C. Vinegoni承认由美国国立卫生研究院（NIH）拨款1 RO1 - EB006432研究院的支持。

成像过程实验装置是在图1中详细所示。 A光源LS供应照明和吸收测量荧光测量作为激发源，它是与窄带通过干扰滤波器BPF的过滤。一组固定和可变的结合自动快门的存在，小号可以控制光的样品量，并保持足够低，以防止任何漂白的中性密度过滤器ND。统一样本照明是通过使用一个扩束合并两个镜头伽利略望远镜和扩散。该样本是沉浸在结算解决方案，并在地方举行的持有人，并沿着它通过一个高速旋转舞台的绝对精度为0.05度（新港，PR50）的垂直轴旋转。三个不同的手动控制器允许的样品上予以倾斜和调整，在正交平面垂直轴。透信号是直接由CCD相机检测到荧光信号是通过一个狭窄的干扰，再加上一个长通滤波器（Omega.光学，伯瑞特波罗（Brattleboro），VT），然后与远心镜头TL收集带通滤波器的过滤。远心镜头提供的提供独特的功能，使他们理想的光学投影层析成像优势。事实上存在光圈停止位于镜头焦距的镜头组装范围内，保证了光线，使光圈的形象，将旅游平行于光轴，从根本上消除任何角度的失真，并提供相同的放大倍率多个平面内的远心深度。 样品制备遵循下列程序是为了解决组织/器官<ol><li>在煤灰样品固定为8个小时在4C。 </li><li>样品在PBS洗涤15分钟</li><li>样品被嵌入在0.8％琼脂糖块</li><li>琼脂糖块是通过一系列的20％至100％的乙醇溶液脱水。这种脱水乙醇的一系列过程，有助于避免任何样品的不对称收缩。 </li><li>最后的样品在100％的乙醇孵育10小时，以便消除任何从样品含水量。 </li><li>样品清除所需的时间，把1:2的苯甲醇和苯甲酸苄酯的解决方案样品。请注意，时间可以有很大的不同，从样品到样品从几个小时到几天。 </li><li>乙醇可在最短的痕迹目前在结算过程中引起断绝由于酒精内结算解决方案本身的热运动的重建项目结束。为了避免这个问题，在建议的第二个结算周期。 </li><li>点4和5应该是在黑暗中进行，以避免任何漂白荧光造影剂或蛋白质。 </li></ol>心脏样品制备重要的是要删除任何血液中的含量从之前的任何组织成像，以避免在重建的高吸收文物。可以遵循以下步骤。 <ol><li>与腹腔（IP）氯胺酮（90毫克/公斤）和甲苯噻嗪（10mg/kg的）的混合注射麻醉鼠标。 注：“从这个角度提出保持避光样本” </li><li>注射肝素（IP）50 U和五分钟后，进行纵向的剖腹探查术。 </li><li>打开左肾静脉（LRV）和下腔静脉注入盐水20毫升。 </li><li>琼脂糖块应该有四个，一小时的孵化脱水，20％。 40％，60％，80％的乙醇，然后最后，样品在100％的乙醇孵育10小时（过夜）。 </li><li>心脏固定后，一般的样品制备过程 注：“从这个角度提出保持避光样本” </li></ol>吸收重建在没有散射光吸收的重建一般类似于X - CT和可使用一个共同的过滤氡反投影算法。吸收图像，然后在透超过360沿垂直轴的1度角的预测，由CCD相机。很方便对齐像素的CCD列样品平行的垂直轴。 <ol><li>琼脂糖块放入充满结算解决方案的一腔</li><li>为激发和传输测量的准直光束照亮样本</li><li>旋转样品超过360个1度角的预测。 </li><li>收购既吸收（内在）和荧光波长透图像。 </li><li>放置一个快门，以避免连续照明和减少漂白。 </li><li>使用AFiltered氡反投影算法tomographically重建样本</li></ol>荧光重建吸收重建所使用的算法不有效期为重建荧光分布，如果不考虑，吸收的贡献会导致严重的文物。 <ol><li>按照“Absoprtion重建”一节中表示。 </li><li>同时收购的吸收光谱和荧光测量</li><li>组合成归出生的场图像数据</li><li>写下的内在价值和荧光波长的光传播的正向模型方程。 </li><li>一个正向模型的格林函数的离散网格计算权重矩阵</li><li>反转的权重矩阵乘以检测归出生的场图像获得重建对象中的荧光分布</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

光学投影层析成像是一个三维成像技术，最近已作为主要在发育生物学和基因表达研究的成像工具推出。该技术首先脱水他们，然后放置在以2:1的比例（BABB或美利小号透明溶液）苯甲醇和苯甲酸苄酯的混合物呈现透明的生物样品。该技术由第一分级乙醇溶液脱水，然后把他们以2:1的比例（BABB或穆雷明显的解决方案），以清除苯甲醇和苯甲酸苄酯的混合物呈现的光学透明的生物样品。后结算过程中，可以大大降低样品中的散射贡献几乎可以忽略不计，而吸收的贡献不能完全消除。当试图重建正在调查样本内的荧光分布，影响重建，并导致这方面的贡献，无可避免的，图像失真和量化误差..虽然可以减少进一步的几周或几个月结算媒体的永久性吸收，这将导致荧光逐渐丧失和不切实际的长的样品处理时间。这才是真正的重建都外生造影剂（分子造影剂）以及内源性对比（如荧光蛋白基因表达的重组）。

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

我们建议一个光学投影层析成像出生的规范化方法（BnOPT），占成像样品的吸收性能，以获得准确，定量荧光断层重建。我们利用该算法重建的荧光分子探针分布在小动物内脏。

生于正常化荧光整个心脏成像的光学投影层析成像

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

born-normalization-for-fluorescence-optical-projection-tomography-for

Research

JoVE Journal

Biology

10.3K 次观看.  Harvard Medical School. 我们建议一个光学投影层析成像出生的规范化方法（BnOPT），占成像样品的吸收性能，以获得准确，定量荧光断层重建。我们利用该算法重建的荧光分子探针分布在小动物内脏。

视频: 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.

适当的照顾和清洁的显微镜

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

科研

生物学

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.

光镜下的主要组成部分

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

相衬，微分干涉对比（DIC）显微镜

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.

半自动小红桃光学心跳分析

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.

为介观荧光体层摄影术在体内成像的发展果蝇</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.

有丝分裂的siRNA转染后的时间推移成像

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.

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

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.

荧光成像与一纳米精度（FIONA）

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.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（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.