Rapid thermal equilibration of differentially heated protein and water in bovine corneal stroma.

We measure and simulate the thermal response of bovine corneal stroma to a picosecond IR heating pulse. A thermal diffusion model is developed for this tissue based on the spatial distribution and properties of protein and water constituents in the stroma. In this idealized model, differentially heated protein and water constituents thermally equilibrate with a thermalization time of 515 ps. Using transient absorption spectroscopy for picosecond protein thermometry, a significantly faster thermalization time of 165 ps is measured. The implications of this faster than expected thermalization for the energy-partition model of short-pulse mid-IR tissue ablation are discussed.

In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse.

Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

Visualizing in vivo liposomal drug delivery in real-time.

Liposomes have tremendous potential for efficient small molecule delivery. Previous studies, however, have been hampered by an inability to monitor their distribution and release of contents. Here, the authors demonstrate the real time monitoring of small molecule delivery using luciferin as a model. To monitor the release of luciferin in vivo, luciferin was packaged in thermosensitive liposomes and delivered into transgenic mice that constitutively express luciferase. Their experiments show the thermally induced release of the liposomal content in real time. In addition, the model provides evidence that the thermosensitive liposomes are stable over a long period of time ( approximately 3 weeks), and still release their content upon heating. These data present a strategy to monitor liposomal drug delivery in vivo with luciferin.

Accelerated bone repair after plasma laser corticotomies.

OBJECTIVE: To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal. SUMMARY BACKGROUND DATA: Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing. METHODS: Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling. RESULTS: Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery. CONCLUSION: In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

Picosecond optical thermometry of protein in H2O.

We demonstrate the use of transient IR absorption measurements for picosecond thermometry of protein in an aqueous environment. For small temperature changes, measured transient absorption changes are shown to be in excellent agreement with the "static" temperature dependence of the protein and water constituents of the sample as measured by FTIR spectroscopy. The thermally induced changes in IR absorption reach equilibrium within a few picoseconds, making this technique an excellent tool for picosecond thermometry.

Mid infrared optical parametric oscillator (OPO) as a viable alternative to tissue ablation with the free electron laser (FEL).

BACKGROUND AND OBJECTIVES: Investigations with a Mark-III free electron laser, tuned to 6.45 microm in wavelength have demonstrated minimal collateral damage and high ablation yield in ocular and neural tissues. While the use of mid-IR light produced by the free electron laser (FEL) has shown much promise for surgical applications, further advances are limited due the high costs of its use. Further investigation and widespread clinical use of six-micron radiation requires the development of an alternative laser source. In this research, we compared a Mark-III FEL and an Er:YAG pumped ZGP-OPO with respect to the effect of pulse duration on ablation efficiency and thermal damage on porcine cornea. STUDY DESIGN/MATERIALS AND METHODS: A five by seven grid of craters was made about the center of each cornea. Craters were made with a 60-microm spotsize with a 500-microm spacing. Ablation craters were made using 50 pulses per crater at approximately three times the ablation threshold (for water). Histological analysis was used to determine crater depth and thermal damage. RESULTS: The average zone of thermal damage at 6.1 microm was found to be 4.1 microm for the optical parametric oscillator (OPO) and 5.4 microm for the FEL. At 6.45 microm, the damaged zone was 7.2 microm for the OPO and 7.2 microm for the FEL. At 6.73 microm, the damaged zone was 6.3 microm for the OPO and 7.6 microm+/-0.3 microm for the FEL. CONCLUSIONS: The OPO caused similar or significantly less thermal damage in porcine cornea when compared with the FEL while generating significantly deeper craters. We determined that the ZGP-OPO has much promise as a bench-top replacement for the FEL for soft tissue ablation.

A genetic reporter of thermal stress defines physiologic zones over a defined temperature range.

We define five unique cellular responses to thermal stress using a reporter construct generated using the stress-inducible promoter from the gene encoding a murine 70 kDa heat shock protein (Hsp70A.1) to express luciferase (luc). Thermal stress was delivered over a range of temperatures (42-68 degrees C) for 5 s to 20 min and luciferase activity was measured in live cells using a cooled CCD camera as a measure of reporter gene transcription. Reporter gene expression was assessed every 2 h for 10 h, and at 24 h post-stress. Expression patterns were validated for selected temperatures. A transition zone where cells lose the ability to produce light and beyond which >50% of cells die was observed to occur within a narrow (2.5 degrees C) temperature window. Although luc and hsp70 mRNA levels in this transition zone were high, there were reduced levels of Luc and Hsp70 protein and ATP levels. Cells treated at these temperatures recovered the ability to produce light in response to a secondary stress at 30 h. This Hsp70-luc reporter gene construct may be useful for defining zones of physiologic responses and assessing collateral thermal damage generated during treatment of biological tissue with lasers and other sources of heat.

Cavity ringdown spectroscopy of thin films in the mid-infrared.

To demonstrate the potential of the cavity ringdown technique in mid-infrared spectroscopy of thin film samples, we measured absorption losses in a C60 film on a BaF2 substrate using a tunable optical parametric amplifier source. With a Brewster angle sample geometry, we achieved a fractional loss sensitivity as small as 1.3 x 10(-7) with 1.5 cm(-1) resolution, an improvement in sensitivity of 2 orders of magnitude compared to standard Fourier transform infrared methods. At an absorption sensitivity of 5 x 10(-7), spectra of several C60 overtone lines were recorded.