Name: fluorescence lifetime macro imager for biomedical applications
Uploaded: 2023-04-07T12:30:00
Description: Watch this Scientific Journal Video about Fluorescence Lifetime Macro Imager for Biomedical Applications at JoVE.com

爱尔兰科学基金会对这项工作的财政支持，SFI/12 / RC / 2276_P2，SFI / 17 / RC-PhD / 3484和18 / SP / 3522，突破性癌症研究（爱尔兰精准肿瘤学）表示感谢。

所有动物程序均根据欧洲共同体理事会指令（2010/63 / EU）在健康产品监管局（HPRA，爱尔兰）颁发的授权下进行，并得到科克大学动物实验伦理委员会的批准。
1. 样品制备
<ol><li>用活组织样品离 体染色<ol><li>对于 离体 应用，使用来自4周龄雌性Balb / c小鼠的新鲜分离的组织样品。</li>
		<li>在实验当天，通过斩首对小鼠实施安乐死，并快速解剖结肠（?...

<ol>
	<li>Papkovsky, D. B., Dmitriev, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+oxygen+and+hypoxia+in+cell+and+tissue+samples.">Imaging of oxygen and hypoxia in cell and tissue samples.</a> Cellular and Molecular Life Sciences. 75 (16), 2963-2980 (2018).</li><li>Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C., Kieda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+the+partial+oxygen+pressure+of+human+tissues+a+crucial+parameter?+Small+molecules+and+hypoxia.">Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia.</a> Journal of Cellular and Molecular Medicine. 15 (6), 1239-1253 (2011).</li><li>Yoshihara, T., Hirakawa, Y., Hosaka, M., Nangaku, M., Tobita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+imaging+of+living+cells+and+tissues+using+luminescent+molecular+probes.">Oxygen imaging of living cells and tissues using luminescent molecular probes.</a> Journal of Photochemistry and Photobiology C: Photochemistry Reviews. 30, 71-95 (2017).</li><li>Papkovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescence+based+oxygen+sensors+essential+tools+for+cell+biology+and+life+science+research.">Phosphorescence based oxygen sensors essential tools for cell biology and life science research.</a> 17th International Meeting on Chemical Sensors - IMCS. , 71-72 (2018).</li><li>Tsytsarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe.">In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.</a> Journal of Neuroscience Methods. 216 (2), 146-151 (2013).</li><li>O'Donovan, C., Hynes, J., Yashunski, D., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescent+oxygen-sensitive+materials+for+biological+applications.">Phosphorescent oxygen-sensitive materials for biological applications.</a> Journal of Materials Chemistry. 15, 2946-2951 (2005).</li><li>Dmitriev, R. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+probes+and+techniques+for+O+2+measurement+in+live+cells+and+tissue.">Optical probes and techniques for O 2 measurement in live cells and tissue.</a> Cellular and Molecular Life Sciences. 69 (12), 2025-2039 (2012).</li><li>Papkovsky, D. B., Zhdanov, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorescence+quenching+method+for+measurement+of+intracellular+PO+2+in+isolated+skeletal+muscle+fibers.">Phosphorescence quenching method for measurement of intracellular PO 2 in isolated skeletal muscle fibers.</a> Journal of Applied Physiology. 86 (2), 720-724 (1999).</li><li>Apreleva, S. V., Wilson, D. F., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+imaging+of+oxygen+by+phosphorescence+lifetime.">Tomographic imaging of oxygen by phosphorescence lifetime.</a> Applied Optics. 45 (33), 8547-8559 (2006).</li><li>Becker, W., Shcheslavskiy, V., R&#252;ck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+phosphorescence+and+fluorescence+lifetime+imaging+by+multi-dimensional+TCSPC+and+multi-pulse+excitation.">Simultaneous phosphorescence and fluorescence lifetime imaging by multi-dimensional TCSPC and multi-pulse excitation.</a> Advances in Experimental Medicine and Biology. 1035, 19-30 (2017).</li><li>Wolfbeis, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescent+sensing+and+imaging+of+oxygen:+Fierce+competition+to+the+Clark+electrode.">Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode.</a> BioEssays. 37 (8), 921-928 (2015).</li><li>Dmitriev, R. I., Zhdanov, A. V., Nolan, Y. M., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neurosphere+oxygenation+with+phosphorescent+probes.">Imaging of neurosphere oxygenation with phosphorescent probes.</a> Biomaterials. 34 (37), 9307-9317 (2013).</li><li>Shcheslavskiy, V. I., Neubauer, A., Bukowiecki, R., Dinter, F., Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+fluorescence+and+phosphorescence+lifetime+imaging.">Combined fluorescence and phosphorescence lifetime imaging.</a> Applied Physics Letters. 108, 091111 (2016).</li><li>Babilas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+phosphorescence+imaging+of+pO2+using+planar+oxygen+sensors.">In vivo phosphorescence imaging of pO2 using planar oxygen sensors.</a> Microcirculation. 12 (6), 477-487 (2005).</li><li>Babilas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcutaneous+pO2+imaging+during+tourniquet-induced+forearm+ischemia+using+planar+optical+oxygen+sensors.">Transcutaneous pO2 imaging during tourniquet-induced forearm ischemia using planar optical oxygen sensors.</a> Skin Research and Technology. 14 (3), 304-311 (2008).</li><li>Golub, A. S., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PO2+measurements+in+the+microcirculation+using+phosphorescence+quenching+microscopy+at+high+magnification.">PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification.</a> American Journal of Physiology-Heart and Circulatory Physiology. 294 (6), 2905-2916 (2008).</li><li>Zhdanov, A. V., Golubeva, A. V., Okkelman, I. A., Cryan, J. F., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+oxygen+gradients+in+giant+umbrella+cells:+An+ex+vivo+PLIM+study.">Imaging of oxygen gradients in giant umbrella cells: An ex vivo PLIM study.</a> American Journal of Physiology - Cell Physiology. 309 (7), 501-509 (2015).</li><li>Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+-+Techniques+and+applications.">Fluorescence lifetime imaging - Techniques and applications.</a> Journal of Microscopy. 247 (2), 119-136 (2012).</li><li>Jenkins, J., Dmitriev, R. I., Papkovsky, D. B., Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+and+tissue+O+2+by+TCSPC-PLIM.">Imaging cell and tissue O 2 by TCSPC-PLIM.</a> Advanced Time-Correlated Single Photon Counting Applications. , 225-247 (2015).</li><li>Becker, W., K&#246;nig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+TCSPC-FLIM+techniques.">Advanced TCSPC-FLIM techniques.</a> Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine. , 23-52 (2018).</li><li>Wei, L., Yan, W., Ho, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+fluorescence+lifetime+analytical+microsystems:+Contact+optics+and+CMOS+time-resolved+electronics.">Recent advances in fluorescence lifetime analytical microsystems: Contact optics and CMOS time-resolved electronics.</a> Sensors. 17 (12), 2800 (2017).</li><li>Hirvonen, L. M., Suhling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+TCSPC:+Methods+and+applications.">Wide-field TCSPC: Methods and applications.</a> Measurement Science and Technology. 28, 012003 (2017).</li><li>Hirvonen, L. M., Festy, F., Suhling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+time-correlated+single-photon+counting+(TCSPC)+lifetime+microscopy+with+microsecond+time+resolution.">Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution.</a> Optics Letters. 39 (19), 5602 (2014).</li><li>Sparks, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+new+gated+optical+image+intensifiers+for+fluorescence+lifetime+imaging.">Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging.</a> Review of Scientific Instruments. 88 (1), 013707 (2017).</li><li>Chelushkin, P. S., Tunik, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in Photon Science: Emerging New Directions. 115, (2017).</li><li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+macro-imager+based+on+Tpx3Cam+optical+camera+for+PLIM+applications.">A new macro-imager based on Tpx3Cam optical camera for PLIM applications.</a> Proceedings of SPIE. , 113591 (2020).</li><li>Fisher-Levine, M., Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TimepixCam:+A+fast+optical+imager+with+time-stamping.">TimepixCam: A fast optical imager with time-stamping.</a> Journal of Instrumentation. 11, (2016).</li><li>Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+time+stamping+of+photons+with+nanosecond+resolution+in+Timepix+based+optical+cameras.">Imaging and time stamping of photons with nanosecond resolution in Timepix based optical cameras.</a> Nuclear Instruments and Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 937. 937, 26-30 (2019).</li><li>Poikela, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timepix3:+A+65K+channel+hybrid+pixel+readout+chip+with+simultaneous+ToA/ToT+and+sparse+readout.">Timepix3: A 65K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout.</a> Journal of Instrumentation. 9, 05013 (2014).</li><li>Nomerotski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+TimepixCam,+a+fast+imager+for+the+time-stamping+of+optical+photons.">Characterization of TimepixCam, a fast imager for the time-stamping of optical photons.</a> Journal of Instrumentation. 12, 01017 (2017).</li><li>Hirvonen, L. M., Fisher-Levine, M., Suhling, K., Nomerotski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+counting+phosphorescence+lifetime+imaging+with+TimepixCam.">Photon counting phosphorescence lifetime imaging with TimepixCam.</a> Review of Scientific Instruments. 88, 013104 (2017).</li><li>Visser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDR:+A+read-out+system+for+Medipix3+&amp;+Timepix3.">SPIDR: A read-out system for Medipix3 &amp; Timepix3.</a> Journal of Instrumentation. 10, 12028 (2015).</li><li>Tsytsarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+brain+metabolism+activity+using+a+phosphorescent+oxygen-sensitive+probe.">In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.</a> Journal of Neuroscience Methods. 216 (2), 146-151 (2013).</li><li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+O2+concentration+in+ex-vivo+tissue+samples+on+a+fast+PLIM+macro-imager.">Mapping O2 concentration in ex-vivo tissue samples on a fast PLIM macro-imager.</a> Scientific Reports. 10, 19006 (2020).</li><li>Kersemans, V., Cornelissen, B., Allen, P. D., Beech, J. S., Smart, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+tumor+volume+measurement+in+the+awake,+manually+restrained+mouse+using+MRI.">Subcutaneous tumor volume measurement in the awake, manually restrained mouse using MRI.</a> Journal of Magnetic Resonance Imaging. 37 (6), 1499-1504 (2013).</li><li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+luminescence+lifetime+macro-imager+based+on+a+Tpx3Cam+optical+camera.">New luminescence lifetime macro-imager based on a Tpx3Cam optical camera.</a> Biomedical Optics Express. 11 (1), 77-88 (2020).</li><li>Papkovsky, D. 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上述协议详细描述了新成像仪的组装及其在微秒级FLIM/PLIM模式下的操作。基于TCSPC的新一代Tpx3Cam相机，通过光机械适配器Cricket与图像增强器，发射滤光片和微距镜头相结合，产生了稳定，紧凑和灵活的光学模块，易于操作。该成像仪在一系列不同的样品和分析任务中表现良好，其中包括磷光材料和活组织O2 成像的表征。成像实验采用近红外Pt（II）-苯并卟啉基细胞渗透可溶性探针NanO2-IR和...

O 2是生命系统的关键环境参数之一，了解O 2的分布及其动力学对于许多生物学研究非常重要1，2，3。通过磷光探针4，5，6，7，8和PLIM 9，10，11，12，13评估组织氧合在生物和医学研究中越来越受欢迎3，9，14，15，16， 17，18，19.这是因为与荧光或磷光强度测量不同，PLIM不受探针浓度、光漂白、激发强度、光学对准、散射和自发荧光等外部因素的影响。
然而，当前的O2 PLIM平台受到其灵敏度、图像采集速度、准确性和一般可用性的限制。时间相关单光子计数（TCSPC）与光栅扫描程序相结合，经常用于PLIM和荧光寿命成像显微镜（FLIM）设备20，21，22。然而，由于PLIM需要较长的像素停留时间（在毫秒范围内），因此图像采集时间比FLIM应用所需的时间要长得多20，22，23。其他技术，如门控CCD / CMOS相机，缺乏单光子灵敏度，帧速率低20，24，25，26。此外，现有的PLIM系统大多以微观形式使用，而宏观系统不太常见27。
基于TCSPC的PLIM宏观成像仪28 的建立是为了克服其中的许多限制。使用新的光机械适配器Cricket极大地促进了成像仪的设计，该适配器具有以下特点：i）两个C接口适配器，可轻松耦合背面的相机模块和正面的物镜;ii） 用于图像增强器的内部外壳和板球外侧的后者的电源插座;iii） 正面 C 接口适配器后面的内部空间，可以在增强器前面安装标准的 25 mm 发射滤光片;iv）带有环形调节器的内置光准直光学元件，允许镜头和相机之间的光学对准/聚焦，以在相机芯片上产生清晰的图像。
在组装的成像仪中，相机模块耦合到Cricket适配器的背面，该适配器还包含一个图像增强器，该增强器由光电阴极组成，后跟微通道板（MCP），放大器和快速闪烁体P47荧光粉。板球内部装有760 nm±50 nm发射滤光片，物镜NMV-50M11'''安装在正面C接口适配器上。最后，镜头和相机与环形调节器光学对齐。
增强器的作用是检测入射光子并将其转换为相机芯片上的快速光爆发，这些光被记录并用于生成发射衰减和寿命图像。该相机模块包括基于TCSPC的先进光学传感器阵列（256像素x 256像素）和新一代读出芯片29，30，31，32，33，允许同时记录成像芯片每个像素的光子爆发的到达时间（TOA）和超过阈值的时间（TOT），时间分辨率为1.6 ns，读出速率为80 Mpixel/s。
在这种配置中，带有增强器的相机具有单光子灵敏度。它是数据驱动的，并且基于快速像素检测器读出（SPIDR）系统34。成像仪的空间分辨率先前用平面磷光O2 传感器和分辨率板模板表征。仪器响应函数（IRF）是通过平面荧光传感器在与所有其他测量相同的设置下成像来测量的。染料的寿命约为2.6 ns，足以用于PLIM模式下的IRF测量。成像仪可以对尺寸最大为 18 mm x 18 mm 的物体进行成像，空间和时间分辨率分别为 39.4 μm 和 30.6 ns（半最大值时全宽），分别为28。
以下协议描述了宏观成像仪的组装及其后续用途，用于绘制用先前表征的近红外O 2探针NanO2-IR35染色的生物样品中的O2浓度。该探针是一种基于铂（II）苯卟啉（PtBP）染料的明亮，可光，细胞渗透的O2传感探针。它在 625 nm 处可激发，在 760 nm 处发射，并在生理范围（0%-21% 或 0-210 μM 的 O 2）内对 O2 提供强大的光学响应。该成像仪还被证明可以表征基于Pt（II）-卟啉染料的不同传感器材料。总体而言，成像器紧凑而灵活，类似于普通的摄影相机。在当前设置中，成像仪适用于不同的宽场PLIM应用。用快速激光源代替LED将进一步提高成像仪的性能，并可能实现纳秒级FLIM应用。

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			<td>627 nm LED</td>
			<td>Parts Express</td>
			<td></td>
			<td>Can be replaced with different LED based on the excitation wavelength of the sensor. Used 390 nm LED for Pt-porphyrin dyes.</td>
		</tr>
		<tr>
			<td>760 &#177; 50 nm emission filter</td>
			<td>Edmund Optics</td>
			<td>84-788</td>
			<td>Can be replaced with different filter based on the emission wavelength of the sensor. Used 650 &#177; 50 nm bandpass filter for Pt-porphyrin dyes.</td>
		</tr>
		<tr>
			<td>Balb/c mice</td>
			<td>Envigo, UK</td>
			<td>Balb/c</td>
			<td></td>
		</tr>
		<tr>
			<td>Black box</td>
			<td>Thorlabs</td>
			<td>XE25C9/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Cricket Adapter</td>
			<td>Photonis</td>
			<td>Cricket-2</td>
			<td></td>
		</tr>
		<tr>
			<td>CT26 cells&#160;</td>
			<td>ATCC</td>
			<td>CT26.WT</td>
			<td>https://www.atcc.org/products/crl-2638</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D0697</td>
			<td>Other media can also be used</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Free Image analysis software. Can be downloaded from: https://imagej.nih.gov/ij/index.html</td>
		</tr>
		<tr>
			<td>MCP-125 image intensifier with P47 phosphor screen</td>
			<td>Photonis</td>
			<td>PP0360EF</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini dishes</td>
			<td>Sarstedt</td>
			<td>83.3900.300</td>
			<td>35 mm diameter&#160;</td>
		</tr>
		<tr>
			<td>Mylar plastic film, 75 micron&#160;</td>
			<td>RS Ireland</td>
			<td>785-0795</td>
			<td>Othe plastic substrates can also be used</td>
		</tr>
		<tr>
			<td>NanO2-IR</td>
			<td>home-made</td>
			<td>n/a</td>
			<td>The probe can be synthesised according to the published method &#39;Tsytsarev V, Arakawa H, Borisov S, Pumbo E, Erzurumlu RS, Papkovsky DB. In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe. J Neurosci Methods. 2013 Jun 15;216(2):146-51. doi: 10.1016/j.jneumeth.2013.04.005. Epub 2013 Apr 25. PMID: 23624034; PMCID: PMC3719178.&#39; or provided by our lab.&#160;</td>
		</tr>
		<tr>
			<td>NMV-50M11&#8221; 50 mm lens</td>
			<td>Navitar</td>
			<td></td>
			<td>Other lenses compatibel with C-mount adators can be used</td>
		</tr>
		<tr>
			<td>Optical breadboard</td>
			<td>Thorlabs</td>
			<td>MB1836</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Sarstedt</td>
			<td>82.1472.001</td>
			<td>92 mm diameter</td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>Tenma</td>
			<td>72-10495</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Generator</td>
			<td>Tenma</td>
			<td>TGP110</td>
			<td></td>
		</tr>
		<tr>
			<td>Sophy</td>
			<td>Amsterdam Scientific Instruments</td>
			<td>n/z</td>
			<td>Provided by ASI together with the Tpx3Cam</td>
		</tr>
		<tr>
			<td>Tpx3Cam</td>
			<td>Amsterdam Scientific Instruments</td>
			<td>TPXCAM</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri2 Software</td>
			<td>University of Oxford</td>
			<td>n/a</td>
			<td>Free Time Resolved Imaging software, can be downloaded from: https://users.ox.ac.uk/~atdgroup/index.shtml</td>
		</tr>
		<tr>
			<td>XYZ Translation Stage</td>
			<td>Thorlabs</td>
			<td>LT3</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence lifetime macro imager for biomedical applications

本文提出了一种新的光致发光寿命成像仪，旨在绘制不同磷光样品中的分子氧（O 2）浓度，范围从固态O 2敏感涂层到用可溶性O2敏感探针染色的活体动物组织样品。特别是，使用了基于纳米粒子的近红外探针NanO2-IR，该探针可与625 nm发光二极管（LED）激发，发射波长为760 nm。成像系统基于Timepix3相机（Tpx3Cam）和光机械适配器，后者还装有图像增强器。O2磷光寿命成像显微镜（PLIM）是各种研究的常用方法，但当前平台在准确性、一般灵活性和可用性方面存在局限性。
这里介绍的系统是一个快速且高灵敏度的成像器，它建立在集成的光学传感器和读出芯片模块Tpx3Cam上。它被证明可以从表面染色的肠组织样本或大肠腔内染色的碎片中产生高强度磷光信号和稳定的寿命值，并允许在大约 20 秒或更短的时间内详细映射组织 O2 水平。还介绍了无意识动物移植肿瘤缺氧成像的初步实验。我们还描述了如何重新配置成像仪以用于基于Pt-卟啉染料的O2敏感材料，使用390 nm LED进行激发，使用带通650 nm滤光片进行发射。总体而言，发现PLIM成像仪可以对所用探头的寿命值以及O2 浓度的相应二维图进行准确的定量测量。它还可用于离体组织模型和活 体 动物的代谢成像。

对于 离体 成像应用，通过在组织的浆膜侧局部应用NanO2-IR探针对肠组织碎片进行染色。为了进行更深的染色，将 1 μL 探针注入管腔中。在后一种情况下，0.2-0.25毫米厚的肠壁将探头与相机隔绝开来。两种染色过程如图 2A所示。
得到的强度和PLIM图像如图2B-G所示。颜色清楚地反映了寿命值的差异，因?...

Watch this Scientific Journal Video about Fluorescence Lifetime Macro Imager for Biomedical Applications at JoVE.com

Fluorescence Lifetime Macro Imager for Biomedical Applications

本文描述了一种新型快速光学成像仪在长衰变发射样品的宏观光致发光寿命成像中的应用。描述了集成、图像采集和分析程序，以及用于成像的传感器材料的制备和表征以及成像仪在研究生物样品中的应用。

用于生物医学应用的荧光寿命宏观成像仪

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

我们的协议通过专用探头和传感材料提供宏观物体（特别是活体动物组织和整个动物）中磷光寿命和氧气浓度的详细二维映射。这些信息对于许多研究领域都很重要。我们的协议使用集成且紧凑的成像模块，该模块在TCSPC模式下运行，并提供复杂生物样品（如生命组织）中寿命和氧气分布的简单准确的可视化。拿起板球适配器并从不同侧面检查它。卸下正面 C 接口适配器以显示安装在板球中的 Photonis PP0360EF增强器，然后将 C 接口适配器放回原处。拆下 Cricket 的正面 C 接口适配器并插入 650 正负 50 纳米发射滤光片。通过放回 C 接口来修复它。然后，通过环形适配器将 TPX 三凸轮摄像头模块连接到板球模块的背面。通过 C 接口适配器将镜头连接到板球模块的正面。将 LED 连接到电源和脉冲发生器。现在，将 390 纳米超亮 LED 安装在连接到黑匣子内面包板上的柱子上。打开 LED 并聚焦，以确保对成像样品进行有效和均匀的激发。将相机组件朝下安装在光学黑匣子顶部，朝向样品成像的载物台。将相机和 LED 连接到双脉冲发生器和四通道示波器。使用示波器和脉冲发生器上的设置，同步发送到相机和 LED 的脉冲。利用 Cricket 装置上的特殊电缆和插座将增压器连接到标准电源，并将增益设置为 2.7 伏。将样品放在相机镜头之前。然后，打开相机和除增强器以外的所有电气模块。激活SOFI软件以调整操作参数，如聚焦和样品对齐。在模块中，将帧曝光设置为 0.01 秒。选择无限帧并将像素操作模式设置为超过阈值的时间。然后，转到预览并选择活动模块以打开 Medipix/Timepix 帧窗口。下一步，开始录制后，调整色标并将图像旋转到所需的方向。之后，关掉房间里的灯并打开增强器。利用镜头和Cricket适配器的对焦功能，将相机光学元件聚焦在样品台上，以生成具有良好对比度和亮度的样品的清晰图像。焦点完成后，关闭 SOFI 软件。然后，转到终端，执行定制设计软件的命令，以二进制格式获取原始数据并关闭终端。打开一个新终端来处理获取的数据。加载数据文件并运行数据缩减器。使用用 C 语言编写的专用程序分析后处理数据，该程序将数据写入 ICS 图像文件。随后，使用免费提供的时间分辨成像软件打开ICS图像文件，并使用两个指数函数来拟合磷光衰减。最后，打开 适合.ICS图像文件与可用的图像分析软件。使用查找表生成磷光寿命图像，并以伪色标对其进行编码。利用测量功能计算整个图像或感兴趣区域的平均生命周期值。显示了含氧和脱氧状态下铂八乙基卟啉染料传感器斑点的磷光强度和磷光寿命成像显微镜图像。确保在板球内部安装了正确的发射滤光片，并使用了正确的 LED、电缆连接和脉冲设置。目前，该协议仅限于动物模型，但有可能应用于诊断和治疗与组织缺氧相关的病理状态，例如癌症，中风和炎症。

Research

JoVE Journal

Biology

BioMEMS and Cellular Biology: Perspectives and Applications

微机电与细胞生物学研究所：展望与应用

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

科研

生物学

Microfluidic Applications for Disposable Diagnostics

微流控一次性诊断的应用

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Time-lapse Imaging of Mitosis 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 ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Absolute Quantum Yield Measurement of Powder Samples

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...