Safety assessments of tissue irradiation with near infrared fs pulses for multi-photon imaging applications

多光子成像正在成为一种具有人类诊断应用变革潜力的模式， 因为它可以提供微米级分辨率的形态和功能组织信息， 避免了对活组织检查和外源性造影剂的需要。然而，这些成像系统依赖于新颖的 以高强度超短脉冲的形式将光传递到组织的激光器，其与组织的相互作用 除了眼睛和皮肤之外，没有得到很好的表征。缺乏确定最大值的既定数据库， 允许暴露（MPE）阈值和一套方法和协议，应用来证明 对于一组特定的辐照条件，患者安全性对这些辐射的临床转化提出了关键障碍。 成像方法我们的目标是建立MPE阈值的NIR，fs脉冲照明的非角质化 人鳞状口腔和宫颈上皮。我们将首先依靠人工合成的人类口腔和阴道- 子宫颈外上皮组织是高度可重复的和商业上可获得的，但模拟重要的 形态和功能人体组织特征。利用这些组织，我们将研究 照明波长、脉冲持续时间、重复率、平均功率、光子密度和总光剂量 通过组织学评估组织形态学，并通过组织学检查增殖、DNA损伤和凋亡。 免疫荧光我们还将使用在实验过程中获得的无标记图像来量化光学代谢活动。 组织照明与感兴趣的参数。我们将确定照明参数的子集， 无、轻微或中度损伤，我们将在人宫颈组织（新鲜切除的）上测试这些条件 和冷冻/解冻）、人角质形成细胞单层和仓鼠口腔上皮（体内，新鲜切除和 冷冻/解冻）。因此，我们希望确定一个协议的类型的标本，有可能产生损害 与体内人鳞状上皮组织最相关的评估以及一系列 预期不会导致任何功能损坏的光传输参数。我们还预计， 基于无标记多光子成像的组织形态和代谢功能表征将 作为独立的高灵敏度组织损伤指示器。我们的发现将直接关系到 实施人体子宫颈在体内的多光子成像;然而，该方法和MPE条件 我们建立了一种简单的方法，可以扩展到其他组织类型，并使这种强大的模式能够转化， to in vivo活体human人imaging成像.

Multiphoton imaging is emerging as a modality with transformative potential for human diagnostic applications, because it can provide morphological and functional tissue information with micron scale resolution, while obviating the need for a biopsy and exogenous contrast agents. However, these imaging systems rely on novel lasers that deliver light to tissue in the form of highly intense, ultrashort pulses, whose interactions with tissues other than the eye and skin are not well-characterized. The lack of established databases that define maximal permissible exposure (MPE) thresholds and a set of methods and protocols that should be used to demonstrate patient safety for a specific set of irradiation conditions presents a critical barrier to the clinical translation of these imaging methods. Our goal is to establish MPE thresholds for NIR, fs pulsed illumination of non-keratinized human squamous oral and cervical epithelia. We will rely initially on engineered human oral and vaginal- ectocervical epithelial tissues that are highly reproducible and commercially available, yet mimic important morphological and functional human tissue characteristics. Using these tissues, we will examine the impact of illumination wavelength, pulse duration, repetition rate, average power, photon density, and total light dose on tissue morphology assessed via histology and proliferation, DNA damage, and apoptosis examined via immunofluorescence. We will also quantify optical metabolic activity using label-free images acquired during tissue illumination with the parameters of interest. We will identify a subset of illumination parameters that yield no, minimal, or moderate damage and we will test these conditions on human cervical tissues (freshly excised and frozen/thawed), human keratinocyte monolayers, and hamster oral epithelia (in vivo, freshly excised and frozen/thawed). Thus, we expect to identify a protocol for the types of specimens that are likely to yield damage assessments that are most relevant to in vivo human squamous epithelial tissues and the combination of a range of light delivery parameters that are not expected to result in any functional damage. We also anticipate that label-free multi-photon imaging-based characterization of tissue morphology and metabolic function will serve as an independent highly sensitive tissue damage indicator. Our findings will be directly relevant to the safe implementation of multi-photon imaging of the human cervix in vivo; however, the approach and MPE conditions we establish will be straightforward to extend to other tissue types and enable translation of this powerful modality to in vivo human imaging.