The first clinical application of near-infrared spectroscopy (NIRS) 11 years ago was on the head of newborn infants under intensive care. Since then much credible and some important data have been accumulated in this area of research. The best data have been obtained using manipulation of arterial oxygen saturation to obtain single or repeated estimates of cerebral blood flow or cerebral blood volume, or interference with cerebral venous return to obtain measures of venous oxygen saturation. It has been more difficult to take advantage of the continuous and noninvasive nature of NIRS. In particular, the value of the cytochrome signal can still be doubted. A role has not yet developed for NIRS in clinical neonatology.

Fetal brain injury resulting from hypoxia and ischemia during labor remains an important cause of death and long-term disability. However, little is known about fetal brain oxygenation and hemodynamics. There are currently no satisfactory clinical techniques for fetal monitoring and there remains a need for a new method to assess brain oxygenation. Fetal near infrared spectroscopy (NIRS) is a new technique that allows noninvasive observation of changes in the cerebral concentrations of oxyhemoglobin and deoxyhemoglobin to be made during labor. A specially designed optical probe is inserted through the dilated cervix and placed against the fetal head. It is then possible to compare changes in NIRS data with other observations of fetal conditions, such as fetal heart rate and acid-base status.

Exercise performance in patients with heart failure is limited primarily due to a reduction in cardiac output. This results in skeletal muscle hypoperfusion. Near infrared spectroscopy provides a simple noninvasive method for assessing skeletal muscle oxygenation during exercise. In this paper we review the application of this technique to patients with heart failure and describe excessive limb and respiratory muscle oxygenation as compared to normal subjects. The potential of this technology for monitoring clinical improvement and therapeutic efficacy also is discussed.

Delayed intracranial hematomas are an important treatable cause of secondary brain injury in patients with head trauma. Early identification and treatment of these mass lesions, which appear or enlarge after the initial CT scan, may improve neurological outcome. Serial examinations using near-infrared spectroscopy (NIRS) to detect the development of delayed hematomas were performed in 305 patients. The difference in optical density (ΔOD) at 760 nm between the normal and the hematoma side was measured on admission and then serially during the first 3 to 5 days after injury. On admission, the ΔOD was highly predictive of the initial findings on CT scan. Patients with an epidural, subdural, or intracerebral hematoma had significantly greater ΔOD in the involved brain region than patients with diffuse brain injury. For the extracerebral hematomas, the ΔOD was significantly related to the size of the hematoma (r²=0.55 and 0.77 for subdural and epidural hematomas, respectively). Fifty-nine (19%) of the patients developed some type of late hematoma: there was an intracerebral hematoma in 29 patients, an extracerebral hematoma in 7 patients, and a postoperative hematoma in 23 patients. Thirty-three of the late hematomas were large enough to require surgical evacuation. The hematomas appeared between 2 and 72 h after admission. In 55 of the 59 patients, an increase in the ΔOD to >0.10 occurred prior to an increase in intracranial pressure or a change in the neurological examination. Early diagnosis using NIRS may allow early treatment and reduce secondary injury caused by delayed hematomas.

Lasers were introduced in digestive endoscopy to stop active gastroduodenal hemorrhages. Their use spread progressively to the treatment of chronic hemorrhages from vascular malformations and sessile tumors. Lasers face competition from other endoscopic techniques such as electrocoagulation, injection techniques, dilation, stents, and brachytherapy. Many series have reported the efficacy of lasers in digestive endoscopy used for their thermal or photochemical effects. However, they were gradually abandoned for the treatment of hemorrhages because of competition from nonlaser techniques. Lasers are still used for ablation of sessile tumors, but their true impact is difficult to evaluate. Modern methods of technology assessment did not allow gastroenterologists to clearly define the place of lasers among surgery, radio-chemotherapy, and other endoscopic techniques, and data on the daily use of lasers are not available. Therefore, the conclusion can only be subjective. The best current application of thermal lasers appears to be in the treatment of rectosigmoid villous adenomas in elderly patients. Small superficial rectal cancers may also become a good subject due to the impact of endoscopic ultrasonography. Early lesions with multifocal or diffuse disease such as early esophageal cancers could be the most promising subject of application for photodynamic therapy in the future.

Fluorescence spectra of dermal lesions on 48 rats were investigated using excitation wavelengths of 275, 300, and 340 nm. Emissions at 340 and 460 nm were measured in both the forequarter and hindquarter lesions as function of time after incision. Unlike the 460-nm emission, intensity at 340 nm increased with time and then saturated. Control studies on intact skin and lesions in dead rats failed to demonstrate any time-dependent changes at these wavelengths. It appears that the increase in the 340-nm intensity is due to changes in the tryptophan level, and may reflect accumulation of fibrinogen in the early wound healing process.

An attempt to develop an optical probe to assess dental pulp vitality led to the conclusion that it would be difficult to construct an effective optical probe for the middle-aged and older patient who is a common candidate for pulpal assessment. In such patients, vascularity of the gingiva is so much greater than that of the pulp that signals originating in the gingiva overwhelm pulpal effects. This conclusion applies to photoplethysmography, pulse oximetry, and laser Doppler flowmetry, and probably to spectrophotometry as well. The effect of the gingiva was first demonstrated by placing opaque plastic film in the gingival sulcus, which caused attenuation of light passing through molar and bicuspid teeth. More dramatically, the remaining pulsatile signal could be much further attenuated by vasoconstricting the gingiva. An unusually sensitive photoplethysmograph was developed (noise floor, with signal averaging, was near 10 fA), as were a variety of optical probes (including two that approached the tooth below the gingival margin). The tools, methods, and conceptual models that were developed are intended also to be of use to investigators who are developing optical probes for use in younger subjects, in whom such probes appear to be useful.

A fluorescence-based immunosensor using a charge-coupled device (CCD) as a detector has been developed for detecting multiple analytes within small (150 μl) sample volumes. Wells approximately 2 mm in diameter were patterned onto glass coverslips using a photoactivated optical adhesive. In a model detection system, four different antigens were covalently attached to the bottoms of these wells, and the polymer subsequently removed to form the sensing surface. The coverslips were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction (GRIN) lens array was used to focus the sensing surface onto the CCD. Solutions of fluorescently labeled antibodies were then placed on the coverslip, and the amount of antibody bound at each location on the coverslip was determined by quantitative image analysis. Concentrations as low as 50 ng/ml of Cy5-labeled antibodies could be detected using the sensor. The small footprint and minimal system requirements of the design should facilitate its incorporation into portable biosensors.

The use of light quenching to selectively eliminate the emission of biochemical fluorophores based on the emission wavelength is described. To demonstrate the possibility of wavelength-selective light quenching, a mixture of two fluorophores, 4-(dimethylamino)-4'-cyanostillene (DCS) and Prodan, emitting at different wavelengths was examined first. The emission spectrum and intensity decay were altered by the 570-nm quenching pulse due to selective quenching of the longer wavelength emission of DCS. Quenching of the solvent-sensitive fluorophore partially bound to human serum albumin and partially in the aqueous phase was then examined. Light quenching with a long wavelength (570 nm) time-delayed pulse selectivelyquenched the Prodan fluorophore in the aqueous phase while in the presence of the Prodan bound to human serum albumin, which emitted at shorter wavelengths, was not quenched. Using one-beam short wavelength excitation and quenching, a selective quenching of the blue-shifted emission of ribonuclease T₁ in the presence of the red-shifted emission of the tryptophan residue in adrenocorticotropic hormone was observed. In both systems wavelength-selective light quenching was demonstrated by a shift in the emission spectra, and by changes in the intensity decay consistent with preferential quenching of one species. Light quenching is instantly reversible by blocking or defocusing of the quenching beam. It can occur for inaccessible residues or in viscous solvents, and thus can be of wide applicability for resolving the complex emission of biological macromolecules.

An artificial eye has been designed and assembled that mimics the focusing geometry of the living eye. The artificial eye's focusing characteristics are measured and compared with those of the in vivo system. The artificial eye is used to measure several nonlinear optical phenomena that may have an impact on the laser damage thresholds of the retina produced by ultrashort laser pulses. We chose a focal length of 17 mm to simulate the rhesus monkey eye, with a visual cone angle of 8.4 deg for a 2.5-mm diameter laser beam input. The measured focal point image diameter was 5.6±1 μm, which was 1.5 times the calculated diffraction-limited image diameter. This focusing system had the best M² of all the systems evaluated. We used the artificial eye to measure the threshold for laser-induced breakdown, stimulated Brillouin scattering, super-continuum generation, and pulse temporal broadening due to group velocity dispersion.

A new mathematical model is proposed for the growth of a small necrosis domain in living tissue caused by local laser irradiation. Laser light is assumed to be delivered to a small internal region where its absorption causes the temperature to attain high values, leading to immediate tissue coagulation. The coagulation is treated in terms of irreversible phase transition, i.e., it is assumed to occur after the tissue temperature exceeds a certain threshold T_cg . The model considers tissue as involving two regions: the necrosis domain, where the blood perfusion rate is equal to zero, and the normal tissue, which responds to temperature variations by increasing the perfusion rate. The model takes into account the fact that in normal tissue changes in temperature are governed by the blood perfusion rate averaged on spatial scales over the length of the vessels directly controlling heat exchange between the tissue and blood rather than the true perfusion rate. Two alternative models, the developed one and a model allied to the classic approach to the mathematical description of local thermal coagulation, are compared. The effects of blood flow nonuniformity and the delay in vessel response on growth of the necrosis domain are analyzed in detail.

The optical theory behind a new technique for reshaping the cornea to correct refractive errors of the eye is presented. This technique is capable of excising both positive and negative meniscus (lenticular)-shaped tissues of predetermined optical power from the anterior cornea. The optical lens bending theory routinely used by optical designers to reduce the amount of spherical and comatic aberrations in a lens system is employed in this technique. This technique uses a microkeratome to remove a planoconvex, or planoconcave-shaped tissue from the cornea while the eye is in a deformed state. When the eye returns to its natural, undeformed state, a lenticular tissue of predetermined optical power has been removed, correcting for myopic or hyperopic refractive errors, respectively. An elementary introduction to the current surgical techniques used for the correction of refractive errors is presented. Meniscus cross sections of the excised tissues are shown to demonstrate the optical theory discussed.

A new experimental setup has been constructed in which a UV laser microbeam and atomic force microscopy (AFM) have been combined on an inverted microscope in order to manipulate and visualize chromosomes with high resolution. The laser beam has been used to dissect Muntjak metaphase chromosomes and was aimed to optimize the physical size of the cuts. The capability of the AFM to visualize biological material with relative ease has been used to characterize the microdissected chromosomes. This work demonstrates that chromosome fiber material can be removed completely at the cut site using appropriate laser power. The minimum cut size achieved with a 337-nm nitrogen UV laser was between 600 and 800 nm. The smallest distance between the cuts was around 500 nm, corresponding to the finest probe for further biochemical use after physical translocation such as the polymerase chain reaction. Limitations on minimizing the cut size due to diffraction-limited focusing and the effects of laser ablation of biomaterial are discussed.

Raman microprobe spectroscopy was used to investigate the effects of post-extraction treatments of human dentin on the penetration of the 4-methacryloxyethyl trimellitic acid (4-MET)/methyl methacrylate (MMA)/ tri-n-butyl borane (TBB) dentin adhesive. Human molar teeth, extracted and stored in distilled water or 70% ethanol solution for 7 or 28 days, were treated with the resin. Spatially resolved Raman microspectroscopy was used to measure penetration. The band intensity ratio 1612 cm^-1 (resin): 962 cm^-1 (dentin) was used as a diagnostic. Diffusion of the resin into dentin increased with the length of storage period. The results indicate that the effects of postextraction treatments on teeth must be considered when laboratory measurements of bonding strengths are employed to predict the clinical performance of dental cements. In addition, it is demonstrated that Raman microprobe spectroscopy is a feasible analytical tool to evaluate the effects of postextraction treatments on teeth.

Dimers of the fluorescent DNA intercalators oxazole yellow and thiazole orange are used for high-sensitivity DNA detection due to their excellent fluorescence properties. Fluorescence lifetime techniques and absorption spectroscopy were used to investigate the DNA binding properties of POPO-1 [4,4,8,8-tetramethyl-4,8- diazaundecamethylene)bis-4-(3-methyl-2,3-dihydrobenzo-1,3-oxazolyl)-2-methylidene] with the double-stranded homopurine-homopyrimidine polynucleotides poly(dA·dT), poly(dG·dC) and calf thymus DNA. The coexistence of different binding modes of POPO-1 with polynucleotides such as bisintercalation and monointercalation was found in connection with minor groove binding as well as electrostatic attachment. At high excess of polynucleotides, bisintercalation is the only existing form of binding whereas an increasing amount of POPO-1 leads to the coexistence of bis- and monointercalated dye molecules. The amount of bound dye increases with decreasing ionic strength of the buffer and is dependent on the polynucleotide itself. The best binding conditions were found with calf thymus DNA, followed by poly(dA·dT) and poly(dG·dC).

The absorption spectrum of aluminum phthalocyanine with a mean degree of sulfonation of 2.1 (AlPcS₂), previously measured in vivo in a murine tumor model (L1210 leukemia), showed a significant (10 to 15 nm) red shift with respect to the spectrum in solution and in vitro. To investigate whether this behavior was due to the peculiar model considered (ascitic tumor), the absorption line shape was evaluated in vivo noninvasively in a solid tumor (murine MS-2 fibrosarcoma) by means of time-resolved reflectance. The spectrum acquired in the range of 650 to 695 nm after the injection of 2.5 or 5 mg/kg body weight AlPcS₂ confirms the red shift to 680 to 685 nm. Experiments performed ex vivo suggest that the spectral shift is related to the interactions of the drug with the extracellular biological substrate and depends on the sensitizer concentration.