Multiphoton excitation is now commonly used to image three-dimensional biological materials (21
), and recently work has focused on using this technique to affect chemical changes (8
). However, little work has extended multiphoton excitation to create chemical changes in tissues with three-dimensional precision (14
). We have previously demonstrated that 2PE is capable of inducing photochemical DNA adduct formation with spatial specificity (9
). The focus of this study was to extend 2PE to target ROS formation to individual cells within an intact biomimetic tissue similar to human dermis.
DCF and its derivatives are commonly used to probe for the presence of ROS. When oxidized to DCF by ROS, the probe becomes a fluorophore (27
). Previous work by others suggests but has not shown that 2PE of DCF and subsequent induction of ROS is possible. First, Chignell and coworkers have demonstrated that DCF is not only a reporter of ROS but also undergoes complex photochemistry, acting as a photosensitizer of both H2
DCF oxidation and ROS formation on absorption of light (22
). Second, the parent molecule of DCF, fluorescein, has been shown to have significant 2PE cross-sections between 690–1050 nm (13
). That fluorescein undergoes 2PE at 800 nm but does not have significant one-photon absorption at 400 nm likely reflects the differing selection rules governing one-photon excitation (1PE) and 2PE. Third, the emission spectrum of fluorescein is the same after 1PE and 2PE (13
), indicating that fluorescein evolves to the same excited state and thus undergoes similar excited-state dynamics after either type of excitation. We exploited these properties and used CM-H2
DCFDA and its oxidative metabolites related to DCF to act as both mediators and reporters of 2PE-directed ROS formation in fibroblasts and fibroblast-derived cells.
In near-confluent cell monolayers incubated with CM-H2DCFDA, pulsed 800 nm irradiation of single fibroblasts resulted in a rise in intracellular fluorescence from DCF, indicating that ROS formation can be targeted to individual cells (). Such increased cellular fluorescence after 2PE was observed in several different cell lines, including both primary and transformed cells, indicating that the mechanism involved is not specific to a particular type of cell. There are two pieces of evidence that ROS formation in target cells results from a multiphoton excitation event and not a one-photon absorption process. First, irradiation of cells with the 800 nm output of the titanium–sapphire laser in continuous wave mode does not produce an increase in ROS, whereas irradiation with the pulsed output of the laser using the same average power but many orders of magnitude larger peak power yields a rapid induction of ROS formation (). Thus, the efficiency of ROS formation with multiphoton excitation is significantly greater than with one-photon absorption at 800 nm. Second, a quadratic relationship exists between the cellular fluorescence intensity and the excitation power (), the hallmark of 2PE. These results unequivocally demonstrate that the observed ROS are generated as a result of 2PE and not simply 1PE at 800 nm of a weakly absorbing photosensitizer.
Our results are consistent with a model in which endogenous ROS from basal cellular metabolism generates a small amount of DCF from CM-H2
DCFDA, which then functions as a photodynamic agent to generate additional ROS after 2PE (23
). ROS formation has been detected by adding H2
DCFDA immediately after broadband ultraviolet irradiation of normal human epidermal keratinocytes and observing fluorescence from DCF, suggesting the presence of endogenous photodynamic chromophores that absorb ultraviolet wavelengths (33
). In contrast, incubation of cells with CM-H2
DCFDA before pulsed 800 nm irradiation resulted in significant increases in fluorescence, whereas addition of CM-H2
DCFDA immediately after 800 nm irradiation did not. Considering that short-lived singlet oxygen does not appear to react with H2
), it seems reasonable to infer that the fluorescence observed in the presence of CM-H2
DCFDA is due to longer-lived ROS. The presence of an endogenous photosensitizer in dermal fibroblasts with a significant near-infrared 2PE cross-section is therefore unlikely, though our experiments cannot exclude that possibility.
There have been reports inferring ROS formation due to 2PE of cells both with and without exogenous photodynamic agents. Chinese hamster ovary cells treated with photofrin or amino-levulinic acid and irradiated with 780 nm pulsed laser showed decreased cloning efficiency (21
). Although the absorption of photofrin is seemingly negligible at 780 nm (34
), the possibility of one-photon absorption by photofrin or an endogenous chromophore giving rise to the observed effects was not considered by the authors. Pulsed 800 nm laser irradiation of rat kangaroo kidney epithelium cells, incubated with the probes of ROS formation, Ni-diaminobenzidine and Jenchrom px blue, produced ROS and led to apoptosis (19
), suggesting that endogenous photodynamic chromophores are capable of multiphoton excitation. However, it is unclear in this work if the probes or their light absorbing reaction products themselves could have acted as the predominant mediators of ROS induction through either a one- or two-photon absorption event. In our experiments, if endogenous chromophores were significantly contributing to long-lived ROS induction, then adding CM-H2
DCFDA immediately after irradiation should have yielded increased fluorescence above that in unirradiated cells, which did not occur.
The generation of ROS by 2PE in two-dimensional cell culture was extended to three-dimensional dermal equivalents. These reconstituted tissue simulants have been shown to have properties that are histologically and physically similar to actual dermis (35
). Individual cells within the dermal equivalent were targeted with 2PE and subsequently developed DCF fluorescence, indicative of ROS formation, whereas nearby cells within the cone of excitation light but not at the focus did not show detectable ROS formation over that in unirradiated cells (). This capability is possible because of the quadratic dependence of ROS formation on laser intensity demonstrated in . This result illustrates the spatial selectivity of ROS formation in a three-dimensional biological tissue that is possible with this technique. As stated earlier, the excitation volume within which 50% of the 2PE effect is expected to occur is 0.4 µm wide and 1.5 µm above and below the focal plane, which is sufficient to target fibroblasts dispersed in dermis and even keratinocytes in epidermis. Although modest when compared with the overall dimensions of human skin, which is typically several millimeters thick, the 25 µm depth at which ROS were targeted in cells makes targeting specific keratinocytes within a typical 50 µm murine epidermis achievable and approaches the ~ 100–200 µm thickness of human foreskin epidermis.
Targeting ROS formation to selected cells within a tissue would be useful for dissecting the damage response of highly ordered structures such as stratified epithelia where the response may vary with cellular position. In this work we demonstrate a general method for generating ROS deep inside a biomimetic tissue while sparing other cells, allowing one to ask questions about the heterogeneous organization of tissues, cell fate and communication among skin cells after genotoxic stress with a precision that is not readily available with current techniques. To our knowledge, this is the first rigorous proof of 2PE of a photodynamic agent with subsequent use of that agent to induce ROS in a biological tissue with three-dimensional specificity.