Three approaches to selective surface fluorescence detection are described. All three of them depend on the use of extremely high numerical aperture (NA) objectives now commercially available (1.45 NA from Zeiss and Olympus and 1.65 NA from Olympus). The first two approaches are elaborations of ‘‘prismless’’ total internal reflection fluorescence (TIRF), one approach with a laser illumination and the second with arc lamp illumination. The new higher NA objectives are much more suitable for TIRF work on biological cells in culture than are 1.4 NA objectives previously described for prismless TIRF. The third approach is not TIRF at all. It uses the high aperture objective to selectively gather the emission of fluorophores located close enough to the substrate for their near-field energy to be captured by the substrate. Schematic diagrams, experimental demonstrations, and practical suggestions for all these techniques are provided.

We provide a basis for automated single-cell sorting based on optical trapping and manipulation using human peripheral blood as a model system. A counterpropagating dual-beam optical-trapping configuration is shown theoretically and experimentally to be preferred due to a greater ability to manipulate cells in three dimensions. Theoretical analysis performed by simulating the propagation of rays through the region containing an erythrocyte (red blood cell) divided into numerous elements confirms experimental results showing that a trapped erythrocyte orients with its longest axis in the direction of propagation of the beam. The single-cell sorting system includes an image-processing system using thresholding, background subtraction, and edge-enhancement algorithms, which allows for the identification of single cells. Erythrocytes have been identified and manipulated into designated volumes using the automated dual-beam trap. Potential applications of automated single-cell sorting, including the incorporation of molecular biology techniques, are discussed.

We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfused isolated mouse hearts, rhod-2 is bolused through the perfusate, resulting in a 6.2±1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33±9%. Quantification of the relative amount of rhod-2 in the heart was done by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 nm (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 µM). Electron microscopy was used to determine the subcellular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain rhod-2. These images demonstrated prominent cytosolic rhod-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 µM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686±237 nM at peak systole, and 360±101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199±215 and 544±53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.

Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogentransformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore. No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coupled instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogentransformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed.

We are developing an imaging system to detect pre-/early cancers in the tracheo-bronchial tree. Autofluorescence might be useful but many features remain suboptimal. We have studied the autofluorescence of human healthy, metaplastic and dysplastic/CIS bronchial tissue, covering excitation wavelengths from 350 to 480 nm. These measurements are performed with a spectrofluorometer whose distal end is designed to simulate the spectroscopic response of an imaging system using routine bronchoscopes. Our data provide information about the excitation and emission spectral ranges to be used in a dual range detection imaging system to maximize the tumor vs healthy and the tumor vs inflammatory/metaplastic contrast in detecting pre-/early malignant lesions. We find that the excitation wavelengths yielding the highest contrasts are between 400 and 480 nm with a peak at 405 nm. We also find that the shape of the spectra of healthy tissue is similar to that of its inflammatory/metaplastic counterpart. Finally we find that, when the spectra are normalized, the region of divergence between the tumor and the nontumor spectra is consistently between 600 and 800 nm and that the transition wavelength between the two spectral regions is around 590 nm for all the spectra regardless of the excitation wavelength, thus suggesting that there might be one absorber or one fluorophore. The use of backscattered red light enhances the autofluorescence contrast.

Emission and excitation spectra of collagen were recorded in the ultraviolet and visible regions. The existence of several types of chromophores absorbing and emitting throughout these spectral regions was observed. It was shown that laser irradiation at 355 and 532 nm caused collagen fluorescence photobleaching by 30%, when the delivered light doses were 9 and 18 J/cm2, respectively. This process of collagen fluorophores photodestruction was found to be a onephoton effect. The effect of hypericin (HYP), a polycyclic quinone, photosensitization on collagen was also studied. Addition of HYP aqueous solution to collagen produced quenching, redshift of the maximum, and broadening of the spectral form of its fluorescence. These effects became more prominent with increasing HYP concentration. The fluorescence of HYP sensitized collagen decreased in a spectrally nonproportional manner during laser irradiation at both 355 and 532 nm.

Enhanced contrast of frequency-domain photon migration (FDPM) measurements for successful biomedical optical imaging may be theoretically achieved with exogenous fluorescence contrast agents. However in practice, the reduced fluorescence signals detected at the air–tissue interface possess significant noise when compared to the signals collected at the incident wavelength. In this study, we experimentally assess signal to noise ratios (SNRs) for FDPM measurements in homogeneous tissue-like scattering media which absorb and fluorescence. At 100 MHz, the SNR for our single-pixel FDPM signals at the incident wavelength is approximately constant at 55 dB while the corresponding fluorescence signal SNR is variable with signal power and is approximately 35 dB. Using these SNR values to guide our studies on the tolerance of absorption and fluorescenceenhanced absorption imaging, we show that the noise tolerance of a Born iterative method for reconstruction of absorption from FDPM measurements at the incident wavelength cannot handle the reduced SNR that is tolerated by a Born iterative type approach for reconstruction of absorption from measurements at the emission wavelength.

Newly developed light-activated surgical adhesives have been investigated as a substitute to traditional protein solders for vascular tissue fusion without the need for sutures. Canine femoral arteries (n=14), femoral veins (n=14), and carotid arteries (n=10) were exposed, and a 0.3-0.6 cm longitudinal incision was made in the vessel walls. The surgical adhesive, composed of a poly(L-lactic-coglycolic acid) scaffold doped with the traditional protein solder mix of bovine serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805 nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The new adhesives were flexible enough to be wrapped around the vessels while their solid nature avoided the problems associated with ‘‘runaway’’ of the less viscous liquid protein solders widely used by researchers. Assessment parameters included measurement of the ex vivo intraluminal bursting pressure 1–2 h after surgery, as well as histology. The acute intraluminal bursting pressures were significantly higher in the laser-solder group (>300 mmHg) compared to the suture control group (<150 mmHg) where four evenly spaced sutures were used to repair the vessel (n= 54). Histological analysis showed negligible evidence of collateral thermal damage to the underlying tissue in the laser-solder repair group. These initial results indicated that laser-assisted vascular repair using the new adhesives is safe, easy to perform, and contrary to conventional suturing, provides an immediate leak-free closure. In addition, the flexible and moldable nature of the new adhesives should allow them to be tailored to a wide range of tissue geometries, thus greatly improving the clinical applicability of laser-assisted tissue repair. ©

The time-dependent temperature distributions produced within thermally homogeneous media heated by a moving laser beam with Gaussian and uniform power density profiles are examined using a time-domain method based on Green’s functions. Regions of finite length, width, and depth within the medium having exponential power absorption are considered. The temperature distribution is written as a single integral with respect to time of simple functions and the resulting expressions have been used to model the heating of blood vessels for birthmark (port-wine stain) removal. The temperature distributions obtained are in good agreement with those produced using Monte Carlo optical and finite difference thermal models.

The goal of this study is to assess the risk of overexposure, when DFB dye laser is used for medical treatment in pulsed mode operation. Results of experimental study showing an unexpected rise of energy in pulses of distributed feedback dye laser (DFDL) output due to temperature phase gratings in dye cell during passively Q switched and mode-locked operation is reported. This unintended increase in the number of pulses, pulse duration, per pulse energy may cause side effects, when used for selective photothermolysis. To probe this phenomenon the most commonly used Rh6G dye was excited with 10–20 pulses of second harmonic of a passively Q switched and mode-locked Nd:yttrium–aluminum–garnet(YAG) laser. The outputs of DFDL and Nd:YAG laser were recorded by an Imacon-675 streak camera. The peak of DFDL output pulses was found delayed proportionally from the peak of the Nd:YAG pulses by more than an interpulse period of excitation laser. A computer program was used to simulate the experimentally measured results to estimate the thermal decay constants and energy retained by medium. The delay between peaks of Nd:YAG (input) and DFDL (output) pulses was found to vary from 10 to 14 ns for various cavity lengths. It was interesting to note that for smaller inter-pulse periods the effect of gradual gain buildup satisfied the threshold conditions for some of the pulses that otherwise cannot lase. This may lead to unintended increase in energy fluence causing overexposure-induced bio effects.

A system that enables the automatic measurement of cardiac rhythm and the quantity of oxygen in the blood has been designed, constructed, and patented. Equipped with the appropriate software, this system registers this information and represents it numerically, in the form of a graph, which can then be printed as a detailed record of cardiac rhythm. This system aids in the determination of cardiac pathologies, and also enables the information to be sent to medical professionals to perform telediagnosis. The apparatus is based on the measurement (sampling) of noninvasive medical parameters. The apparatus is intended to cover a broad range of requirements and needs, as can be used by medical professionals (to detect pathologies related to the pumping and circulation of blood in the body) as well as by lay people who might wish to monitor or gain information concerning their cardiac rhythm and the general functioning of their heart. Thus, the system is designed to be clear and concise in its information as well as easy to use, especially for people unrelated to the medical profession. The way to constructing this system is explained in detail.