우 수동으로 개별 이미지를 조정합니다. </li><li> 새 파일 이름으로 정렬 된 이미지를 저장합니다. </li><li> 3 차원 시각화 소프트웨어를 사용하여 시험편의 형태 학적 특징을 분석한다. </li></ol>

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The authors have nothing to disclose.

여기서는 급격한 차원 시각화 및 복잡한 구조의 형태 학적 분석을 위해 호환되는 직렬 HREM 화상 취득 루틴 실험실 장비를 사용하여 변형 된 프로토콜을 제시한다. 고해상도 이미지 대신 개별 섹션의 블록 표면에서 직접 수행되기 때문에, 미세한 형태 적 특징은 유지하고 신속 차원 시각화 프로그램에서 디지털로 복원된다. 3D 영상을 시각화하고 복잡한 형태 적 특징을 이해하...

The advent of 3D imaging technologies has opened the possibility of systemic analysis of detailed morphological features during fetal development. A variety of 3D imaging modalities is now available include micro-magnetic resonance imaging (micro-MRI), micro computed tomography, high frequency ultrasound, optical coherence tomography, optical project tomography and episcopic microscopy1-4. Each modality has its own unique features in resolution, contrast, speed, cost, in utero capability and availability.
Episcopic fluorescence image capture (EFIC) and high-resolution episcopic microscopy (HREM) are episcopic microscopy imaging methods4. Here, high-resolution serial images are captured continuously from the block face instead of tissue sections. The resulting images faithfully reflect detailed morphological features with minimal or no tissue distortion. The acquired volumetric data is readily converted to high-resolution 3-D images suitable for accurate morphometric analysis. Because of relative low cost and high resolution, HREM and EPIC have become the superior alternatives for systemic assessment of mouse models of human birth defects2,4-8.
Both EFIC and HREM methods require specimens to be embedded in the suitable medium. EFIC detects autofluorescence emitted from the embedded tissue. Because of this, it is limited to specimens emitting high levels of autofluorescence but not those with relatively weak autofluorescence such as early stage embryos9. To overcome the dependency of autofluorescence, specimens are stained with a fluorescent dye such as eosin for HREM imaging. The choices of dyes and staining methods also make HREM more compatible to detect molecular signals in the context of tissue architecture and morphology4,10.
In this article, we present an improved HREM protocol suitable for whole body assessment of the late stage mouse embryos. To facilitate staining, we include a pretreatment step to increase tissue penetration, thereby enabling HREM imaging of older mouse embryos.

<table><tbody><tr><td>Mice</td><td>The Jackson Laboratory</td><td>C57BL6</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Pharmco-Aaper</td><td>111000200</td><td>200 Proof, Absolute, ACS/USP/Kosher Grade</td></tr><tr><td>Formalin</td><td>Sigma</td><td>HT501128-4L</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U5128</td><td>Clearing Solution</td></tr><tr><td>Glycerol</td><td>Fisher scientific</td><td>G33-4</td><td>Clearing Solution</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Invitrogen</td><td>15525-017</td><td>Clearing Solution</td></tr><tr><td>Eosin Y Disodium Salt</td><td>Fisher scientific</td><td>BP241925</td><td>Infiltration Solution</td></tr><tr><td>Acridine Orange hemi (zinc chloride) salt</td><td>Sigma</td><td>A6014</td><td>Infiltration Solution</td></tr><tr><td>Whatman paper</td><td>GE</td><td>3030-153</td><td></td></tr><tr><td>JB-4 Embedding Kit - Solution A</td><td>Polysciences, Inc.</td><td>0226A-800</td><td>Infiltration Solution</td></tr><tr><td>JB-4 Embedding Kit - Solution B</td><td>Polysciences, Inc.</td><td>0226B-30</td><td>Embedding Solution</td></tr><tr><td>JB-4&amp;nbsp;Embedding Kit - Catalyst</td><td>Polysciences, Inc.</td><td>02618-12</td><td>Infiltration Solution</td></tr><tr><td>Embedding Molds</td><td>Polysciences, Inc.</td><td>23185-1</td><td>16 x 8 mm</td></tr><tr><td>Peel-A-Way Sharp Embedding Molds</td><td>Polysciences, Inc.</td><td>18986-1</td><td>22 mm x 22 mm square, truncated to 12 mm x 12 mm</td></tr><tr><td>JB-4&amp;nbsp;Plastic Block Holders</td><td>Polysciences, Inc.</td><td>15899</td><td></td></tr><tr><td>Disposable Graduated Transfer Pipes</td><td>VWR</td><td>414004-014</td><td></td></tr><tr><td>Petrl Dish</td><td>VWR</td><td>25384-088</td><td>Embryo dissection</td></tr><tr><td>Forceps Inox Tip</td><td>&amp;nbsp;ROBOZ</td><td>RS-5015</td><td>Embryo dissection</td></tr><tr><td>Graefe Tissue Forcep</td><td>&amp;nbsp;ROBOZ</td><td>RS-5153</td><td>Embryo dissection</td></tr><tr><td>Delicate Operating Scissor</td><td>&amp;nbsp;ROBOZ</td><td>RS-6700</td><td>Embryo dissection</td></tr><tr><td>14 ml Polypropylene Round-Bottom Tubes</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>50 ml Polypropylene Conical Tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Ice Bucket</td><td>Magic Touch Iceware International Corp.</td><td>R19-343</td><td></td></tr><tr><td>100 ml (3.4 oz.) Antistatic Polystyrene Weigh Boats</td><td>VWR</td><td>89106-766</td><td></td></tr><tr><td>330 ml (11.2 oz.) Antistatic Polystyrene Weigh Boats</td><td>VWR</td><td>89106-770</td><td></td></tr><tr><td>Sodium Chloride</td><td>MP Biomedicals</td><td>102892</td><td>PBS Solution</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P0662</td><td>PBS Solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5405</td><td>PBS Solution</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma-Aldrich</td><td>S9390</td><td>PBS Solution</td></tr><tr><td>Digital Camera</td><td>Hamamatsu Photonics</td><td>C11440-10C</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>Illuminator</td><td>Carl Zeiss</td><td>HXP 200C</td><td></td></tr><tr><td>Fluorescence Stereo Zoom Microscope</td><td>Carl Zeiss</td><td>Axio Zoom.V16</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Fiber Optic Light Source</td><td>Leica</td><td>KL 1500 LCD</td><td></td></tr><tr><td>Disposable Microtome Blade</td><td>VWR</td><td>95057-832</td><td></td></tr><tr><td>Single Edge Dispenser</td><td>Personna</td><td>62-0330-0000</td><td></td></tr><tr><td>ZEN</td><td>Carl Zeiss</td><td>pro 2012</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>CS6 v13.0</td><td></td></tr><tr><td>Amira 3D Software</td><td>Visage Imaging</td><td>5.4</td><td></td></tr><tr><td>Computer</td><td>Dell T7600, 2x900GB SAS hard drive and 64 GB DDR3 RDIMM memory</td><td></td><td></td></tr><tr><td>Rocking Platform Shakers</td><td>VWR</td><td>40000-304</td><td></td></tr><tr><td>Standard Hot Plate Stirrer</td><td>VWR</td><td>12365-382</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo</td><td>AB54-S</td><td></td></tr></tbody></table>

rapid acquisition of 3d images using high-resolution episcopic microscopy

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) morphometric data. Here, we describe the detailed protocol to image the entire mouse embryo. The protocol consists of four major sections: sample preparation, embedding, image acquisition and finally, 3D visualization. The technology requires specimens to be stained with a fluorescent dye, which can be problematic for large or dense specimens. To overcome this limitation, we have improved the existing protocol to enhance tissue penetration of the dye by pretreating the specimen with a solution containing urea and sodium dodecyl sulfate. The protocol uses only routine laboratory equipment and reagents for easy adaptation in standard laboratory settings. We show that the resulting high-resolution 3D images faithfully recapitulate the detailed morphologic features of the internal organs of mouse embryos, thereby permitting morphometric analyses. Together, we present a detailed and improved protocol using standard laboratory equipment to acquire high-resolution 3D images of small and large sized specimens.

우리는 E9.5과 E13.5 8 사이의 마우스 배아의 높은 품질의 HREM 이미지를보고했다. 후반 개최 배아의 이미지 품질은, 그러나, 크게 때문에 형광 염료 에오신의 제한된 조직 침투의 손상. 염색 효율을 높이기 위해, 형광 영상 (11)에 호환되는 여러 전처리 방법을 시험 하였다. 특히, E15.5 마우스 배아는 무서 / 전자 A2 (12) 또는 전신 13 CUBIC 중 하나를...

Watch this Scientific Journal Video about Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy at JoVE.com

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

3 차원 영상의 신속한 획득 고해상도 Episcopic 현미경을 사용하여

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) ...

rapid-acquisition-3d-images-using-high-resolution-episcopic

Research

JoVE Journal

Developmental Biology

7.6K 조회수.  Wenzhou Medical University. We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

비디오: Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

고해상도 3D 영상의 전 VIVO</em마이크로 중부 표준시로> 생물 학적 샘플

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

연구

생체공학

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

고해상도 무시 무시 현미경 (HREM) - 유기 재료의 시각화 및 처리를위한 간단하고 강력한 프로토콜

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

발생학

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

어린 싹 센터의 높은 해결의 intravital 스트라이프 - 조명 현미경

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

면역학 및 감염병학

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

네 차원 형광 이미징과 신경 말단 생활에 엔도 좀 역학의 시각화

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

신경과학

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

척추동물 축 신장 및 세분화를 연구하기 위한 세 가지 및 네 차원 시각화 및 분석 접근법

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

정확한 세포 표현과 정확한 단백질 국소화를 위한 세균 세포의 3차원 이미징

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

고급 3D 형광 현미경을 사용하여 자식 작용의 정량 분석

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

생물학

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

고속 3D 구조 조명 현미경을 사용하여 라이브 박테리아의 Cytokinetic Z 반지의 슈퍼 해상도 이미징 (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

입체 슈퍼의 간섭 광 활성화 현지화 현미경으로 F-액틴 필라멘트의 해상도 현미경 (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

머리에 고정되고 자유롭게 움직이는 동물에서 Microprism을 사용한 다층 이미징

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

저자 스포트라이트: Comparative Imaging of Neural Activity in Awake and Freedom Moving States(깨어 있고 자유롭게 움직이는 상태에서의 신경 활동 비교 이미징)

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

자유롭게 움직이고 머리가 고정된 동물에서 마이크로프리즘을 사용하여 식별된 신경 집단의 장기 이미징

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...