Of the large variety of technologies that can be used to study mammalian physiology and pathophysiology, confocal and two‐photon intravital microscopy (IVM) is unique. For the past three decades, it has enabled researchers to monitor biological processes, live and in their true environment, all at high temporal and spatial resolutions. It is the only technology that provides single‐cell (and even subcellular) information, while maintaining the complex environment that exists only in living organisms. As such, IVM has revealed not only how cells move and communicate in various healthy tissues, but also how these behaviors change under pathological conditions. The impact of IVM has been felt within many biomedical research areas, but particularly in fields of neuroscience, immunology, and in cancer research, where the technology has helped to elucidate the key mechanisms of immune cell communication and metastatic progression and has revealed new targets for therapeutic strategies.

However, even with this success, advances that expand the utility of the technique and enable IVM to provide more complete information about in vivo pathophysiological phenomena at the single‐cell level were still necessary and continue to be made.1 These recent developments include (1) expanding the spectral and spatiotemporal boundaries of the current technology,2-10 (2) retrieving more detailed molecular information on cellular and tissue functions within living organisms,11-18 and (3) translating basic science knowledge into clinically relevant information.19-21 The publications in this special issue, Intravital Microscopy: Innovations and Applications, review these recent achievements, as well as present new advances in the IVM of osteoimmunological cross talk, musculoskeletal development, infection biology, and cancer research.

To begin this special issue, Coste et al. provide a comprehensive review of the wide variety of intravital imaging techniques used to access various tissues, from transdermal imaging to implantable chronic imaging windows. They then discuss how new advances in IVM have led to novel biological insights in the fields of immunology, metabolomics, and cancer metastasis, and how new instrumentation and clinical protocols are allowing the direct application of IVM to patient care.

Next, Handschuh et al. review the ways in which IVM goes beyond looking at the cells of a single organism, but instead elucidates the mechanisms of host–pathogen interactions. They describe how IVM, in combination with advances in labeling and tracking of specific cell types and individual pathogens, has helped to dissect the sequence of events during the course of infections: from the early events of tissue invasion, where pathogens employ different strategies to establish themselves within the host and circumvent or evade immune defenses, to the induction of the immune response by pathogen–immune cell and immune cell–immune cell interactions, and finally to events implicated in the clearance of infections or the induction of immunopathology. Finally, the authors discuss the new advances, such as fluorescent reporter‐based techniques, that will provide tools for extracting functional information on immune cell signaling and metabolism that could enhance our understanding of the mechanistic aspects of pathogen‐mediated infections.

As an example of this type of development, Okkelman et al. combine two commercially available dyes with FLIM imaging of live cultured cells and 3D organoids to create and validate fluorescent probes capable of the quantitative assessment of cell metabolism and mitochondrial function through the measurement of mitochondrial membrane potential. This technology offers a new way of viewing the metabolic processes of all cells, independent of their origin, phenotype, or state of differentiation, as respiration is fundamental to every cell type. The technology presented by Okkelman et al. may complement two other readouts of nutrition and respiration already employed in intravital imaging: the ubiquitous coenzyme‐dependent (NAD(P)H and FAD) metabolism and oxygen availability.9, 13-15, 17, 18 However, the performance of the current technology has not been yet applied intravitally, the results presented by the authors convincingly demonstrate its power and feasibility for use with intravital imaging.

In the manuscript by Stefanowski et al., the authors further develop and expand the utility of their recently published microendoscope for longitudinal two‐photon intravital imaging of the murine femur.3 By using a modular construction, they make the technique capable of visualizing the spatiotemporal dynamics of bone healing and regeneration after osteotomy—a generally accepted long bone fracture model in rodents. This new technology, named LIMBOSTOMY, has a significantly increased observation volume compared to the original design3 as it uses a larger gradient refractive index (GRIN) lens (600 μm diameter). Particular attention was paid to correct the wave front distortions that typically compromise the optical performance of GRIN lenses. This approach results in subcellular resolution throughout the imaged cylindrical volume (400 μm diameter and 100 μm in height). The authors apply LIMBOSTOMY to monitor and quantify the neovascularization process within the callus of fracture sites after osteotomy. Being a chronic imaging technique capable of investigating the entire process of bone healing in one and the same individual, LIMBOSTOMY enables the authors to describe in detail the course of two phases of angiogenesis: first, rapid vessel sprouting pervades the field of view within 3–4 days after osteotomy, and second, the vessel network continues to be dynamically remodeled up to 14 days after osteotomy. Applications of this technology have the potential to go beyond bone healing research, to impact research into developmental bone biology as well as hematopoiesis within the marrow of long bones.

Putting this work into its broader context is the review by Kim et al., which presents recent advances in multiphoton imaging technologies that have led to a greater understanding of bone tissue homeostasis, remodeling, and regeneration. This work thoroughly discusses the technologies that have helped to identify cellular phenomena, and their underlying signaling pathways, involved in the communication between different types of bone and bone marrow cells. As stated by Kim et al., bone is “a dynamic connective and supportive tissue”, and is “constantly sensing and responding to both external mechanical forces and internal systemic and local signals.” This specific feature of bone is eminently impactful not only when bone fractures occur (as was discussed in Stefanowski et al.), but also during the key processes of normal bone physiology. Kim et al. specifically focus on the dynamic interplay of osteoblasts, osteocytes, and osteoclasts, as well as on the challenges which a continuously changing vasculature and extracellular matrix impose on these cells. The authors present key IVM applications that have led to a better understanding of the dynamic molecular and cellular mechanisms underlying bone tissue homeostasis, remodeling, and regeneration under physiological and pathological conditions.

The original research article by Servin‐Vences et al. focuses on the application of two‐photon microscopy (TPM) to investigate the molecular origins of osteoarthritis. The authors use linearly and circularly polarized two‐photon excitation to look at the microscopic structure of femoral cartilage in 4–5 days old wild‐type mice, and those lacking the polymodal ion‐channel TRPV4 (TRPV4^−/−). By looking at nonambulatory pups, the investigators aimed to gain insight into whether there are structural changes in the cartilage in Trpv4^−/− mice at an age before the femoral heads have been affected by mechanical loading due to weight bearing and walking. Utilizing polarized excitation and detection, second‐harmonic generation signals from all orientations of collagen fibers within the articular cartilage were collected and compared, enabling an in‐depth characterization of the collagen's supramolecular alignment, morphology, and organization. Through these experiments, no statistically significant difference between the two structures was observed, indicating that the impact of a loss of TRPV4 on cartilage biology is likely not due to malformation of the cartilage but due to a slower process that occurs over time.

The combination of in vivo TPM with other imaging modalities is an interesting, yet largely unexplored field, with relatively few, mostly not coregistered, examples.22, 23 In their publication, Rakhymzhan et al. present a coregistered nearly simultaneous large‐volume multimodal IVM approach that combines TPM with optical coherence tomography (OCT), a technology which has already found broad application in the clinic, as exemplary highlighted by.24 They employ their imaging approach to visualize and link the morphology and specific cellular phenotypes in a broad variety of organs (i.e., lymph node, spleen, retina, and paws) in mice. Using this technology, the authors find that the morphology and motility of tissue‐resident macrophages (CX₃CR1⁺ cells), as revealed by TPM, correlates with the tissue organization, as captured by the OCT and second‐harmonic generation signals. Their approach of coregistered OCT and TPM has the potential to simultaneously provide molecule‐specific high‐resolution imaging (TPM) and label‐free visualization of tissue morphology (OCT), and thus build a bridge between basic research knowledge and clinical observations.

The relevance of IVM for clinical applications is emphasized by the original article of Li et al., which is focused on the development and monitoring of therapeutics. Cellular heterogeneity may modify drug responses, as in the case of monoclonal antibody therapies within solid tumors. To address this question, the authors develop a confocal IVM approach that allows, within the solid tumor environment, visualization and tracking of the delivery and action of trastuzumab: a monoclonal antibody (against the growth factor receptor HER2) that is widely used in the clinic for treating breast and gastric cancer patients. To mimic the clinically observed cellular heterogeneity in tumors of patients the researchers implanted in mice mosaicked xenografts composed of a mixture of cancer cells with variable HER2 expression. In this way, the authors were able to show that, while trastuzumab accumulates to a greater extent in tumor cells expressing high levels of HER2 (as compared to HER2‐low tumor cells), over time, most of the drug delivered to the animal actually accumulates within tumor‐associated phagocytes. The findings presented here provide strong evidence of how environmental conditions lead to variations of drug therapy efficacy, demonstrating the potential of IVM in translational research.

Taken together, the collection of articles within this special issue demonstrates the wide‐ranging impact that IVM has in the biomedical sciences and in the clinic. Advances such as those presented here will provide the tools and the basic science knowledge to impact the next generation of clinical care.

在可用于研究哺乳动物生理学和病理生理学的多种技术中，共聚焦和双光子活体显微镜 (IVM) 是独一无二的。在过去的三年中，它使研究人员能够以高时间和空间分辨率监测生活和真实环境中的生物过程。它是唯一提供单细胞（甚至亚细胞）信息，同时保持仅存在于生物体中的复杂环境的技术。因此，IVM 不仅揭示了细胞如何在各种健康组织中移动和交流，而且揭示了这些行为在病理条件下如何变化。许多生物医学研究领域都感受到了 IVM 的影响，尤其是在神经科学、免疫学和癌症研究领域，

然而，即使取得了这一成功，扩大该技术的实用性并使 IVM 能够在单细胞水平上提供关于体内病理生理现象的更完整信息的进展仍然是必要的，并将继续进行。1这些最近的发展包括 (1) 扩展当前技术的光谱和时空边界，2-10 (2) 检索有关生物体内细胞和组织功能的更详细的分子信息，11-18和 (3) 翻译基础科学知识到临床相关信息。19-21本期特刊中的出版物，活体显微镜：创新和应用，回顾这些最近的成就，以及目前在骨免疫交叉对话、肌肉骨骼发育、感染生物学和癌症研究的 IVM 方面的新进展。

为了开始这个特刊，科斯特等人。提供对用于访问各种组织的各种活体成像技术的全面审查，从透皮成像到可植入的慢性成像窗口。然后，他们讨论了 IVM 的新进展如何在免疫学、代谢组学和癌症转移领域产生了新的生物学见解，以及新的仪器和临床协议如何允许 IVM 直接应用于患者护理。

接下来，Handschuh 等人。回顾 IVM 超越观察单个生物体细胞的方式，而是阐明宿主 - 病原体相互作用的机制。他们描述了 IVM 如何结合特定细胞类型和单个病原体的标记和跟踪方面的进步，帮助剖析感染过程中的事件序列：从组织侵袭的早期事件开始，病原体采用不同的策略来建立自身在宿主体内并规避或逃避免疫防御，通过病原体-免疫细胞和免疫细胞-免疫细胞相互作用诱导免疫反应，最后是与感染清除或免疫病理学诱导有关的事件。最后，作者讨论了新的进展，例如基于荧光报告基因的技术，

作为此类开发的一个例子，Okkelman 等人。将两种市售染料与活培养细胞和 3D 类器官的 FLIM 成像相结合，以创建和验证能够通过测量线粒体膜电位对细胞代谢和线粒体功能进行定量评估的荧光探针。这项技术提供了一种观察所有细胞代谢过程的新方法，独立于它们的起源、表型或分化状态，因为呼吸是每种细胞类型的基础。Okkelman 等人提出的技术。可以补充已经在活体成像中使用的另外两个营养和呼吸读数：无处不在的辅酶依赖性（NAD（P）H 和 FAD）代谢和氧气可用性。9、13-15、17、18 然而，当前技术的性能尚未应用于活体成像，作者提供的结果令人信服地证明了其用于活体成像的能力和可行性。

在 Stefanowski 等人的手稿中，作者进一步开发和扩展了他们最近发表的显微内窥镜在鼠股骨纵向双光子活体成像中的应用。3通过使用模块化结构，他们使该技术能够可视化截骨后骨愈合和再生的时空动态——这是一种普遍接受的啮齿动物长骨骨折模型。这项名为 LIMBOSTOMY 的新技术与原始设计相比具有显着增加的观察量3因为它使用更大的梯度折射率 (GRIN) 透镜（直径 600 μm）。特别注意校正通常会影响 GRIN 透镜光学性能的波前畸变。这种方法导致整个成像圆柱体积（直径为 400 微米，高度为 100 微米）的亚细胞分辨率。作者应用 LIMBOSTOMY 来监测和量化截骨术后骨折部位愈伤组织内的新生血管形成过程。作为一种慢性成像技术，能够研究同一个人的整个骨愈合过程，LIMBOSTOMY 使作者能够详细描述血管生成的两个阶段的过程：首先，快速的血管发芽在 3–截骨术后 4 天，第二次，在截骨术后长达 14 天，血管网络继续动态重塑。这项技术的应用有可能超越骨骼愈合研究，影响发育骨骼生物学以及长骨骨髓内的造血研究。

Kim 等人的评论将这项工作置于更广泛的背景下，该评论介绍了多光子成像技术的最新进展，这些技术使人们对骨组织稳态、重塑和再生有了更深入的了解。这项工作彻底讨论了有助于识别细胞现象及其潜在信号通路的技术，这些技术涉及不同类型的骨和骨髓细胞之间的通信。正如 Kim 等人所说，骨骼是“一种动态的结缔组织和支持组织”，并且“不断地感知和响应外部机械力以及内部系统和局部信号。” 骨骼的这一特定特征不仅在发生骨折时（如 Stefanowski 等人所讨论的）而且在正常骨生理学的关键过程中都具有显着影响。金等人。特别关注成骨细胞、骨细胞和破骨细胞的动态相互作用，以及不断变化的脉管系统和细胞外基质对这些细胞施加的挑战。作者介绍了关键的 IVM 应用，这些应用有助于更好地了解生理和病理条件下骨组织稳态、重塑和再生的动态分子和细胞机制。

Servin-Vences 等人的原始研究文章。专注于应用双光子显微镜 (TPM) 研究骨关节炎的分子起源。作者使用线性和圆极化双光子激发来观察 4-5 天大的野生型小鼠和那些缺乏多峰离子通道 TRPV4 (TRPV4 ^-/- )的股骨软骨的微观结构。通过观察不能走动的幼崽，研究人员旨在深入了解 Trpv4 ^-/-的软骨中是否存在结构变化股骨头之前年龄的小鼠因负重和行走而受到机械负荷的影响。利用极化激发和检测，收集并比较来自关节软骨内胶原纤维所有方向的二次谐波生成信号，从而能够深入表征胶原的超分子排列、形态和组织。通过这些实验，没有观察到两种结构之间的统计学显着差异，表明 TRPV4 缺失对软骨生物学的影响可能不是由于软骨畸形，而是由于随着时间的推移发生的较慢的过程。

体内 TPM 与其他成像方式的结合是一个有趣的领域，但在很大程度上是未开发的领域，例子相对较少，大多没有共同注册。22, 23在他们的出版物中，Rakhymzhan 等人。提出了一种几乎同时配准的大体积多模态 IVM 方法，该方法将 TPM 与光学相干断层扫描 (OCT) 相结合，该技术已经在临床中得到了广泛应用，作为示例。24他们采用他们的成像方法来可视化和关联小鼠多种器官（即淋巴结、脾脏、视网膜和爪子）的形态学和特定细胞表型。使用这项技术，作者发现组织驻留巨噬细胞 (CX ₃ CR1 ⁺细胞），如 TPM 所揭示的，与组织组织相关，如由 OCT 和二次谐波生成信号捕获的。他们的联合 OCT 和 TPM 方法有可能同时提供分子特异性高分辨率成像 (TPM) 和组织形态学的无标记可视化 (OCT)，从而在基础研究知识和临床观察之间架起一座桥梁。

Li 等人的原始文章强调了 IVM 与临床应用的相关性，该文章侧重于治疗方法的开发和监测。细胞异质性可能会改变药物反应，就像实体瘤中的单克隆抗体疗法一样。为了解决这个问题，作者开发了一种共聚焦 IVM 方法，允许在实体瘤环境中可视化和跟踪曲妥珠单抗的递送和作用：一种广泛用于临床的单克隆抗体（针对生长因子受体 HER2）用于治疗乳腺癌和胃癌患者。为了模拟临床观察到的患者肿瘤细胞异质性，研究人员将嵌合异种移植物植入小鼠体内，该异种移植物由具有可变 HER2 表达的癌细胞混合物组成。这样，作者能够证明，虽然曲妥珠单抗在表达高水平 HER2 的肿瘤细胞中积累的程度更大（与 HER2 低的肿瘤细胞相比），但随着时间的推移，递送给动物的大部分药物实际上在肿瘤内积累。相关吞噬细胞。这里提出的发现提供了强有力的证据，证明环境条件如何导致药物治疗功效的变化，证明了 IVM 在转化研究中的潜力。

总之，本期特刊中的文章集展示了 IVM 在生物医学科学和临床中的广泛影响。诸如此处介绍的进展将提供工具和基础科学知识，以影响下一代临床护理。