用于生物医学应用的荧光寿命宏观成像仪
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 627 nm LED | Parts Express | Can be replaced with different LED based on the excitation wavelength of the sensor. Used 390 nm LED for Pt-porphyrin dyes. | |
| 760 ± 50 nm emission filter | Edmund Optics | 84-788 | Can be replaced with different filter based on the emission wavelength of the sensor. Used 650 ± 50 nm bandpass filter for Pt-porphyrin dyes. |
| Balb/c mice | Envigo, UK | Balb/c | |
| Black box | Thorlabs | XE25C9/M | |
| Cricket Adapter | Photonis | Cricket-2 | |
| CT26 cells | ATCC | CT26.WT | https://www.atcc.org/products/crl-2638 |
| DMEM | Sigma-Aldrich | D0697 | Other media can also be used |
| ImageJ Software | ImageJ | Free Image analysis software. Can be downloaded from: https://imagej.nih.gov/ij/index.html | |
| MCP-125 image intensifier with P47 phosphor screen | Photonis | PP0360EF | |
| Mini dishes | Sarstedt | 83.3900.300 | 35 mm diameter |
| Mylar plastic film, 75 micron | RS Ireland | 785-0795 | Othe plastic substrates can also be used |
| NanO2-IR | home-made | n/a | The probe can be synthesised according to the published method '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.' or provided by our lab. |
| NMV-50M11” 50 mm lens | Navitar | Other lenses compatibel with C-mount adators can be used | |
| Optical breadboard | Thorlabs | MB1836 | |
| Petri Dishes | Sarstedt | 82.1472.001 | 92 mm diameter |
| Power Supply | Tenma | 72-10495 | |
| Pulse Generator | Tenma | TGP110 | |
| Sophy | Amsterdam Scientific Instruments | n/z | Provided by ASI together with the Tpx3Cam |
| Tpx3Cam | Amsterdam Scientific Instruments | TPXCAM | |
| Tri2 Software | University of Oxford | n/a | Free Time Resolved Imaging software, can be downloaded from: https://users.ox.ac.uk/~atdgroup/index.shtml |
| XYZ Translation Stage | Thorlabs | LT3 |
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