루시페라제 표현 암 세포 라인의 다양한는 마우스 모델에서 미리 임상 연구에 사용될 수 있습니다. 이러한 세포는 쉽게 선택 마커에 대한 필요가 표준 미디어 성장하는 병원체가없는 냉동 문화로 제공됩니다. 우리의 실험을 위해, 우리는 생체내의 유전자 발현이나 tumorgenesis의 광학 지표 역할을 루시페라제 유전자를 표현 4T1 - luc2 murine 내유 종양 세포 라인을 사용합니다. 우리는 비 invasively 기본 종양의 성장을 추적하는 루시페라제 표현을 사용하지만, 그들은 또한 전이성 병변을 찾아 모니터링하는 데 사용할 수 있습니다. 루시페라제 활동 주입하기 전에, 그리고이 작업을 수행하기 위해서는 검증해야 90% 합류 플라스크는 trypsinization에 의해 수확 후 계산됩니다. 수행하는 50,000 셀이 다음 microtiter 접시 및 시리얼 dilutions의 단일 우물에 적절하고 있습니다. Luciferin는 ML 당 150ug에있는 우물에 추가 2 분 동안 incubated입니다. microplate는 IVIS이나 표현 수준을 결정하는 발광 판 Reader에서 몇 군데 있습니다. 이 세포 라인은 초당 세포 당 6,500 광자까지 표현하고 있지만, 초당 셀 당 500 광자 이상 어떤 표현 수준은 생체내에서 성공적으로 몇 군데 있습니다. 이제 우리는 최적의 활동의 세포를 가지고, 우리는 피하 주입 단계로 진행할 수 있습니다. 종양의 최적의 검출을 용이하게하기 위해, 우리는 athymic immunocompromised 누드 마우스 변형을 사용하고 있습니다. 주사하기 전에, 동물 깊은 마취에 대한 효과 anesthetized 있습니다. 다음 우리가 100ul PBS에서 250,000 세포까지 주사할 수는 측면으로 subcutaneously 주입하고 있습니다. 1 ML의 주사기에있는 세포를로드하고 26 게이지 바늘을 첨부합니다. 포셉는 &quot;텐트&quot;하고 기지에 세포를 주입 함께 부드럽게 피부를 들어 봐요. 새로 주입된 세포는 즉시 몇 군데 있습니다. 각 이미지 세션에서 kg의 체중 당 Luciferin의 150mg의 총 후 복막 구멍 두개의 주사를 통해 관리합니다. 본 연구에서는 동물은 일관성있는 광자 유량을 보장하기 위해 Luciferin 주입 후 10 분 몇 군데 있습니다. 우리는 다음 단계에서 당신에게 보여 드리죠. Luciferin 동력학은 매일 다를 수 있습니다. 이 특정 모델에서 Luciferin 주입 후 10 분 측정하면 15 %의 다양성의 범위 내에서 결과를 주었다. 우리 실험에서는 우리는 다시 - thinned 요금 결합 소자 ° C 최대 감도를 달성하기 위해 -90으로 냉각을 사용하여 생체내 이미징 시스템에서 IVIS 스펙트럼을 사용합니다. 절대 quantitation를 지원하기 위해, 시스템 다운 시간 동안 어두운 요금을 측정하고 초기화하는 동안 자체 보정을 실행합니다. 이미징을 시작하려면, IVIS 시스템을 초기화 (한 번의 클릭) 및 실험을위한 이미징 매개 변수를 설정합니다. 몇 군데되는 동물의 숫자에 대한보기의 필드를 선택합니다. 최대 5 동물은 필수 마취 매니폴드를 사용하여 장비 유지하실 수 있습니다. 무대는 상수입니다 37 ° C 동물​​의 체온을 유지하기 위해. EKG 포트가 스트레스 절차 기간 동안 동물의 건강을 모니터링하기 위해 제공됩니다. 이미지 소프트웨어, 노출 시간, F - 중지하고 픽셀을 생활 비닝은 세포주의 표현 수준에 따라 최적화할 수 있습니다. 이러한 설정은 양적 결과에 영향을주지 않고 실험 도중 언제든지 변경할 수 있습니다. IVIS 흰 빛과 이미지를 중첩되는 정량 발광 생물이나 형광 신호, 아래에있는 동물의 사진 이미지를 얻습니다. 발광 생물 신호는 초당 광자로 표현하고 강도지도로 표시됩니다. 이미지 디스플레이는 quantitation에 영향을주지 않고 이미지에 최적의 콘트라스트와 해상도를 제공하기 위해 조정됩니다. 세포에서 발광이 관심을 도구의 영역을 사용하여 주사의 사이트에서 측정할 수 있습니다. 측정 데이터는 저장하거나 분석을 위해 내보낼 수있는 이미지 캡처에 관한 모든 실험 매개 변수와 함께 테이블에 표시됩니다 여러 이미지를 획득하여 실험의 특성에 따라 초 개월에 걸쳐 종단 연구에 비교할 수 있습니다. 우리는 시간 제로에서 종양에서 광자 자속을 측정하고 격주 간격으로 이미지와 함께 4 주 동안 모니터합니다. 종양에서 광자 유량은 bioluminescence는 종양의 크기와 직접 연결합니다 그래서 루시페라제을 표현하는 라이브 세포의 수에 비례합니다. 오일 포스트 주입에 종양이 아직 만져서 알 수있는 것이 아니라 세포가 bioluminescence과 종양이 적극적으로 성장을 보아입니다 통해 계량하실 수 있습니다. 이 단계에서는 bioluminescence 신호는 훨씬 stronge입니다R. 노출 시간, F - 중지하고 픽셀 비닝는 이미지가 명확하고 카메라가 포화되지 않도록 조정할 수 있습니다. 이러한 측정은 실험에 이전 이후 수집들에게 비교할 수 있도록 IVIS 자동으로 집광의 변화에​​ 대한 보상. 칠일에 게시 주입 종양 측정은 이미 데이터가 7 일 발생한 것은 이번이 처음과 bioluminescence에 대한 만져서 알 수있는 수 있습니다. 28일 포스트 주입에서 종양 괴사되고 있으며, 세포는 죽지 시작합니다. 캘리퍼스 측정하여 예상 종양 크기는 appreciably 변경되지 않습니다하지만, 종양의 발광은 세포 죽음을 나타내는 줄어 듭니다. 인도적 종점에 도달할 때까지 캘리퍼스 측정 및 bioluminescence 측정 계속하실 수 있습니다. hypoxia 또는 치료 정권에 의한 종양 괴사들은 종양 질량을 감소하지 않더라도 감소 bioluminescence로 표시됩니다.

나는 시각 실험이 문서의 저널을 생산하기 위해 수행 모든 실험이 기관 애니멀 케어 승인한 동물 프로토콜 060을 준수 수행 있다고 확신하고 칼리퍼 생명 과학에서위원회 (IACUC), 알라 메다 CA 재 김 박사를 사용하고 싶습니다 . D., IACUC 위원장, 칼리퍼 생명 과학.

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1 마우스 내유 종양 세포는 15~20일에 만져서 알 수있는 종양을 형성 뉴 / 뉴 생쥐의 하위 cutaneously 이식 수 있습니다. 이 이종 이식 종양 모델 시스템 putative antitumor 화합물의 생체내 평가 미리 임상을 위해 중요합니다. 4T1 세포 라인은 constitutively 반딧불 루시페라제 유전자 (luc2)을 표현하기 위해 설계되었습니다. 4T1 - luc2 종양을 나르는 생쥐는 종양 IVIS 스펙트럼 같은 민감한 광학 이미징 시스템을 사용하여 모니터링할 수있는 영상 조명 신호를 방출 Luciferin로 주입하는 경우. 종양에서 광자 플럭스은 발광 세포의 개수에 비례하여 신호가 종양 성장과 발전을 모니터 측정할 수 있습니다. IVIS는 발광 생물 신호 및 종단 연구의 절대 quantitation이 양적 결과를 손상시키지 않고 몇 개월 동안 신호 크기의 여러 명령을 통해 수행할 수 있도록 조정합니다. 종양 크기가 기존의 물리적인 방법으로 만져서 알 수있는 또는 측정되기 전에 종양 성장 bioluminescence으로 며칠 동안 모니터링할 수 있습니다. 이것은 급속한 모니터링 종양 발달 초기에 이벤트에 대한 통찰력을 제공하거나 짧은 실험 절차를 초래할 수 있습니다. 종양 세포 죽음과 hypoxia 또는 약물 치료에 의한 괴사는 발광 생물 신호의 감소에 의해 초기 표시됩니다. 이 세포 죽음을 물리적인 방법으로 측정으로 종양의 크기 감소에 의해 동반되지 않을 수도 있습니다. 종양 괴사의 초기 이벤트를 볼 수있는 능력은 치료 요원의 선택과 개발에 상당한 영향을 미치고 있습니다. IVIS를 사용하여 종양 성장의 양적 영상은 정확한 quantitation를 제공하고 결과를 생성하기 위해 실험 과정을 가속.

Watch this Scientific Journal Video about In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum at JoVE.com

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

루시페라제 표현 내유 종양 세포는 생쥐의 subcutaneously 이식 및 비 invasively 종단 연구에 종양이 성장과 발전을 모니터 광학 이미징을 사용하여 시각입니다.

IVIS 스펙트럼을 사용하여 내유 종양의 생체내 이미징에서 발광 생물

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

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In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

46.7K 조회수.  Caliper Life Sciences.  中文 

비디오: In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

내유 이미징 창문 Dendra2 Photoswitching

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

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생물학

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

Microprism를 사용하여 딥 두피 레이어의 생체내 이미징에서

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

내유 상피 이식 절차

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

의 전체 순서의 NGS 분석을위한 Transposase 기반 기술에 의해 gDNA를 농축<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA 손상 복구에 참여&gt;, 9 유전자

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Screenable<em&gt; 생체</em&gt; 형질 전환 발광 생물을 사용 미토콘드리아 변조기에 대한 분석<em&gt; 예쁜 꼬마 선충</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

의 강성 측정<em&gt; 전의 VIVO</em원자 힘 현미경을 사용하여&gt; 마우스 대동맥

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

초파리 준비 및 빛 간섭 현미경을 사용하여 생체 내에서 심장 기능의 세로 이미징 (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...