C. Vinegoni 건강 (NIH) 부여 1 RO1 - EB006432 국립 연구소의 지원을 인정한다.

이미징 절차 실험 설정은 그림 1에 자세히 표시됩니다. 광원 LS는 흡수 측정을위한 조명으로, 형광 측정 여기 소스로 모두 제공되며, 그것은 좁은 밴드 통과 필터 BPF와 간섭 필터입니다. S는 샘플에 빛의 양을 제어하고 photobleaching을 방지하기 위해 충분히 낮은 유지하도록 자동으로 셔터의 존재와 함께 고정 및 가변 중립 밀도 필터 ND의 설정하십시오. 통일 샘플 조명은 빔 확장기가 결합된이 렌즈 갈릴리 망원경과 디퓨저와 함께 사용하여 이루어진다. 예제 S는 비운의 솔루션에 포장되어 있으며, 0.05 도의 절대 정확도와 고속 회전 단계의 웨이 (뉴 포트, PR50)하여 수직 축을 따라 소유자 및 회전에 장소에서 개최됩니다. 세 개의 서로 다른 수동 컨트롤러는 직교 평면에 기울여서 조정해야 샘플 수직 축에 대한 수 있습니다. transillumination 신호 직접 CCD 카메라에 의해 감지되며 형광 신호는 다음 longpass 필터 (Omega. 광학, 브레 틀 보로, VT)과 telecentric 렌즈 TL과 수집과 함께 좁은 대역 통과 간섭 필터를 통해 필터링됩니다. Telecentric 렌즈는 광학 프로젝션 tomography에 이상적 만드는 독특한 기능을 제공하는 장점을 제공합니다. 사실 조리개의 존재는 렌즈의 초점에서 렌즈 어셈블리 내에있는 중지는 조리개 중지의 이미지를 만들 광선은, 거의 모든 관점 왜곡을 제거하고 같은 배율을 제공하는 광학 축에 평행 여행 것이라는 보장 telecentric 깊이 내에서 여러 대의 비행기. 샘플 준비 다음 절차는 조직 / 기관 문제를 해결하기 위해 다음이다 <ol><li> 샘플은 4C 8 시간 PFA에서 수정되었습니다. </li><li> 샘플은 다음 15 분 동안 PBS에 씻은입니다 </li><li> 샘플은 0.8 % 아가로 오스 블록에 포함된 </li><li> 아가로 오스 블록 에탄올 솔루션의 20 % ~ 100 %의 시리즈를 통해 탈수 있습니다. 이 dehydrating 에탄올 시리즈 절차는 샘플의 비대칭 수축을 피하는에 도움이됩니다. </li><li> 마지막 예제는 샘플로부터 수분을 제거하기 위해 100 % 에탄올에 10 시간 incubated입니다. </li><li> 취소한 샘플에 필요한 시간 벤질 알코올 및 벤질 벤조 산의 솔루션 1시 2분에 샘플을 넣어. 시간이 몇 시간에서 며칠이 샘플에서 샘플을 상당히 달라질 수 있습니다. </li><li> 최소한의 흔적에 에탄올은 삭제 솔루션 자체 내에서 알코올의 열 운동에 의한 reconstructions의 유물을 도려 상승을주는 개간 과정의 끝에 존재하실 수 있습니다. 추천이 문제를 방지하기 위해, 두 번째 개간주기. </li><li> 포인트 4와 5는 모든 형광등 대비 대리인 또는 단백질의 표백을 피하기 위해 어둠 속에서 수행되어야합니다. </li></ol> 심장 샘플 준비 reconstructions 높은 흡수 유물을 피하기 위해 몇 군데가 이전에 어떤 조직에서 어떤 혈액 콘텐츠를 제거하는 것이 중요합니다. 다음 절차는 다음 수 있습니다. <ol><li> 마취제 (90 MG / kg)과 xylazine (10mg/kg)의 혼합의 intraperitoneally (IP) 주입으로 마우스를 마취. 참고 : &quot;.이 시점에서 앞으로 빛으로부터 보호 샘플을 유지&quot; </li><li> 헤파린 (IP)의 50 U를 주사하고 5 분 후에, 세로 개복술을 수행합니다. </li><li> 왼쪽 신장 정맥 (어브)를 열고 IVC에 생리 식염수 20ml의 달이다. </li><li> 아가로 오스 블록은 20 %, 넷, 한 시간 incubations로 탈수한다. 다음 40 %, 60 % 및 80 % 에탄올, 그리고 결국, 샘플 10 시간 (야간)에 대한 100 % 에탄올에 incubated 수 있습니다. </li><li> 심장은 다음 일반 샘플 준비 절차를 다음과 고정됩니다 참고 : &quot;.이 시점에서 앞으로 빛으로부터 보호 샘플을 유지&quot; </li></ol> 흡수 Reconstructions 산란의 부재에서 광 흡수의 재건 일반적으로 X - CT로 유사하며 일반적인 필터링 라돈의 backprojection 알고리즘을 사용하여 얻을 수있다. 흡수 이미지는 다음 세로 축을 따라 1도 각도 360 계획을 통해 transillumination에 CCD 카메라로 찍은 있습니다. 그것은 CCD의 픽셀의 열 샘플 병렬의 수직 축을 정렬하는 것이 편리합니다. <ol><li> 삭제 솔루션 가득한 챔버에 아가로 오스의 블록을 플레이스 </li><li> 여기 및 전송 측정 모두에 대해 collimated 빔을 시료를 조명 </li><li> 1도 각도 360 계획을 통해 샘플을 회전합니다. </li><li> 모두 흡수 (내장) 및 형광 파장에 transillumination의 이미지를 획득. </li><li> 지속적인 조명을 피하고 표백 줄이기 위해 셔터를 놓습니다. </li><li> AF를 사용하여iltered 라돈의 backprojection 알고리즘은 tomographically 샘플을 재구 성할 </li></ol> 형광 Reconstructions 흡수 reconstructions에 사용되는 알고리즘은 고려하지 않을 경우, 흡수 기여가 심각한 유물로 이어질 것이며, 형광 유통의 재건을 위해 유효하지 않습니다합니다. <ol><li> 로 &quot;Absoprtion의 Reconstructions&quot;섹션에 표시된 진행합니다. </li><li> 동시에 흡수 및 형광 측정을 모두 취득 </li><li> 표준 출생 필드 이미지로 데이터를 결합 </li><li> 내장과 형광 파장에 대한 빛의 전파의 전달 모델의 방정식을 적어 두십시오. </li><li> 앞으로 모델의 그린 기능 discretized 메쉬에 체중 매트릭스를 계산 </li><li> 체중 매트릭스를 반전하고 개체의 형광 유통의 재건을 얻기 위해 감지된 표준화 출생 현장 이미지에 의해 그것을 곱하면 </li></ol>

<ol>
	<li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R., Davidson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-544 (2002).</li><li>Lee, K., Avondo, J., Morrison, H., Blot, L., Stark, M., Sharpe, J., Bangham, A., Coen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+plant+development+and+gene++in+three+dimensions+using+optical+projection+tomography.">Visualizing plant development and gene in three dimensions using optical projection tomography.</a> Plant Cell. 18, 2145-2156 (2006).</li><li>Oldham, M., Sakhalkar, H., Wang, Y. M., Guo, P. Y., Oliver, T., Bentley, R., Vujaskovic, Z., Dewhirst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+whole+rodent+organs+using+optical+computed+and+emission+tomography.">Three-dimensional imaging of whole rodent organs using optical computed and emission tomography.</a> J. Biomed. Opt. 12, 1-10 (2007).</li><li>Ntziachristos, V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+three-dimensional+fluorescence+reconstruction+of+diffuse+media+by+use+of+a+normalized+Born+approximation.">Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation.</a> Opt. Lett. 26, 893-895 (2001).</li></ol>

born normalization for fluorescence optical projection tomography for whole heart imaging

광학 프로젝션 tomography는 최근 주로 발달 생물학 및 유전자 발현 연구에 이미징 도구로 소개되어 3 차원 영상 기술입니다. 기술이 먼저 그들을 dehydrating 후 벤질 알코올 및 2시 1분 비율 (밥 또는 머리의 클리어 솔루션)의 벤질 벤조 산의 혼합물에 배치하여 광학 투명 생물 학적 샘플을 렌더링. 기술 먼저 다음 벤질 알코올 및 취소 2시 1분 비율 (밥 또는 머리의 클리어 솔루션)의 벤질 벤조 산의 혼합물에서 그들을 배치 등급 에탄올 솔루션에서 그들을 dehydrating하여 광학 투명 생물 학적 샘플을 렌더링. 흡수 기여 완전히 제거될 수 없다면서 삭제 처리 후 시료의 산란 공헌을 획기적으로 줄일 수와 거의 무시할 만들 수 있습니다. 조사 샘플 내에 형광 분포를 재구성하려고하면,이 기여 이미지 유물 및 부량 오류로 불가피하게, reconstructions 및 리드에 영향을 .. 흡수가 주 또는 지운 미디어 개월 영속성과 함께 추가로 줄일 수 있지만, 이것은 형광의 진보적인 손실과 unrealistically 긴 샘플 처리 시간으로 이어질 것입니다. 두 외인성 대비 요원 (분자 대비 대리인)뿐만 아니라 내생 대비 (유전자 표현 형광 단백질의 예 reconstructions)을 재구성하면 마찬가지입니다.

Watch this Scientific Journal Video about Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging at JoVE.com

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

우리는 정확하고 양적 형광 단층 reconstructions를 얻기 위해 몇 군데 샘플의 흡수 특성에 대한 계정 광학 프로젝션 Tomography 위해 태어난 표준 접근 (BnOPT)를하는 것이 좋습니다. 우리는 작은 동물의 기관 내에있는 형광 분자 프로브 분포를 재구성하기 위해 제안된 알고리즘을 사용합니다.

전체 심장 이미징에 대한 형광 광 프로젝션 Tomography에 대한 출생 정규화

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

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10.3K 조회수.  Harvard Medical School. 우리는 정확하고 양적 형광 단층 reconstructions를 얻기 위해 몇 군데 샘플의 흡수 특성에 대한 계정 광학 프로젝션 Tomography 위해 태어난 표준 접근 (BnOPT)를하는 것이 좋습니다. 우리는 작은 동물의 기관 내에있는 형광 분자 프로브 분포를 재구성하기 위해 제안된 알고리즘을 사용합니다.

비디오: Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

현미경의 올바른 관리 및 청소

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

연구

생물학

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

라이트 현미경의 주요 구성 요소

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

단계 대비 및 차등 간섭 대비 (DIC) 현미경

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

작은 하트의 세미 - 자동 광학 도면 분석

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

에 대한 Mesoscopic 형광 Tomography 인 - 생체내</em개발> 이미징 Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

광학 프로젝션 Tomography를 사용하여 인간 유방암 표본과 그 분석의 장기 문화

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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 ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.