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

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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.