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对人类诊断应用具有变革潜力的方式， 因为它可以提供微米级分辨率的形态和功能组织信息，而 不需要活检和外源性造影剂。然而，这些成像系统依赖于新的 以高强度超短脉冲的形式将光传输到组织的激光，其与组织的相互作用 除了眼睛和皮肤外，都没有很好的特征。缺乏已建立的数据库来定义最大值 允许暴露(MPE)阈值和一套应用于演示的方法和方案 在一组特定的照射条件下，患者的安全性是临床翻译这些条件的关键障碍 成像方法。我们的目标是建立非角化性组织的近红外、飞秒脉冲照射的MPE阈值 人口腔和宫颈鳞状上皮。我们最初将依靠基因工程的人类口腔和阴道- 宫颈外上皮组织，可高度复制和商业使用，但模仿重要的 人体组织的形态和功能特征。使用这些组织，我们将检查 照明波长、脉冲持续时间、重复频率、平均功率、光子密度和总光剂量 通过组织学和增殖、DNA损伤和细胞凋亡检测来评估组织形态。 免疫荧光。我们还将使用在以下过程中获取的无标记图像来量化光学代谢活动 具有感兴趣的参数的组织照明。我们将确定照明参数的子集，以产生 不，轻微或中度损伤，我们将在人类宫颈组织(新鲜切除的)上测试这些情况 和冷冻/解冻)，人角质形成细胞单层，以及仓鼠口腔上皮(活体内，新鲜切除和 冻结/解冻)。因此，我们希望为可能产生损害的标本类型确定一项协议 与活体人类鳞状上皮组织最相关的评估以及一系列 预计不会导致任何功能损害的光传输参数。我们还预计， 基于组织形态和代谢功能的无标记多光子成像表征将提供服务 作为一个独立的高度敏感的组织损伤指标。我们的发现将与外管局直接相关 活体人体宫颈多光子成像的实现方法和条件 我们确定将直接扩展到其他组织类型，并使这一强大的模式能够转换 到活体人体成像。

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.