2.1.

Animal Model

All experiments were conducted in accordance with guidelines specified by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University. A transgenic mouse strain, in which the hsp70 promoter drives the expression of luciferase and GFP reporter genes was provided by the Contag Laboratory (Stanford University). A detailed description of this mouse strain can be found in Ref. 32. In brief, these mice have an FVB background and contain an hsp70 cassette that is located upstream from GFP and luciferase (luc) vectors. In this system, whenever hsp70 mRNA transcription occurs, these bicistronic reporters are also transcribed and translated. As a result, the GFP and luciferase gene products emit light that can be used as a surrogate marker for hsp70 gene activity levels.³¹

Real-time polymerase chain reaction and bioluminescent imaging (BLI) were used to select an optimum breeding pair from an original cohort of eight hsp70A1-L2G founder mice. Male and female founders were paired and their offspring were screened using bioluminescent imaging and genotyping methods. Only female mice between 8 and $10\text{weeks}$ old were used to minimize age and gender variability. $48\mathrm{h}$ prior to laser experiments, mice were anesthetized with isoflurane in a vaporizer (Ohmeda, BOC Health Care, United Kingdom). After the mice were sedated, a rectangular area of the dorsal fur was removed using clippers, and remaining hair was removed using depilatory cream. After thoroughly cleaning off any remaining cream using saline moistened gauze, the mice were then returned to animal care for $2\text{days}$ .

2.2.

Free Electron Laser Operating Parameters

The FEL was used to make four $6.5\times 6.5\text{-}\mathrm{mm}$ square lesions on the dorsum of each mouse. Wounds were separated by at least $4\mathrm{mm}$ . The laser was operated at $30\mathrm{Hz}$ at wavelengths of 2.94, 6.1, or $6.45\mu \mathrm{m}$ . Energy was delivered in $5\text{-}\mu \mathrm{s}$ macropulses with pulse energies of 2, 4, 6, 8, 10, 12.5, 15, 20, 22.5, and $25\pm 0.5\mathrm{mJ}$ . At each wavelength, the collimated beam was focused to a $200\text{-}\mu \mathrm{m}$ Gaussian beam radius using a $50\text{-}\mathrm{cm}$ focal length $\mathrm{Ca}{\mathrm{F}}_2$ lens, resulting in radiant exposures of 1.59, 3.17, 4.76, 6.35, 7.96, 9.95, 11.94, 15.91, 17.91, and $19.89\mathrm{J}∕{\mathrm{cm}}^2$ . In a previous study, the ablation threshold on mouse skin was found to be $2.5\mathrm{J}∕{\mathrm{cm}}^2$ at $6.45\mu \mathrm{m}$ , $1.0\mathrm{J}∕{\mathrm{cm}}^2$ at $6.10\mu \mathrm{m}$ , and $0.2\mathrm{J}∕{\mathrm{cm}}^2$ at $2.94\mu \mathrm{m}$ .²⁶ In terms of threshold multiples, $19.89\mathrm{J}∕{\mathrm{cm}}^2$ equates to roughly 100 times threshold at $2.94\mu \mathrm{m}$ , 20 times threshold at $6.10\mu \mathrm{m}$ , and 8 times threshold at $6.45\mu \mathrm{m}$ .

A standard knife-edge method was conducted to measure the spot size of the Gaussian $\left({\mathrm{TEM}}_{00}\right)$ laser beam at the surface of the mouse dorsum. This method was necessary to correct for spot size variations due to chromatic aberrations by ensuring that a consistent beam radius of $200\mu \mathrm{m}\pm 10\mu \mathrm{m}$ was achieved for each experiment. For each wavelength tested, ablation was performed at or immediately past the beam waist (i.e., using a constant spot size with the beam “diverging” inside the tissue).

Mice were positioned on a three-axis translation stage equipped with a black circular mouse holding plate [Fig. 1a ]. Pulse energies were measured using an EnergyMax 400A energy meter with an LP-50 detector head (Coherent/Molectron, Portland, Oregon). At least four lesions were made $(n⩾4)$ for each radiant exposure at each wavelength.

Fig. 1

Schematic representation of experimental methodology. (a) Free electron laser (FEL) optical positioning setup and parameters ( $\lambda$ : 2.94, 6.10, $6.45\mu \mathrm{m}$ ; H: 1.59, 3.17, 4.76, 6.35, 7.96, 9.95, 11.94, 15.92, 17.91, $19.89\mathrm{J}∕{\mathrm{cm}}^2$ ; ${\omega }_r$ : $200\mu \mathrm{m}\pm 10\mu \mathrm{m}$ ; ${\tau }_p$ : $5\mu \mathrm{s}$ , repetition rate $30\mathrm{Hz}$ . A $10\text{week}$ old hsp70A1-L2G transgenic mouse was placed and treated on a $20\mathrm{cm}$ diameter holding plate. (b) Computer-assisted-surgical-technique (CAST) guided FEL wounding.

2.3.

Bioluminescent Imaging

After the FEL lesions were made, the mice were imaged using a Xenogen IVIS 200 imaging system (Caliper/Xenogen, Alameda, California). $15\min$ before each imaging session, the mice were injected i.p. with a $15\text{-}\mathrm{mg}∕\mathrm{ml}$ solution of luciferin substrate to yield a dose of $150\mathrm{mg}∕\mathrm{kg}$ . Mice were imaged at 0, 2, 5, 7, 9, 12, 15, 17, 21, 24, 36, and $48\mathrm{h}$ after the start of the FEL procedure. During each imaging session, mice were placed in the imaging chamber on a heated $37°\mathrm{C}$ stage. Both photographic and bioluminescent images were acquired, and each bioluminescent image was superimposed over its corresponding photographic image. Bioluminescent images were integrated over $30\mathrm{s}$ and the data were represented with a false color scheme representing the regions of varying light emission. Regions of interest (ROIs) encompassed each $6.5\times 6.5\text{-}\mathrm{mm}$ square laser lesion [Fig. 1b]. Light emissions from the specified ROIs were then quantified in units of total number of p/s using LivingImage analysis software (v2.12, Xenogen). The light emission from each ROI was then normalized to the light emission recorded from an unwounded section of tissue. This fold-induction value indicated the magnitude of hsp70 expression generated by the laser treatment.

2.4.

Histology

A subset of mouse skin samples was harvested for histological analysis. Tissues were examined for cell morphology, degree of damage, and early wound repair response at 12, 48, and $120\text{-}\mathrm{h}$ postsurgery. An Olympus Vanox-T AHZ microscope equipped with a Pixera Pro 600 ES camera was used to capture the images. For standard histology, a section of the laser-treated tissue was fixed in formalin, embedded in paraffin, sectioned and stained using hemotoxylin and eosin (HE) and Gomori’s trichrome (green) stains.³¹ The depth of damage was evaluated using a change in tincture in the Gomori’s trichrome stains and was measured using Image-Pro Plus software at a standardized magnification. The magnitude of epidermal hyperplasia was also measured from tissues evaluated at $120\text{-}\mathrm{h}$ postsurgery. A portion of each wound was prepared for frozen sectioning in Tissue-Tek® O.C.T.™ compound (Sakura Finetek, Torrance, California), flash frozen in liquid nitrogen, and stored at $-70°\mathrm{C}$ . These sections were used for fluorescence imaging of GFP.

2.5.

Confocal Fluorescence Imaging of hsp70 Green Fluorescent Protein

Fluorescence imaging of the second bicistronic reporter gene, GFP, was conducted to obtain depth resolved hsp70 expression. Frozen tissue samples were sectioned with a microtome ( $10\mu \mathrm{m}$ thickness) and imaged using the $5\times$ , $0.15\text{-}\mathrm{NA}$ Plan-Neofluar objective lens of a Zeiss LSM510 inverted confocal microscope. Samples were excited using a $488\text{-}\mathrm{nm}$ argon laser, and the hsp70-associated GFP fluorescence was detected using a $505\text{-}\text{to}550\text{-}\mathrm{nm}$ bandpass filter. Metamorph (Molecular Devices, Sunnyvale, California) was used to quantify signals from the skin’s surface to $250\mu \mathrm{m}$ below the surface. The GFP signal was measured using rectangular regions of interest $(13\times 100\mu \mathrm{m})$ . Values represent the mean fluorescence intensity values at three locations for ten different samples. On serial sections of tissue (HE stained), cell counts were measured using Image-Pro Plus (MediaCybernetics, Bethesda, Massachusetts). Cell density was determined using regions of interest with an area $600\mu {\mathrm{m}}^2$ . The fluorescent integrated intensity was then normalized by dividing by the cell density.

3.1.

Visualization and Quantification of hsp70 Promoter Activity in Laser-Treated Mouse Dorsum

A sample of the BLI time-course study of skin treated with the FEL using a wavelength of $6.10\mu \mathrm{m}$ and radiant exposures of 7.96, 11.94, 15.92, and $19.89\mathrm{J}∕{\mathrm{cm}}^2$ is shown in Fig. 2 . Prior to surgery, the untreated dorsal skin showed relatively low background bioluminescent intensities, with only minor traces of signal on the skin over the spine [Fig. 2a]. Five hours after surgery, significant increases in BL intensities were observed for all laser treatments [Fig. 2b]. The 15.92- and $19.89\text{-}\mathrm{J}∕{\mathrm{cm}}^2$ exposure sites showed significantly higher expression than sites exposed to 7.96 and $11.94\mathrm{J}∕{\mathrm{cm}}^2$ . For all lesions, the bioluminescent intensities progressively increased after surgery to a maximum intensity at $\sim 12\text{-}\mathrm{h}$ postsurgery [Figs. 2c and 2d].

Fig. 2

Visualization of hsp70 promoter activity on laser-treated mouse skin. Bioluminescent imaging time course of a FEL wounded hsp70-luc mouse dermis. (a) $\text{Time}=0\mathrm{h}$ . (b) $\text{Time}=5\text{-}\mathrm{h}$ postsurgery. (c) $\text{Time}=7\text{-}\mathrm{h}$ postsurgery. (d) $\text{Time}=12\text{-}\mathrm{h}$ postsurgery. The FEL parameters used to make the wounds were the following: $\lambda =6.10\mu \mathrm{m}$ , ${\omega }_r=200\mu \mathrm{m}$ , ${\tau }_p=5\mu \mathrm{s}$ , $30\mathrm{Hz}$ ; H: 7.96, 11.94, 15.92, and $19.89\mathrm{J}∕{\mathrm{cm}}^2$ . Laser-treated tissue has $\sim 18\times$ higher bioluminescence intensities (scale $\text{bar}=6\mathrm{mm}$ ).

Figures 3a, 3b, 3c show quantitative BLI plotted versus time for each wavelength and radiant exposure tested. hsp70 expression exhibited dose dependency and generally increased with increasing laser radiant exposure, as long as damage was not excessive. For all samples, an initial peak of hsp70 expression was seen $9\text{-}\text{to}12\text{-}\mathrm{h}$ postsurgery. At $6.45\mu \mathrm{m}$ , a biphasic peak of hsp70 expression was observed with maxima occurring both 9 and $24\mathrm{h}$ after laser treatment [Fig. 3a]. Increases in radiant exposures from $7.96\text{to}19.89\mathrm{J}∕{\mathrm{cm}}^2$ showed 12 to 28 fold-increases in initial hsp70 expression. At $6.10\mu \mathrm{m}$ , a maximum of an 18-fold increase in hsp70 was obtained with a radiant exposure of $17.91\mathrm{J}∕{\mathrm{cm}}^2$ [Fig. 3b]. Compared to the other wavelengths, the $2.94\text{-}\mu \mathrm{m}$ treatment induced markedly more macroscopic damage to the tissue that manifested itself predominantly as bleeding (data not shown). Therefore, treatments using radiant exposures less than $7.96\mathrm{J}∕{\mathrm{cm}}^2$ were also tested [Fig. 3c]. The magnitude of hsp70 expression increased as the radiant exposure was increased from $1.59\text{to}3.17\mathrm{J}∕{\mathrm{cm}}^2$ ; however, higher radiant exposures resulted in a decrease in hsp70 expression.

Fig. 3

Quantification of hsp70 expression using bioluminescent imaging (BLI). Hsp70 fold-induction (BLI relative to control) plotted versus time. Bioluminescent images were integrated over $30\mathrm{s}$ . Values are reported as $\text{mean}\pm \mathrm{SEM}$ . FEL at (a) $\lambda =6.45\mu \mathrm{m}$ , (b) $\lambda =6.10\mu \mathrm{m}$ , and (c) $\lambda =2.94\mu \mathrm{m}$ .

3.2.

Depth-Resolved hsp70 Expression in Laser-Treated Tissues

To examine the spatial distribution of hsp70 expression, GFP signal was evaluated using fluorescence microscopy. Such analyses allowed us to develop a 3-D representation of hsp70 expression after FEL ablation. Sample confocal fluorescence images (cross sections) of each laser-treated tissue are shown in Figs. 4a, 4b, 4c . The tissue treated with $6.45\mu \mathrm{m}$ had modest signal throughout, and the highest signals appeared in the reticular dermis and cutaneous muscle (panniculus carnosus). The epidermis and papillary dermis were lethally damaged to a depth of approximately $100\mu \mathrm{m}$ , as evidenced by the absence of GFP expression. This dark area where GFP expression is minimal correlates to the region of observed tissue damage. Compared to the $6.45\text{-}\mu \mathrm{m}$ treated samples, significantly lower GFP expression was observed in the tissues treated with $6.10\mu \mathrm{m}$ [Fig. 4b]. In the $2.94\text{-}\mu \mathrm{m}$ irradiated samples, considerable expression was observed in the epidermis, superficial dermis, and subdermal muscle [Fig. 4c].

Fig. 4

Morphological methods to measure depth of damage and fluorescent imaging to measure hsp70 expression. Histology and confocal fluorescence imaging of sections of laser-irradiated dermal tissue. For a consistent comparison, exposures that induced comparable (12-fold) increases in hsp70 expression are shown. Radiant exposures of 11.94, 15.92, and $1.59\mathrm{J}∕{\mathrm{cm}}^2$ were used for the wavelengths of 6.45, 6.10, and $2.94\mu \mathrm{m}$ , respectively. Tissue was harvested $12\text{-}\mathrm{h}$ postinjury. (a), (b), and (c) Fluorescence imaging of hsp70 induced GFP expression (scale $\text{bar}=100\mu \mathrm{m}$ , $\text{epidermis}=\mathrm{epi}$ , $\text{dermis}=\mathrm{d}$ , $\mathrm{a}=\text{adipose}$ tissue, and $\mathrm{pc}=\text{panniculus}$ carnosus). (d), (e), and (f) HE stained sections (scale $\text{bar}=75\mu \mathrm{m}$ ). (g), (h), and (i) Gomori’s (green) trichrome stain is used to visualize the depth of damage of laser-treated tissue (scale $\text{bar}=75\mu \mathrm{m}$ ).

To compare the laser treatments, the magnitude of GFP reporter expression was quantified with respect to depth [Fig. 5 ]. In Fig. 5a, the integrated fluorescent intensities are plotted versus tissue depth. At $2.94\mu \mathrm{m}$ , peak GFP emission was biphasic, with maxima at 40 and $150\mu \mathrm{m}$ below the skin surface (marked with an ${}^{*}$ ). In contrast, at $6.45\text{-}\mu \mathrm{m}$ , GFP expression was lowest closest to the skin surface and increased with depth to a maximum at $150\mu \mathrm{m}$ . Compared to the other wavelengths, the $6.10\text{-}\mu \mathrm{m}$ wavelength had significantly less signal at all depths. In Fig. 5b, the cell density is plotted versus depth. At $6.10\mu \mathrm{m}$ , the highest density of cells appeared approximately $40\mu \mathrm{m}$ below the surface. In contrast, the 2.94- and $6.45\text{-}\mu \mathrm{m}$ treatments showed the highest cell densities approximately $100\text{to}150\mu \mathrm{m}$ below the surface. The data suggest that $6.10\mu \mathrm{m}$ promoted less cell death or more cellular migration to the skin surface than the other two wavelengths. Quantitative fluorescence intensities were then normalized to cell density, and these normalized fluorescence values are plotted versus depth in Fig. 5c. The wavelengths of 2.94 and $6.45\mu \mathrm{m}$ showed biphasic peaks occurring at 50 and $250\mu \mathrm{m}$ below the surface, while the $6.10\text{-}\mu \mathrm{m}$ treatment had peak expression from $100\text{to}150\mu \mathrm{m}$ below the skin surface.

Fig. 5

Quantification of fluorescence signal. (a) Fluorescent integrated intensity versus depth of tissue $\left(\mu \mathrm{m}\right)$ . Fluorescent intensity was integrated within rectangular regions of interest $(13\times 100\mu \mathrm{m})$ going from the skin surface $(z=0\mu \mathrm{m})$ to the muscle $(z=250\mu \mathrm{m})$ . Mean fluorescence intensity is plotted and the SD $<5\%$ (SD not plotted for clarity). (b) Average cell number per area versus depth of tissue. (c) The fluorescent intensity values plotted in (a) were normalized to the cell numbers in (b) and plotted as function of tissue depth.

3.3.

Histological Analysis of Laser-Induced Damage

Tissues were compared histologically at laser conditions that showed a 12-fold induction of hsp70 expression $9\text{to}12\mathrm{h}$ after laser surgery, based on the results from [Figs. 3a, 3b, 3c]. Radiant exposures of 11.94, 15.92, and $1.59\mathrm{J}∕{\mathrm{cm}}^2$ were compared for wavelengths of 6.45, 6.10, and $2.94\mu \mathrm{m}$ , respectively. Cellularity and damage (mechanical and thermal) were visualized using an HE stain [Figs. 4d, 4e, 4f] and a Gomori’s trichrome stain [Figs. 4g, 4h, 4i]. Figures 4d, 4e, 4f indicate marked differences between the histological characteristics of samples exposed to the three wavelengths. The $6.45\text{-}\mu \mathrm{m}$ treatment showed the thickest superficial layer of coagulation (see asterisk) [Fig. 4d]. Figures 4g, 4h, 4i reveal marked differences in tissue damage patterns. The intense green staining of collagen with trichrome is indicative of normal nondisrupted collagen, and the transition to red is indicative of collagen denaturation. The tissues exposed to $6.45\mu \mathrm{m}$ showed the deepest zones of collagen denaturation, and the $6.10\text{-}\mu \mathrm{m}$ wavelength showed the shallowest depth of collagen damage.

For all of the wavelengths tested, the depth of damage was dose-dependent, with higher radiant exposures showing deeper damage. The measured depth of damage was plotted versus FEL radiant exposure $(\mathrm{J}∕{\mathrm{cm}}^2)$ [Fig. 6a ]. Interestingly, at a given radiant exposure, the depth of damage with $2.94\mu \mathrm{m}$ was $\sim 3\times$ that at $6.10\mu \mathrm{m}$ , and $\sim 2\times$ that at $6.45\mu \mathrm{m}$ .

Fig. 6

Collateral thermal damage. (a) The max depth of damage at $12\mathrm{h}$ plotted versus radiant exposure $(\mathrm{J}∕{\mathrm{cm}}^2)$ . Higher FEL radiant exposures cause deeper tissue damage. At a given radiant exposure the $2.94\text{-}\mu \mathrm{m}$ treatment damages $\sim 2\times$ deeper than $6.45\mu \mathrm{m}$ and $\sim 3\times$ deeper than $6.10\mu \mathrm{m}$ . Depth of damage was measured from tissue sections using histological stains (Gomori’s trichrome). The max depth of damage is expressed as $\text{mean}\pm \mathrm{SD}$ , $\mathrm{n}=90$ . (b) Peak hsp70 fold-induction is plotted versus radiant exposure $(\mathrm{J}∕{\mathrm{cm}}^2)$ for 2.94, 6.10, and $6.45\mu \mathrm{m}$ . (c) Peak hsp70 fold-induction plotted versus depth of tissue damage $\left(\mu \mathrm{m}\right)$ . The data for 6.45 and $6.10\mu \mathrm{m}$ lie on the same line (black dotted line). The $2.94\text{-}\mu \mathrm{m}$ data are shifted (i.e., more hsp70 expression for a given depth of damage) for radiant exposures up to $4.76\mathrm{J}∕{\mathrm{cm}}^2$ (gray dotted line), and then shows a decrease in hsp70 expression for higher radiant exposures (and deeper depths) that is not seen in either 6.10- or $6.45\text{-}\mu \mathrm{m}$ irradiated tissues.

3.4.

Effects of Laser Wavelength and Radiant Exposure on the Magnitude of hsp70 Expression

The peak magnitudes of hsp70 expression were measured and plotted versus laser radiant exposure $(\mathrm{J}∕{\mathrm{cm}}^2)$ [Fig. 6b]. For all wavelengths tested, increased radiant exposures led to increased hsp70 expression. However, at $2.94\mu \mathrm{m}$ , a maximum hsp70 expression of 23-fold occurred at $4.76\mathrm{J}∕{\mathrm{cm}}^2$ , while exposures $⩾4.76\mathrm{J}∕{\mathrm{cm}}^2$ showed decreased hsp70 expression. This suggested that significant cell damage was induced in the tissue above this threshold. At $6.45\mu \mathrm{m}$ , a maximum of 28-fold occurred with the $19.89\text{-}\mathrm{J}∕{\mathrm{cm}}^2$ exposure, and with $6.10\mu \mathrm{m}$ an 18-fold induction was observed with $17.91\mathrm{J}∕{\mathrm{cm}}^2$ . Of the wavelengths tested, $6.10\mu \mathrm{m}$ generated the lowest levels of hsp70 expression.

3.5.

Correlation Between hsp70 Expression and Collateral Thermal Damage

In Fig. 6c, the hsp70 induction levels are plotted versus the depth of tissue damage. The data showed that 6.10 and $6.45\mu \mathrm{m}$ exhibited similar responses (see black lines). The $2.94\text{-}\mu \mathrm{m}$ plot revealed a markedly different trend (see dotted gray line) [Fig. 6c]. Compared to the $6\text{-}\mu \mathrm{m}$ wavelengths, the $2.94\text{-}\mu \mathrm{m}$ curve is shifted to the left for lower radiant exposures. Furthermore, at low radiant exposures, the depth of damage increased with increases in hsp70 expression, but for higher radiant exposures, the depth of damage increased while hsp70 levels decreased.

3.6.

Epidermal Hyperplasia in Laser-Treated Tissues

Pronounced thickening of the epidermis, or epidermal hyperplasia, can be used as an indicator of a more intense or persistent repair response, presumably caused by a more damaging insult. At $120\text{-}\mathrm{h}$ postsurgery, tissue treated with a wavelength of $6.10\mu \mathrm{m}$ showed the least amount of epidermal thickening. For all wavelengths tested, the degree of epidermal hyperplasia was generally dose-dependent. Laser-treated tissues for each treatment wavelength using a radiant exposure of $11.94\mathrm{J}∕{\mathrm{cm}}^2$ are illustrated [Figs. 7a, 7b, 7c, 7d ]. The tissue treated with $6.10\mu \mathrm{m}$ showed minimal epidermal hyperplasia; however, at the same radiant exposure, both 6.45- and $2.94\text{-}\mu \mathrm{m}$ treatments showed $2\times$ more hyperplasia than the $6.10\text{-}\mu \mathrm{m}$ treatment [Figs. 7b, 7c, 7d]. In Fig. 7e, the magnitude of epidermal thickening is plotted versus the laser radiant exposure. At the lowest radiant exposure $(7.96\mathrm{J}∕{\mathrm{cm}}^2)$ , each treatment wavelength demonstrated similar degrees of epidermal hyperplasia ( $\sim 4–5\times$ thicker). Maximal epidermal thickening of 10-fold resulted from a wavelength of $2.94\mu \mathrm{m}$ and a radiant exposure of $19.89\mathrm{J}∕{\mathrm{cm}}^2$ . The magnitude of hyperplasia at $6.10\mu \mathrm{m}$ was statistically lower than the other wavelengths.

Fig. 7

Epidermal hyperplasia: an early wound repair response in laser-treated tissues. (a–d) Sample representation of Gomori trichrome stains of tissues evaluated $120\text{-}\mathrm{h}$ postsurgery (scale $\text{bar}=50\mu \mathrm{m}$ ). (a) Untreated tissue, (b) $\lambda =6.10\mu \mathrm{m}$ , (c) $\lambda =6.45\mu \mathrm{m}$ , and (d) $\lambda =2.94\mu \mathrm{m}$ . All tissues were treated with a radiant exposure of $11.94\mathrm{J}∕{\mathrm{cm}}^2$ . The $6.10\mu \mathrm{m}$ generated the least amount of epidermal thickening. (Note: arrow demarcates the zone of epidermal hyperplasia.) (e) The magnitude of epidermal thickening $120\mathrm{h}$ after FEL exposure is plotted versus the radiant exposure for each wavelength. (Note: untreated tissue has an average thickness of $20\mu \mathrm{m}$ ). The magnitude of epidermal thickening is expressed as the $\text{mean}\pm \mathrm{SEM}$ . Samples were compared using a student’s t-test ( ${}^{*}=\mathrm{P}<0.01$ , ${}^{**}=\mathrm{P}<0.005$ , ${}^{***}=\mathrm{P}<0.001$ , and $\mathrm{n}=25$ ).

4.1.

Laser Surgical Ablation

Laser sources in the IR $\left(2\text{to}10\mu \mathrm{m}\right)$ are well suited for ablation procedures, and studies have shown that wavelengths that target protein absorption (6.10 and $6.45\mu \mathrm{m}$ ) may be more effective than wavelengths that target nonspecific water absorption $\left(2.94\mu \mathrm{m}\right)$ . However, in most tissues including skin, it is still unclear which IR wavelength is superior. Many biophysical studies have been conducted to elucidate the mechanism governing the removal of tissue in ablation procedures.^{9, 10, 21, 34, 35} However, few studies have examined the repair response of the tissue that remains after ablation.

In this study, we have used a unique approach to examine the biological effects that are associated with IR laser ablation. In contrast to traditional studies, which typically have used only histological tools to assess collateral damage on excised tissues, we have used optical imaging techniques to quantify damage on the skin of an intact live organism.^{8, 21}

In addition to providing repair response information, the optical imaging tools used in this study also gave us the ability to quantify the cellular response to thermal stress in a sensitive and noninvasive manner. Traditionally, histological tools are used to examine the severe damage that is generated in the tissue’s extracellular matrix (ECM). Bearing in mind that the cell ultimately dictates the fate of the laser-treated tissue, we suggest that it may be more informative to focus on the thermal response of the cell rather than that of the ECM. In addition, intracellular protein damage, which drives the transcriptional activation of heat shock genes, only requires temperature increases of $\sim 5°\mathrm{C}$ ; while peak transition temperatures $⩾20°\mathrm{C}$ are required for ECM proteins to denature.^{28, 36} In contrast to conventional histology alone, the BLI of the hsp70-luc-GFP reporter gene in a transgenic mouse model, in combination with histology, gave us the additional ability to distinguish lethal from sublethal thermal damage.

4.2.

Advantages of Using Multimodal Optical Imaging Techniques

To ascertain a greater wealth of biological information in this study, we used both bioluminescence imaging (BLI) and fluorescence imaging (FLI) to visualize hsp70 expression. The primary advantage of BLI is that it can be used to detect and quantify very low levels of light from relatively deep tissue sources. In fact, studies have reported that as few as 200 cells can be detected at a depth of $\sim 2\mathrm{cm}$ in an in vivo mouse model.³⁷ Mouse dermis is roughly $300\mu \mathrm{m}$ thick, so for this study bioluminescence techniques are well suited to measure the emission of light from the entire FEL-treated tissue zone. However, since the BLI images in this study are 2-D and lack depth information, FLI was useful for examining the hsp70 levels with respect to precise lateral spatial location $(x,y)$ and depth $\left(z\right)$ . An additional advantage of FLI is unlike BLI, it does not require a live tissue and can be measured postmortem on fixed tissues. Overall, these properties make combined BLI and FLI ideal for visual representation, spatio-temporal characterization, and quantification of hsp70 driven reporter activity in FEL wounds.

4.3.

Collateral Tissue Damage

Tissue temperatures are reported to exceed $400°\mathrm{C}$ during laser ablation procedures.³⁸ These elevated temperatures cause collagen to denature, water to vaporize, and organic matter to decompose. Collagen denaturation, an irreversible kinetic process, is primarily governed by the temperature-time history and specimen hydration level.³⁹ Collagen typically denatures at $\sim 60°\mathrm{C}$ within minutes and at $\sim 68°\mathrm{C}$ within seconds.⁴⁰ Traditionally in ablation studies, the degree of collateral damage is reported using the measured depth of collagen denaturation. In this study, we have shown that for all wavelengths, increases in the radiant exposure caused increases in collateral damage. Of the wavelengths tested, the $6.10\text{-}\mu \mathrm{m}$ wavelength induced the least collateral damage, roughly half that of $6.45\mu \mathrm{m}$ and a third that of $2.94\mu \mathrm{m}$ . This finding is consistent with that observed by other authors in the cornea.^{8, 26} The finding that $2.94\mu \mathrm{m}$ caused the deepest damage yet has the smallest $\delta$ seems counterintuitive, but other studies have also reported similar results.^{17, 19, 20} Spectroscopic investigations have shown that the ${\mu }_a$ of water changes during laser treatment, thus the $\delta$ at $2.94\mu \mathrm{m}$ may actually be 5 to $10\times$ higher than predicted.^{17, 19, 20} Thus, the dynamic optical properties of tissue may contribute to the deeper damage induced at $2.94\mu \mathrm{m}$ . Also, since $2.94\mu \mathrm{m}$ targets the OH-stretch mode (water absorption peak), this wavelength may also cause greater tissue dehydration, increasing the rate of collagen denaturation and the degree of damage.³⁹ Lastly, given that the pulse duration equals $5\mu \mathrm{s}$ , conditions for thermal confinement are not met for the $2.94\text{-}\mu \mathrm{m}$ wavelength $({\tau }_{\mathit{th}}=1.67\times {10}^{-6}\mathrm{s})$ , but are met for the 6.10- and $6.45\text{-}\mu \mathrm{m}$ wavelengths ( $2.67\times {10}^{-5}\mathrm{s}$ and $2.4\times {10}^{-4}\mathrm{s}$ , respectively). Thermal superposition owing to multiple pulses may further complicate the temperature-time history dynamics.

4.4.

Using hsp70 Luciferase Green Fluorescent Protein Systems to Measure Sublethal Thermal Effects

Since the more subtle effects of cellular thermal damage are not distinguishable with histological methods, the noninvasive readout of hsp70 levels were used to assess the magnitude of sublethal thermal damage. In a previous study with a skin equivalent model, we showed that the hsp70 levels rose to a maximum with increasing laser exposures.^{30, 31, 41} Here we also found a similar biphasic behavior. Consistent with the histological data, the BLI data showed that $6.45\mu \mathrm{m}$ $(\delta \sim 12\mu \mathrm{m})$ induced $\sim 2\times$ higher hsp70 expression than $6.10\mu \mathrm{m}$ $(\delta \sim 4\mu \mathrm{m})$ . Besides the $\delta$ , the direct targeting of extracellular and/or intracellular proteins could also account for the higher hsp70 levels. Since $6.45\mu \mathrm{m}$ targets the amide-II band of proteins, both intra- and extracellular, this targeting may cause greater protein denaturation than $6.10\mu \mathrm{m}$ , which targets the amide-I band. Such targeting may generate more stress to the cells that remain after ablation.

Nevertheless, a Beer’s law approximation using the estimated $\delta$ in Table 1 ⁴² cannot account for the exceedingly high hsp70 levels observed at $2.94\mu \mathrm{m}$ using $\mathrm{H}⩽4.76\mathrm{J}∕{\mathrm{cm}}^2$ . Based on the lower δ, we anticipated that the hsp70 levels at $2.94\mu \mathrm{m}$ would be the lowest of the wavelengths tested. The data suggest that tissue damage and hsp70 expression are not directly proportional for all wavelengths. Both mid-IR wavelengths (6.45 and $6.10\mu \mathrm{m}$ ) had similar linear relationships between these two parameters; however, for $2.94\mu \mathrm{m}$ , hsp70 expression and depth of damage showed a markedly different relationship. The curve is shifted to the left for low radiant exposures $(<4.76\mathrm{J}∕{\mathrm{cm}}^2)$ , indicating that greater cellular injury (hsp70 magnitude) is triggered for a given depth of tissue damage. For instance, to generate $90\mu \mathrm{m}$ of tissue damage, $5\mathrm{J}∕{\mathrm{cm}}^2$ is needed at $2.94\mu \mathrm{m}$ , while $8\mathrm{J}∕{\mathrm{cm}}^2$ is required at $6.45\mu \mathrm{m}$ [Fig. 6a]. Thus, for the same depth of measured histological damage, $2.94\mu \mathrm{m}$ had $\sim 2\times$ higher hsp70 expression than $6.45\mu \mathrm{m}$ [Fig. 6c]. In addition, at higher radiant exposures, the $2.94\text{-}\mu \mathrm{m}$ curve shows a decline in hsp70 expression, while the depth of damage increases. This change is presumably due to severe thermal effects that impair hsp70 transcription or translation. Taken together, these findings suggest that $2.94\mu \mathrm{m}$ causes more severe thermal insult to cells than 6.10 and $6.45\mu \mathrm{m}$ . Overall, it appears that hsp70 expression is influenced by both the $\delta$ and the specific constituent (biomolecule) that a laser wavelength targets.

4.5.

Defining Lethal Expression Thresholds with hsp70 Expression

Due to the more severe and deeper damage, radiant exposures $⩾4.76\mathrm{J}∕{\mathrm{cm}}^2$ at $2.94\mu \mathrm{m}$ resulted in decreases in hsp70 expression. Unlike the other mid-IR wavelengths, $2.94\mu \mathrm{m}$ generated visual damage that included bleeding and coagulation, during and immediately after laser exposure. Therefore, the decreasing hsp70 levels may be due to lethal cell damage or generation of more chromophores (e.g., blood or eschar) that absorb the emitted underlying BLI signal $({\lambda }_{\text{emission}}=590\pm 70\mathrm{nm})$ . At a given radiant exposure, the $2.94\text{-}\mu \mathrm{m}$ wavelength is expected to generate higher tissue temperatures than the other wavelengths, owing to the higher ${\mu }_a$ since $\Delta T$ is proportional to ${\mu }_a$ .⁴³ These higher temperatures drive heat conduction and may cause greater photothermal damage. Such damage may impair the cell and perhaps the cellular machinery that makes the bioluminescence reaction possible. Since the bioluminescence reaction requires ATP as a cofactor, damage to the mitochondria (which occurs at $T⩾50°\mathrm{C}$ ) may reduce the efficiency of the reaction and thereby reduce the light emission.^{29, 44}

To classify these findings, we defined a lethal hsp70 expression threshold value, hereafter referred to as $hsp{\mathit{70}}_{L\text{-}\mathrm{Th}}$ . We define the $hsp{\mathit{70}}_{L\text{-}\mathrm{Th}}$ as the radiant exposure required to cause a decrease in hsp70 levels. For the radiant exposures listed, the $2.94\text{-}\mu \mathrm{m}$ wavelength had an $hsp{\mathit{70}}_{L\text{-}\mathrm{Th}}$ at $(\mathrm{H}⩾4.76\mathrm{J}∕{\mathrm{cm}}^2)$ , while the protein targeting wavelengths did not have observable thresholds, at least not for the maximum radiant exposures achievable with the FEL. Overall, the BLI approach is not applicable at extremely destructive laser exposures, and in those cases histology provides a proven method of assessing tissue damage.

4.6.

Wound Repair Response

The finding that the temporal expression kinetics of hsp70 are affected by the laser wavelength suggests that the tissue’s repair response is wavelength-dependent. Tissues treated with $6.45\mu \mathrm{m}$ exhibited bimodal peaks occurring $\sim 9$ to 12 and $\sim 24\mathrm{h}$ after surgery, while the tissues treated with 6.10 and $2.94\mu \mathrm{m}$ had only one maximum occurring $9\text{to}12\mathrm{h}$ after surgery. This could be due to the greater $\delta$ or to the relatively higher absorption by proteins at $6.45\mu \mathrm{m}$ . Later wound repair also appears to be wavelength-dependent. Five days after laser surgery, we evaluated the magnitude of epidermal hyperplasia, which is a transient characteristic of the resurfacing phase of skin repair. In the context of the present study, reduced hyperplasia was indicative of a less severe wound, and the $6.10\text{-}\mu \mathrm{m}$ wavelength was superior in this aspect.

4.7.

Broader Implications of Findings

Nearly $15\text{years}$ ago, the $6.45\text{-}\mu \mathrm{m}$ wavelength was shown to cause minimal collateral damage in excised tissue.²¹ More recent evidence has suggested that $6.1\mu \mathrm{m}$ is superior to $6.45\mu \mathrm{m}$ .^{8, 9, 26} In this study, we have also found that $6.10\mu \mathrm{m}$ was superior to 6.45 and $2.94\mu \mathrm{m}$ . Previous studies have reported that the difference between 6.45 and $6.10\mu \mathrm{m}$ was not clinically significant (7 versus $5\mu \mathrm{m}$ , respectively).²⁶ In contrast, in this work, where we conduct ablation on intact live animals, we have shown that the differences in tissue damage between 6.45 and $6.10\mu \mathrm{m}$ are of practical importance (90 versus $50\mu \mathrm{m}$ , respectively). Since dermal stratum are roughly $20\mu \mathrm{m}$ thick, using $6.10\mu \mathrm{m}$ may allow for more controlled ablation. In addition, this work has also shown that $2.94\mu \mathrm{m}$ was markedly inferior to both $6\text{-}\mu \mathrm{m}$ wavelengths.

Considering the limited clinical utility of the FEL, the implications of these findings are significant. The FEL requires high costs to operate and has practical limitations in terms of beam delivery. The development of low-cost benchtop laser sources, based on a variety of solid-state platforms, may increase the clinical use of lasers for surgical ablation. Since surgical performance is primarily governed by the selection of the laser’s pulse structure, wavelength, and radiant exposure,^{8, 9, 10} studies like the work presented here help assist in the development of these alternative sources. Recent reports have shown that the micropulse structure is not a critical parameter that needs to be duplicated in benchtop laser sources.⁹ The data in this report suggest that efforts in developing laser sources using $6\text{-}\mu \mathrm{m}$ radiation, such as using the zinc germanium phosphide optical parametric oscillator or systems based on stimulated Raman scattering, are useful cost-effective alternatives to the FEL, and should be further pursued.^{26, 45}

In a broader sense, this study also demonstrates that using multimodal optical imaging techniques in a transgenic mouse model (hsp70-L2G) can aid in the assessment of the biological response in surgical ablation procedures. The bioluminescence data demonstrates that the magnitude of hsp70 expression was both dose and wavelength-dependent. While many previous studies have used optical imaging techniques to visualize the promoter activity of genes involved in cutaneous wound repair, this study reveals that the hsp70 mouse is a particularly useful tool for investigating laser-tissue interactions.^{46, 47, 48} This type of model may be effective in connecting our knowledge of the biophysics of the ablation process to the histological assessments of laser-treated tissues. Ultimately, this model can be used to improve laser surgical procedures, including a variety of dermatological aesthetic laser procedures, such as skin resurfacing.