A femtosecond laser, normally used for LASIK eye surgery, is used to perforate cadaveric human stapes. The thermal side effects of bone ablation are measured with a thermocouple in an inner ear model and are found to be within acceptable limits for inner ear surgery. Stress and acoustic events, recorded with piezoelectric film and a microphone, respectively, are found to be negligible. Optical microscopy, scanning electron microscopy, and optical coherence tomography are used to confirm the precision of the ablation craters and lack of damage to the surrounding tissue. Ablation is compared to that from an Er:YAG laser, the current laser of choice for stapedotomy, and is found to be superior. Ultra-short-pulsed lasers offer a precise and efficient ablation of the stapes, with minimal thermal and negligible mechanical and acoustic damage. They are, therefore, ideal for stapedotomy operations.
fermtosecond phenomena; lasers in medicine; electron microscopy; thermal effects
We demonstrate the feasibility of a time-resolved fluorescence spectroscopy (TRFS) technique for intraluminal investigation of arterial vessel composition under intravascular ultrasound (IVUS) guidance. A prototype 1.8-mm (5.4 Fr) catheter combining a side-viewing optical fiber (SVOF) and an IVUS catheter was constructed and tested with in vitro vessel phantoms. The prototype catheter can locate a fluorophore in the phantom vessel wall, steer the SVOF in place, perform blood flushing under flow conditions, and acquire high-quality TRFS data using 337-nm wavelength excitation. The catheter steering capability used for the coregistration of the IVUS image plane and the SVOF beam produce a guiding precision to an arterial phantom wall site location of 0.53±0.16 mm. This new intravascular multimodal catheter enables the potential for in vivo arterial plaque composition identification using TRFS.
fluorescence spectroscopy; atherosclerosis; fluorescence lifetime; intravascular ultrasound
We demonstrate noninvasive near-infrared diffuse optical spectroscopy (DOS) measurements of tissue hemoglobin contents that can track progressive reductions in central blood volume in human volunteers. Measurements of mean arterial blood pressure (MAP), heart rate (HR), stroke volume (SV), and cardiac output (Q) are obtained in ten healthy human subjects during baseline supine rest and exposure to progressive reductions of central blood volume produced by application of lower body negative pressure (LBNP). Simultaneous quantitative noninvasive measurements of tissue oxyhemoglobin (OHb), deoxyhemoglobin (RHb), total hemoglobin concentration (THb), and tissue hemoglobin oxygen saturation (StO2) are performed throughout LBNP application using broadband DOS. As progressively increasing amounts of LBNP are applied, HR increases, and MAP, SV, and Q decrease (p<0.001). OHb, StO2, and THb decrease (p <0.001) in correlation with progressive increases in LBNP, while tissue RHb remained relatively constant (p=0.378). The average fractional changes from baseline values in DOS OHb (fOHb) correlate closely with independently measured changes in SV (r2=0.95) and Q (r2=0.98) during LBNP. Quantitative noninvasive broadband DOS measurements of tissue hemoglobin parameters of peripheral perfusion are capable of detecting progressive reductions in central blood volume, and appear to be sensitive markers of early hypoperfusion associated with hemorrhage as simulated by LBNP.
hemorrhagic shock; lower body negative pressure; hemodynamic decompensation
The short working distance of microscope objectives has severely restricted the application of optical micromanipulation techniques at larger depths. We show the first use of fiber-optic tweezers toward controlled guidance of neuronal growth cones and stretching of neurons. Further, by mode locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes and thus allowing study of the subsequent response of neurons to localized injury. At high average powers, lysis of a three-dimensionally trapped cell was accomplished.
optical tweezers; laser microbeam; optical stretcher; optical micromanipulation; optical fiber; neuron growth; cellular lysis
The purpose of this work was to validate the use of Pluronic fluorescent nanomicelles for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium was compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles showed, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 hour after intravenous injection. Their two-photon imaging depth was similar in normal mouse brain using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20–100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block co-polymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.
Animals; Blood Vessels; anatomy & histology; Blood Volume Determination; Brain; blood supply; Cell Line, Tumor; Dextrans; diagnostic use; Diagnostic Imaging; Fluorescent Dyes; diagnostic use; Humans; Mice; Mice, Nude; Micelles; Microscopy, Confocal; methods; Nanostructures; Neoplasms, Experimental; blood supply; Optical Phenomena; Photons; Poloxamer; Rhodamines; diagnostic use; Styrenes; diagnostic use; Intravital two-photon microscopy; cerebral and tumor microvasculature; Pluronic nanomicelles.
Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the β2-adrenergic receptor (β2AR). The VT2R/β2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/β2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2 / β2AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.
hypothalamic-pituitary-adrenal axis; neurohormones; G-protein coupled receptors; receptor interactions
A board-level broadband frequency domain photon migration (mini-FDPM) instrument has been constructed to replace a conventional network-analyzer-based FDPM instrument. The mini-FDPM instrument with four wavelengths (681, 783, 823, and 850 nm), matches conventional FDPM instrument in performance (−88 dBm noise level, 100 dB dynamic range) and bandwidth (1 GHz), and recovers the same optical properties within about 6% in absorption and 4% in reduced scattering for liquid phantoms covering a wide range of relevant optical properties. Compared to the conventional FDPM instrument, the mini-FDPM instrument is more than 5× faster (~200 ms per 401 modulation frequencies) and several orders of magnitude less in size and cost. Standard fiber-optic-based probes can be used with the mini-FDPM instrument, which increases applications in a number of clinically relevant measurement scenarios. By drastically reducing size and cost, FDPM miniaturization lowers barriers to access and will help promote FDPM in clinical research problems. The mini-FDPM instrument forms the core of a modular broadband diffuse optical spectroscopy instrument that can be used for a variety of clinical problems in imaging and functional monitoring (i.e., breast/skin cancer, brain activation, and exercise physiology).
diffuse optical spectroscopy; near-infrared; photon migration; tissue spectroscopy; frequency domain photon migration (FDPM)
A multispectral digital microscope (MDM) is designed and constructed as a tool to improve detection of oral neoplasia. The MDM acquires in vivo images of oral tissue in fluorescence, narrowband (NB) reflectance, and orthogonal polarized reflectance (OPR) modes, to enable evaluation of lesions that may not exhibit high contrast under standard white light illumination. The device rapidly captures image sequences so that the diagnostic value of each modality can be qualitatively and quantitatively evaluated alone and in combination. As part of a pilot clinical trial, images are acquired from normal volunteers and patients with precancerous and cancerous lesions. In normal subjects, the visibility of vasculature can be enhanced by tuning the reflectance illumination wavelength and polarization. In patients with histologically confirmed neoplasia, we observe decreased blue/green autofluorescence and increased red autofluorescence in lesions, and increased visibility of vasculature using NB and OPR imaging. The perceived lesion borders change with imaging modality, suggesting that multimodal imaging has the potential to provide additional diagnostic information not available using standard white light illumination or by using a single imaging mode alone.
oral cancer; diagnosis; noninvasive; optical imaging; fluorescence; decreased autofluorescence; reflectance; polarized; orthogonal; porphyrin; vasculature; monochromatic
We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC2(3), that reports the MP across the inner membrane of the mitochondria located in the sperm’s midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system’s capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.
sperm motility; laser tweezers; escape laser power; mitochondrial membrane potential; 3,3′-diethyloxacarbocyanine iodide; carbonyl cyanide 3-chlorophenylhydrazone
In the recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical, easy to couple to commercial microscopes and provide powerful and stable light that can be triggered by TTL pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole field illumination which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localised illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411 nm) filter with appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole field visible illumination and spot UV illumination of 15–20 μm. We tested our system by performing calcium imaging experiments combined with L-glutamate or NMDA photo-release in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budget to run similar experiments in a variety of physiological applications.
Photolysis; Calcium imaging; LED illumination; Epifluorescence microscope
Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 μm), protein (amide-II band, 6.45 μm), and both water and protein (amide-I band, 6.10 μm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 μm have approximately 4× higher hsp70 expression than 6.10 μm. Histology shows that under comparable fluences, tissue injury at the 2.94-μm wavelength was 2× and 3× deeper than 6.45 and 6.10 μm, respectively. The 6.10-μm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-μm wavelength is a superior wavelength for laser ablation of skin.
ablation; free-electron laser; heat shock protein expression; skin; wound healing; bioluminescence; optical imaging
Multispectral near-infrared (NIR) tomographic imaging has the potential to provide information about molecules absorbing light in tissue, as well as subcellular structures scattering light, based on transmission measurements. However, the choice of possible wavelengths used is crucial for the accurate separation of these parameters, as well as for diminishing crosstalk between the contributing chromophores. While multispectral systems are often restricted by the wavelengths of laser diodes available, continuous-wave broadband systems exist that have the advantage of providing broadband NIR spectroscopy data, albeit without the benefit of the temporal data. In this work, the use of large spectral NIR datasets is analyzed, and an objective function to find optimal spectral ranges (windows) is examined. The optimally identified wavelength bands derived from this method are tested using both simulations and experimental data. It is found that the proposed method achieves images as qualitatively accurate as using the full spectrum, but improves crosstalk between parameters. Additionally, the judicious use of these spectral windows reduces the amount of data needed for full spectral tomographic imaging by 50%, therefore increasing computation time dramatically.
iomedical optics; image reconstruction; inverse problems
Fluorescence measurements have been used to track the dosimetry of photodynamic therapy (PDT) for many years, and this approach can be especially important for treatments with aminolevulinic-acid-induced protoporphyrin IX (ALA-PpIX). PpIX photobleaches rapidly, and the bleaching is known to be oxygen dependent, and at the same time, fractionation or reduced irradiance treatments have been shown to significantly increase efficacy. Thus, in vivo measurement of either the bleaching rate and/or the total bleaching yield could be used to track the deposited dose in tissue and determine the optimal treatment plans. Fluorescence in rat esophagus and human Barrett’s esophagus are measured during PDT in both continuous and fractionated light delivery treatment, and the bleaching is quantified. Reducing the optical irradiance from 50 to 25 mW/cm did not significantly alter photobleaching in rat esophagus, but fractionation of the light at 1-min on and off intervals did increase photobleaching up to 10% more (p value=0.02) and up to 25% more in the human Barrett’s tissue (p value<0.001). While two different tissues and two different dosimetry systems are used, the data support the overall hypothesis that light fractionation in ALA-PpIX PDT esophageal treatments should have a beneficial effect on the total treatment effect.
fluorescence; photodetection; fiber optic sensors; photodynamic therapy; fiber optics; targets
The recycling of G-protein-coupled receptors (GPCR) to the cell surface after internalization plays an important role in the regulation of overall GPCR activity. The angiotensin II type I receptor (AT1R) belongs to class B GPCRs that recycle slowly back to the cell surface. Previous studies have proposed that Rab11 controls the recycling of AT1R; however, recent reports show that Rab4, a rapid recycling regulator, co-localizes also with internalized AT1R. Different from the sub-cellular co-localization provided by fluorescence microscopy, fluorescence resonance energy transfer (FRET) microscopy provided the spatial relationship of AT1R with Rab4 and Rab11 in the nanometer-range proximity during the entire course of AT1R recycling. During the early recycling stage, internalized AT1Rs were mainly associated with Rab4 in the cytoplasm. During the mid-recycling stage, AT1Rs were associated with both Rab4 and Rab11 in the perinuclear compartments. However, during the late-recycling stage, AT1Rs were mainly associated with Rab11, both in the perinuclear compartments and the plasma membrane. Co-immunoprecipitation data confirmed these dynamic associations, which were disrupted by silencing of either the Rab4 or Rab11 gene. Based on these observations, we propose a Rab4 and Rab11 coordinated model for AT1R recycling.
angiotensin II type I receptors; recycling; fluorescence resonance energy transfer; Rab4; Rab11; G-protein-coupled receptors
We propose a novel method for speckle reduction for optical coherence tomography based on angular compounding by B-scan Doppler-shift encoding (AngularCBD). By de-centering the probe beam from the pivot of a scanning mirror, the illumination angle represented by different components of the beam can be encoded in Doppler shift. Compounding multiple images reconstructed from different Doppler-shift bands, we can suppress speckle without sacrificing image acquisition speed. Speckle reduction with AngularCBD is demonstrated by imaging a phantom and tissue sample in vitro and in vivo.
Speckle reduction; Optical Coherence Tomography; Doppler shift; Angular compounding
Controlling two-photon molecular fluorescence leading to selective fluorophore excitation has been a long sought after goal in fluorescence microscopy. In this letter, we thoroughly explore selective fluorescence suppression through simultaneous two-photon absorption by two different fluorophores followed by selective one-photon stimulated emission for one particular fluorophore. We achieve this by precisely controlling the time delay between two identical ultrafast near infrared laser pulses.
two-photon fluorescence; selective stimulated emission; one color scheme; laser-scanning microscopy
The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe here a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near infrared (NIR) laser into the scanhead and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon software without modification.
The cochlea is the mammalian organ of hearing. Its predominant vibratory element, the basilar membrane, is tonotopically tuned, based on the spatial variation of its mass and stiffness. The constituent collagen fibers of the basilar membrane affect its stiffness. Laser irradiation can induce collagen remodeling and deposition in various tissues. We tested whether similar effects could be induced within the basilar membrane. Trypan blue was perfused into the scala tympani of anesthetized mice to stain the basilar membrane. We then irradiated the cochleas with a 694-nm pulsed ruby laser at 15 or 180 J /cm2. The mice were sacrificed 14 to 16 days later and collagen organization was studied. Polarization microscopy revealed that laser irradiation increased the birefringence within the basilar membrane in a dose-dependent manner. Electron microscopy demonstrated an increase in the density of collagen fibers and the deposition of new fibrils between collagen fibers after laser irradiation. As an assessment of hearing, auditory brainstem response (ABR) thresholds were found to increase moderately after 15 J/cm2 and substantially after 180 J /cm2. Our results demonstrate that collagen remodeling and new collagen deposition occurs within the basilar membrane after laser irradiation in a similar fashion to that found in other tissues.
collagen; resonant frequency; hearing; photocoagulation; remodeling
Iron oxide particles are becoming an important contrast agent for MRI cell tracking studies using MRI. Simultaneous delivery of fluorescence indicators with the particles to individual cells offers the possibility of correlating optical and MRI. In this paper, it was demonstrated that micron sized iron oxide particles (MPIOs) can be used as a carrier to deliver fluorescent probes to cells in culture as well as to migrating neural progenitors in vivo. Migrating progenitors were tracked with MRI and easily identified by histology because of the fluorescent probe. These data suggest that using MPIOs to deliver fluorescent probes should make it possible to combine MRI and optical imaging for in vivo cell tracking.
The ability to inject exogenous material as well as to alter subcellular structures in a minimally invasive manner using a laser microbeam has been useful for cell biologists to study the structure-function relationship in complex biological systems. We describe a quantitative phase laser microsurgery system, which takes advantage of the combination of laser microirradiation and short-coherence interference microscopy. Using this method, quantitative phase images and the dynamic changes of phase during the process of laser microsurgery of red blood cells (RBCs) can be evaluated in real time. This system would enable absolute quantitation of localized alteration/damage to transparent phase objects, such as the cell membrane or intracellular structures, being exposed to the laser microbeam. Such quantitation was not possible using conventional phase-contrast microscopy.
laser scissors; short-coherence interference microscopy; quantitative phase imaging; optical micromanipulation; cellular damage
We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5–5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.
nonlinear microscopy; adaptive optics; three-dimensional microscopy; medical and biological imaging
Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.
endothelial cell; membrane; total internal reflection fluorescence; mechanotransduction; fluorescence correlation spectroscopy; time-correlated single-photon counting
FTIR spectroscopy is sensitive to the molecular composition of tissue and has the potential to identify pre-malignant tissue (dysplasia) as an adjunct to endoscopy. We demonstrate collection of mid-infrared absorption spectra with a silver halide (AgCl0.4Br0.6) optical fiber and use spectral pre-processing to identify optimal sub-ranges that classify colonic mucosa as normal, hyperplasia, or dysplasia. We collected spectra (n = 83) in the 950 to 1800 cm−1 regime on biopsy specimens obtained from human subjects (n = 37). Subtle differences in the magnitude of the absorbance peaks at specific wavenumbers were observed. The best double binary algorithm for distinguishing normal-versus-dysplasia and hyperplasia-versus-dysplasia was determined from an exhaustive search of spectral intervals and pre-processing techniques. Partial least squares discriminant analysis was used to classify the spectra using a leave-one-subject-out cross-validation strategy. The results were compared with histology reviewed independently by two gastrointestinal pathologists. The optimal thresholds identified resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively. These results indicated that mid-infrared absorption spectra collected remotely with an optical fiber can be used to identify colonic dysplasia with high accuracy, suggesting that continued development of this technique for the early detection of cancer is promising.
FTIR; dysplasia; optical fiber; early detection; cancer; endoscopy
A mechanically scanned CO2 laser operated at high laser pulse repetition rates can be used to rapidly and precisely remove dental decay. This study aims to determine whether these laser systems can safely ablate enamel and dentin without excessive heat accumulation and peripheral thermal damage. Peripheral thermal damage can adversely impact the mechanical strength of the irradiated tissue, particularly for dentin, and reduce the adhesion characteristics of the modified surfaces. Samples were derived from noncarious extracted molars. Pulpal temperatures were recorded using microthermocouples situated at the pulp chamber roof of samples (n=12), which were occlusally ablated using a rapid-scanning, water-cooled 300 Hz CO2 laser over a two minute time course. The mechanical strength of facially ablated dentin (n=10) was determined via four-point bend test and compared to control samples (n=10) prepared with 320 grit wet sand paper to simulate conventional preparations. Composite-to-enamel bond strength was measured via single-plane shear test for ablated/non-etched (n=10) and ablated/acid-etched (n=8) samples and compared to control samples (n=9) prepared by 320 grit wet sanding.
Thermocouple measurements indicated that the temperature remained below ambient temperature at 19.0°C (s.d.=0.9) if water-cooling was used. There was no discoloration of either dentin and enamel, the treated surfaces were uniformly ablated and there were no cracks observable on the laser treated surfaces. Four-point bend tests yielded mean mechanical strengths of 18.2 N (s.d.=4.6) for ablated dentin and 18.1 N (s.d.=2.7) for control (p>0.05). Shear tests yielded mean bond strengths of 31.2 MPa (s.d.=2.5, p<0.01) for ablated/acid-etched samples, 5.2 MPa (s.d.=2.4, p<0.001) for ablated/non-etched samples, and 37.0 MPa (s.d.=3.6) for control. The results indicate that a rapid-scanning 300 Hz CO2 laser can effectively ablate dentin and enamel without excessive heat accumulation and with minimal thermal damage. It is not clear whether the small (16%) but statistically significant reduction in the shear bond strength to enamel is clinically significant since the mean shear bond strength exceeded 30 MPa.
enamel; CO2 laser; adhesion; heat accumulation; peripheral thermal damage