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.
