Improving multiphoton imaging with shaped ultrashort laser pulses Multiphoton imaging has become the standard for deep tissue imaging and has recently been approved for human clinical trials in Germany for the non-invasive diagnosis of skin cancer such as melanoma. There is a pressing need to determine the laser pulse duration dependence of different laser-induced damage mechanisms in order to find the pulse parameters that yield optimal signal with minimal laser damage. For example, pigmented tissue has significant absorption in the near-infrared, which in turn causes thermal damage;on the other extreme, instantaneous three-photon absorption leads to DNA damage in living tissue. For this study we propose using a laser system capable of delivering pulses as short as 10 fs (fifteen times shorter than typical two-photon microscopes) outfitted with a pulse shaper that allows us to change the pulse duration from 10 to 1000 fs by restricting the spectral bandwidth (amplitude shaping) or by stretching the pulse in the time domain (phase shaping). The goal is to evaluate laser induced damage first in tissue phantoms and then in skin tissue specimens and to plot its dependence on laser intensity and pulse duration. These measurements will allow us to define a window of 'optimal performance'for the different tissue samples, conditions for which signal is maximized and damage is minimized.
Specific aims of the proposed research are: 1. Evaluate the dependence of laser-induced damage in skin phantoms (melanin-rich samples, cell culture models) on pulse duration and laser power. Quantify the expected yield in signal and mitigation of photodamage due to the optimization of laser pulse duration (10-1000 fs) at the sample. Seek for alternative pulse shaping strategies, other than linear chirp and spectral narrowing, to be used for optimization of multiphoton microscopy imaging (e.g., pulse sequence generation). Compare best results versus those obtained by conventional two-photon microscopes using 100-150 fs pulses. 2. Validate the 'laser-safe operational window'model on various skin tissues and other biological samples. Provide guidelines as to what are the optimum pulse duration and energy conditions for nonlinear optical imaging in those tissues. The use of ultrafast laser sources (with broader-than-conventional spectral bandwidth) and pulse shaping to assess phototoxicity and optimize laser parameters for safe multiphoton imaging of pigment-rich tissues is novel. The long term goal of this project is to further facilitate data acquisition in the biomedical imaging field with minimal perturbation to the living system. This research will impact a number of nonlinear biomedical imaging modalities as well as future diagnostic techniques. In particular, it will benefit to the noninvasive optical biopsy of skin lesions such as melanoma, the studies of wound healing processes, and transdermal drug delivery.
The proposed work will improve biomedical imaging by achieving greater signal, improved contrast and less laser induced damage when imaging living cells. Richer biological information can thus be obtained non- invasively which will help researchers and doctors better understand the morphology and pathology of the cell and disease progression.
|Arkhipov, Sergey N; Saytashev, Ilyas; Dantus, Marcos (2016) Intravital Imaging Study on Photodamage Produced by Femtosecond Near-infrared Laser Pulses In Vivo. Photochem Photobiol 92:308-313|
|Saytashev, Ilyas; Arkhipov, Sergey N; Winkler, Nelson et al. (2012) Pulse duration and energy dependence of photodamage and lethality induced by femtosecond near infrared laser pulses in Drosophila melanogaster. J Photochem Photobiol B 115:42-50|