“Effects of ultrashort laser pulse (USLP) irradiation on melanocyte viability”

1. Santiago Camacho
2. Francisco Perez
3. Guillermo Aguilar

DESCRIPTION

During the time this grant was effective, we focused on the experimental development of artificial skin models which involve living cells. These models are commonly known as “RAFTs” because of their appearance (Fig. 1) and are normally composed of human keratinocytes and melanocytes in the epidermal layer and human fibroblasts in the dermal layer, which are all suspended within a rat-tail collagen matrix. RAFTs mimic in vivo human skin in terms of structure, cellular activity, and function. Therefore, RAFTs offer an ideal test media to evaluate the effects of laser-tissue interactions on cell viability as well as the subsequent wound healing process.

Francisco Pérez-Gutiérrez was the graduate student leading this project. To master the techniques required for culturing cells and fabricating RAFTs, Francisco and Felipe Godínez, an undergraduate student, underwent a long training process including several cell-lines culturing and model preparation at different locations, such as the Beckman Laser Institute (BLI) at UCI and Dr. Kathryn DeFea’s laboratory at the UCR/UCLA Biomedical Sciences program at UCR.

The purpose of preparing these skin models was to irradiate them with short and ultrashort laser pulses (USLP) and study the effect of the laser parameters on cell integrity, hence, the feasibility of including this state-of-the-art technology to current dermatologic therapies.
To achieve this, Francisco had to build from scratch a complex optical system which now allows us to irradiate tissue samples with pulsed nanosecond laser light ranging from 355 to 2500 nanometers in wavelength, while simultaneously visualizing with a CCD camera the ablation induced on the sample. The technical assistance of Drs. Santiago Camacho and Gerardo Gutierrez for the installation of this system was invaluable. Figure 2 shows a schematic and Figures 3a and 3b show pictures of the optical setup which is now available at our laboratory.

Several experiments were carried out testing different laser parameters and making scans of short and ultrashort laser pulses on top of various RAFTs, trying to identify damage at tissue level using standard live/dead cell florescent assays and imaging provided by a confocal microscope in Dr. Merixtell Riquelme-Pérez’s laboratory at CICESE, Ensenada BC, Mexico. The purpose was to evaluate the cell viability by comparing an irradiated RAFT with an unused control RAFT and generating viability plots such as those generated by Diaz et al. [1], described in Figure 4 for cartilage chondrocyte viability.

The original hypothesis was that a localized area of damaged cells would be found as a result of the scans with the pulsed laser. However, in our first attempts, we found total ablation of the collagen matrix that holds the cells to the RAFT. Subsequently, we tried to irradiate the RAFT with single laser pulses, but the region of damage was too small to track due to the small area in which the laser beam is focused (~ 6 micrometers in diameter).
Based on these observations, we concluded that a confocal microscope coupled to a nanosecond laser system would allow us to obtain a real-time image of the RAFT before, during and after the laser pulse, which would thus enable us to identify the laser effects at the cellular rather than whole tissue level.

With this purpose in mind, RAFTs were irradiated using a dye laser pumped by a nitrogen laser, 5 ns, 50 μJ, single laser pulse at a wavelength of 440 nm focused through a 0.9 NA microscope objective. Figures 5a-d clearly illustrate a live (green) cell—seen inside the yellow circle—dying as time progresses (tainting red) in the confocal microscopy images sequenced below.

This work has led us to realize that the nonlinear absorption of short and USLP focused through microscope objectives can be used to achieve very fine and highly localized laser effects inside biological media, which are otherwise transparent at low irradiances. Therefore, short and USLP constitute a very powerful tool for applications where laser damage at cell or subcellular level is required, without imprinting collateral damage. Such is the case of genomic or proteomic studies, laser microdissection, and subsequent laser catapulting. We will soon pursue related biomedical applications, such as the effects of laser irradiation on circulating melanoma cells, which has proven to be a viable detection method of melanoma and breast cancer.

References:
1. SH Diaz, JS Nelson, BJF Wong, Rate process analysis of thermal damage in cartilage. Physics in Medicine and Biology, 2003. 48(1): p. 19-29.

Other Participants

Felipe Godinez

Rodger Evans

FUNDING

1. UCMEXUS: “Studies of ultrashort laser pulse interaction with vascular and pigmented dermatologic lesions”

2. UCMEXUS: “Effects of ultrashort laser pulse (USLP) irradiation on melanocyte viability”

3. National Science Foundation (NSF): “Thermo-mechanical interactions of ultrashort laser pulses with subsurface targets of tissue models”

PUBLICATIONS

1. F. Pérez-Gutiérrez, R. Evans, S. Camacho-López, and G. Aguilar, “Short and ultrashort laser pulse-induced bubbles on transparent and scattering tissue models”, in Proceedings of SPIE, San Jose CA, Jan 20-25, 2007, V.6435, pp.64350V-1-8.