Journal of Biomedical Optics

Published by Society of Photo-optical Instrumentation Engineers
Online ISSN: 1560-2281
Print ISSN: 1083-3668
Dependence of the source-size correction factor C E on the retinal irradiance diameter.
System for exposure of monkey retina in vivo to a laser beam having selectable irradiance diameters at the retina. 
ED 50 for laser-induced retinal injury in monkey eyes as a function of the retinal irradiance diameter for 100-ms-duration exposures to laser radiation from an argon laser operating at 514 nm: open triangle, 1-h endpoint, extramacular exposures; closed triangle, 24-h endpoint, extramacular exposures; open circles, 1-h endpoint, macular exposures; closed circles, 24-h endpoint, macular exposures.
Comparison of the ED 50 for retinal injury induced by 100-ms- 
Dependence of ED 50 on the retinal irradiance diameter for a 
The retinal injury threshold dose for laser exposure varies as a function of the irradiated area on the retina. Zuclich reported thresholds for laser-induced retinal injury from 532 nm, nanosecond-duration laser exposures that varied as the square of the diameter of the irradiated area on the retina. We report data for 0.1-s-duration retinal exposures to 514-nm, argon laser irradiation. Thresholds for macular injury at 24 h are 1.05, 1.40, 1.77, 3.58, 8.60, and 18.6 mJ for retinal exposures at irradiance diameters of 20, 69, 136, 281, 562, and 1081 microm, respectively. These thresholds vary as the diameter of the irradiated retinal area. The relationship between the retinal injury threshold and retinal irradiance diameter is a function of the exposure duration. The 0.1-s-duration data of this experiment and the nanosecond-duration data of Zuclich show that the ED(50) (50% effective dose) for exposure to a highly collimated beam does not decrease relative to the value obtained for a retinal irradiance diameter of 100 microm. These results can form the basis to improve current laser safety guidelines in the nanosecond-duration regime. These results are relevant for ophthalmic devices incorporating both wavefront correction and retinal exposure to a collimated laser.
There is an increasing use of high-power fiber lasers in manufacturing and telecommunications industries operating in the infrared spectrum between 1000 and 2000 nm, which are advertised to provide as much as 10 kW continuous output power at 1070 nm. Safety standards have traditionally been based on experimental and modeling investigations with scant data available for these wavelengths. A series of studies using 1070-nm infrared lasers to determine the minimum visible lesion damage thresholds in skin using the Yucatan miniature pig (Sus scrofa domestica) for a range of beam diameters (0.6, 1.1, 1.9, 2.4, 4.7, and 9.5 cm) and a range of exposure durations (10 ms to 10 s) is presented. Experimental peak temperatures associated with each damage threshold were measured using thermal imaging. Peak temperatures at damage threshold for the 10-s exposures were ∼10°C lower than those at shorter exposures. The lowest and highest experimental minimum visible lesion damage thresholds were found to have peak radiant exposures of 19 and 432 J/cm2 for the beam diameter-exposure duration pairs of 2.4 cm, 25 ms and 0.6 cm, 10 s, respectively. Thresholds for beam diameters >2.5 cm had a weak to no effect on threshold radiant exposure levels for exposure times ≤0.25 s, but may have a larger effect on thresholds for exposures ≥10 s.
Schematic illustration of the biofunctionalization steps applied for the immobilization of the influenza A virus on GaAs ͑ 001 ͒ substrate ͑ a ͒ that having been etched with NH 4 OH was passivated using a 2 mM solution of mixed PEG thiols ͑ 1:15 ͒ ͑ b ͒ and incubated in a solution of neutravidin ͑ c ͒ . Biotinylated antibodies against influenza A were immobilized on the neutravidin-coated substrate ͑ d ͒ , which enabled immobilizing virus particles from a solution ͑ e ͒ . 
FTIR spectra of SAMs deposited on three different GaAs ͑ 001 ͒ samples ͓ lines ͑ a ͒ , ͑ b ͒ , and ͑ c ͔͒ , demonstrating the reproducibility of the thiolation process. 
AFM images and cross-sectional scans of ͑ a ͒ a reference GaAs ͑ 001 ͒ sample, ͑ b ͒ the GaAs ͑ 001 ͒ sample biofunctionalized with PEG thiols and neutravidin, and ͑ c ͒ the sample that following the function- alization with the influenza A antibody was exposed to a solution of inactivated influenza A virus. 
Fluorescence microscopy image of a GaAs ͑ 001 ͒ sample that, following the specific immobilization of the influenza A virus, was exposed to FITC-conjugated antibodies against the influenza A virus. Inset shows a control fluorescence image observed if FITC-conjugated influenza A antibodies were replaced with FITC conjugated hepatitis B antibodies. 
In the quest for the development of an all-optical biosensor for rapid detection and typing of viral pathogens, we investigate biosensing architectures that take advantage of strong photoluminescence emission from III-V quantum semiconductors (QS). One of the key elements in the development of such a biosensor is the ability to attach various analytes to GaAs--a material of choice for capping III-V QS of our interest. We report on the study of biofunctionalization of GaAs (001) with polyethylene-glycol (PEG) thiols and the successful immobilization of influenza A virus. A diluted solution of biotinylated PEG thiols in OH-terminated PEG thiols is used to form a network of sites for the attachment of neutravidin. Biotinylated polyclonal influenza A antibodies are applied to investigate the process of the immobilization of inactivated influenza A virus. The successful immobilization is demonstrated using atomic force microscopy and fluorescence microscopy measurements.
First introduced in the 1980s, laser speckle contrast imaging is a powerful tool for full-field imaging of blood flow. Recently laser speckle contrast imaging has gained increased attention, in part due to its rapid adoption for blood flow studies in the brain. We review the underlying physics of speckle contrast imaging and discuss recent developments to improve the quantitative accuracy of blood flow measures. We also review applications of laser speckle contrast imaging in neuroscience, dermatology and ophthalmology.
A new method is described for obtaining a 3-D reconstruction of a bioluminescent light source distribution inside a living animal subject, from multispectral images of the surface light emission acquired on charge-coupled device (CCD) camera. The method uses the 3-D surface topography of the animal, which is obtained from a structured light illumination technique. The forward model of photon transport is based on the diffusion approximation in homogeneous tissue with a local planar boundary approximation for each mesh element, allowing rapid calculation of the forward Green's function kernel. Absorption and scattering properties of tissue are measured a priori as input to the algorithm. By using multispectral images, 3-D reconstructions of luminescent sources can be derived from images acquired from only a single view. As a demonstration, the reconstruction technique is applied to determine the location and brightness of a source embedded in a homogeneous phantom subject in the shape of a mouse. The technique is then evaluated with real mouse models in which calibrated sources are implanted at known locations within living tissue. Finally, reconstructions are demonstrated in a PC3M-luc (prostate tumor line) metastatic tumor model in nude mice.
The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.
Optical-resolution photoacoustic microscopy (OR-PAM) is applied to functional brain imaging in living mice. A near-diffraction-limited bright-field optical illumination is employed to achieve micrometer lateral resolution, and a dual-wavelength measurement is utilized to extract the blood oxygenation information. The variation in hemoglobin oxygen saturation (sO(2)) along vascular branching has been imaged in a precapillary arteriolar tree and a postcapillary venular tree, respectively. To the best of our knowledge, this is the first report on in vivo volumetric imaging of brain microvascular morphology and oxygenation down to single capillaries through intact mouse skulls. It is anticipated that: (i) chronic imaging enabled by this minimally invasive procedure will advance the study of cortical plasticity and neurological diseases; (ii) revealing the neuroactivity-dependent changes in hemoglobin concentration and oxygenation will facilitate the understanding of neurovascular coupling at the capillary level; and (iii) combining functional OR-PAM and high-resolution blood flowmetry will have the potential to explore cellular pathways of brain energy metabolism.
For bioluminescence imaging (BLI) of small animals, the most commonly used luciferase is Fluc from the firefly, but recently, green (CBGr99) and red (CBRed) click beetle luciferases became available. Because signal attenuation by tissues is lower for red light, red luciferases appear to be advantageous for BLI, but this has not been thoroughly tested. We compare different luciferases for BLI. For this purpose, cell transfectants are generated expressing comparable amounts of CBGr99, CBRed, or Fluc. This is achieved by coexpression of the luciferase with eGFP using the bicistronic 2A system, which results in stoichiometric coexpression of the respective proteins. In vitro, the CBGr99 transfectant exhibits the strongest total photon yield. For in vivo BLI, the transfectants are injected into mice at different locations. At a subcutaneous position, CBGr99 is clearly superior to the other luciferases. When the tumor cells are located in the peritoneum or lung, where more absorption by tissue occurs, CBGr99 and CBRed transfected cells emit a comparable number of red photons and are superior to Fluc, but CBGr99 reaches the maximum of the light emission faster than CBRed. Thus, although CBGr99 emits mainly green light, the high yield of total and red photons makes it an excellent candidate for BLI.
In the last two decades, both diffuse optical tomography (DOT) and blood oxygen level dependent (BOLD)-based functional magnetic resonance imaging (fMRI) methods have been developed as noninvasive tools for imaging evoked cerebral hemodynamic changes in studies of brain activity. Although these two technologies measure functional contrast from similar physiological sources, i.e., changes in hemoglobin levels, these two modalities are based on distinct physical and biophysical principles leading to both limitations and strengths to each method. In this work, we describe a unified linear model to combine the complimentary spatial, temporal, and spectroscopic resolutions of concurrently measured optical tomography and fMRI signals. Using numerical simulations, we demonstrate that concurrent optical and BOLD measurements can be used to create cross-calibrated estimates of absolute micromolar deoxyhemoglobin changes. We apply this new analysis tool to experimental data acquired simultaneously with both DOT and BOLD imaging during a motor task, demonstrate the ability to more robustly estimate hemoglobin changes in comparison to DOT alone, and show how this approach can provide cross-calibrated estimates of hemoglobin changes. Using this multimodal method, we estimate the calibration of the 3 tesla BOLD signal to be -0.55%+/-0.40% signal change per micromolar change of deoxyhemoglobin.
We investigate whether the functional near-infrared spectroscopic (fNIRS) signal includes a signal from the changing skin blood flow. During a locomotor task on a treadmill, changes in the hemodynamic response in the front-parietal area of healthy human subjects are simultaneously recorded using an fNIRS imaging system and a laser Doppler tissue blood flow meter. Independent component analysis (ICA) for fNIRS signals is performed. The skin blood flow changes during locomotor tasks on a treadmill. The activated spatial distribution of one of the components separated by ICA reveals an overall increase in fNIRS channels. To evaluate the uniformity of the activated spatial distribution, we define a new statistical value-the coefficient of spatial uniformity (CSU). The CSU value is a highly discriminating value (e.g., 2.82) compared with values of other components (e.g., 1.41, 1.10, 0.96, 0.61, and 0.58). In addition, the independent component signal corresponding to the activated spatial distribution is similar to changes in skin blood flow measured with the laser Doppler tissue blood flow meter. The coefficient of correlation indicates strong correlation. Localized activation areas around the premotor and medial somatosensory cortices are shown more clearly by eliminating the extracted component.
The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 - 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼ 200 μm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.
Certain substituted naphthalimides have been shown to produce, on photochemical activation, mechanically viable bonds between a variety of tissue surfaces. It is believed that these compounds act as photochemically activated oxidants, catalyzing the formation of reactive intermediates in the extracellular matrices of approximated tissue surfaces. The condensation of these intermediates results in the formation of crosslinks between the intimate surfaces. Of particular interest is the application of this technique to the repair of tears in the typically unrepairable avascular zone of menisci. The menisci are collagen-rich fibrocartilaginous tissues that support up to 90% of the load across the knee joint and participate in important functions including shock absorption, joint stabilization, hyperextension prevention, and lubrication of the knee. Preliminary ex vivo and in vivo work in our laboratories has demonstrated that photochemically activated naphthalimides have significant potential for the repair of meniscal lesions. We describe preliminary ex vivo studies assessing the relative abilities of a variety of water-soluble monomeric 4-amino-1,8-naphthalimides to bond bovine knee meniscal tissue on visible light irradiation.
We propose high-speed spectral domain polarization-sensitive optical coherence tomography (SD-PS-OCT) using a single camera and a 1x2 optical switch at the 1.3-microm region. The PS-low coherence interferometer used in the system is constructed using free-space optics. The reflected horizontal and vertical polarization light rays are delivered via an optical switch to a single spectrometer by turns. Therefore, our system costs less to build than those that use dual spectrometers, and the processes of timing and triggering are simpler from the viewpoints of both hardware and software. Our SD-PS-OCT has a sensitivity of 101.5 dB, an axial resolution of 8.2 microm, and an acquisition speed of 23,496 A-scans per second. We obtain the intensity, phase retardation, and fast axis orientation images of a rat tail tendon ex vivo.
The optical inhomogeneity of flowing blood, which appears as a waisted double fan-shaped intensity pattern inside vessels in cross-sectional optical coherence tomography (OCT) images, was investigated for the first time. High resolution spectral domain OCT in the 1.3 μm wavelength region is used to assess this inhomogeneous intravascular backscattering of light in an in vivo mouse model and flow phantom measurements. Based on a predicted alignment of the red blood cells toward laminar shear flow, an angular modulation of the corresponding backscattering cross-section inside the vessels is assumed. In combination with the signal attenuation in depth by absorption and scattering, a simple model of the intravascular intensity modulation is derived. The suitability of the model is successfully demonstrated in the in vivo experiments and confirmed by the in vitro measurements. The observed effect appears in flowing blood only and shows a strong dependency on the shear rate. In conclusion, the shear-induced red blood cell alignment in conjunction with the vessel geometry is responsible for the observed intensity distribution. This inherent effect of blood imaging has to be considered in attenuation measurements performed with OCT. Furthermore, the analysis of the intravascular intensity pattern might be useful to evaluate flow characteristics.
Cartilage reshaping jig. (a) The cartilage specimen is held in compression between two aluminum rods 2.5 cm apart mounted on a rotational stage. In reshaping experiments only I(t) and S c (t) are monitored. I(t) is monitored on the lock-in amplifier display, and when d(I(t))/dt0 laser irradiation is stopped. (b) Following laser irradiation, the stage is then rotated to a new position so that nonirradiated tissue can be brought into the laser irradiation zone.
Serial montage of a cartilage specimen that has undergone reshaping across the entire length: (a) the cartilage specimen before reshaping, (b) immediately after laser radiation and 15 min rehydration in normal saline solution (while wrapped around a plastic dowel), and (c) the same specimen in normal saline solution (c) after 7 days and (d) after 21 days.
Laser-assisted cartilage reshaping is mediated by thermally induced stress relaxation, and may be used to alter cartilage morphology for reconstructive surgical procedures in the upper airway and face without carving, morselizing, or suturing. Internal stress σ(t), integrated backscattered light intensity I(t) from a He-Ne probe laser (λ=632.8 nm), and radiometric surface temperature Sc(t) were measured during the reshaping of porcine nasal septal cartilage using a pulsed Nd:YAG laser (λ=1.32 μm). Internal stress and integrated backscattered light intensity were observed to increase, plateau, and then decrease in similar ways during laser irradiation. The plateau region occurred when the cartilage front surface temperature approached 65 °C. I(t) was utilized in a feedback control procedure to reshape cartilage specimens from a flat to a curved geometry. Immediately following laser irradiation, the tissues were rehydrated in normal saline for 15 min while wrapped around a small dowel. A stable shape change was retained for 21 days while the specimens were stored in normal saline at 5 °C. The backscattered light intensity signal mirrors underlying changes in internal stress, and further rate of change or slope of I(t) is nearly zero when the surface temperature reaches about 65 °C. Measurements of I(t) (or, equivalently, the fractional change in integrated backscattered light intensity ΔI(t)/I0) may be used to control the process of laser-assisted cartilage reshaping and minimize nonspecific thermal injury due to uncontrolled heating. © 1998 Society of Photo-Optical Instrumentation Engineers.
Design of the mPlum-infrared fluorescent protein (IFP) 1.4 fusion gene construct. (a) A schematic representation of the mPlum-IFP 1.4 fusion construct. (b) In-frame structure of the mPlum-IFP 1.4 fusion protein demonstrated by the encoded amino acids of the fusion protein's sequence.  
Visualization of IFP 1.4 signals from the mPlum-IFP 1.4 fusion protein exposed to the mPlum excitation channel. (a) 293T cells expressing mPlum-IFP 1.4 (middle panel) fusion protein exhibited stronger signals than similar cells expressing IFP 1.4 (upper panel) after they were exposed to the 543-nm laser and detected using the IFP emission channel (680 to 740 nm). Co-expressed GFP was detected by 488-nm excitation. Panel A–D: IFP 1.4 transfected cells; panel E–H: mPlum-IFP 1,4 fusion construct transfected cells; panel I–L: mPlum transfected cells; ch: channel. (b) The Western blot analysis for co-expressed GFP in IFP1.4 and mPlum-IFP1.4 fusion protein. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (c) Semi-quantification of the brightness in IFP 1.4 or the mPlum-IFP 1.4 fusion protein normalized against GFP. ***: p < 0.005. (d) The reverse transcription-polymerase chain reaction (RT-PCR) for comparing the mRNA level of IFP 1.4 and mPlum-IFP 1.4 genes that were transfected to cells. The target of the RT-PCR was an IFP 1.4 DNA fragment with a 190 bp length. β-actin (96 bp) was used as an internal control.  
The Förster resonance energy transfer (FRET)-like properties of the mPlum-IFP 1.4 fusion protein. (a) The excitation and emission spectra of mPlum and IFP 1.4. (b) Detection of the brightness of mPlum donor after photobleaching of IFP 1.4 acceptor in the mPlum-IFP 1.4 fusion protein using confocal microscopy. (c) Quantification of fluorescent intensity of the mPlum donor before and after photobleaching of the acceptor in the mPlum-IFP 1.4 fusion protein and separately expressed mPlum and IFP 1.4 plasmids (mPlum + IFP). (d) Analysis of the distance between the donor and acceptor in relation to FRET efficiency in single cells. A higher FRET efficiency corresponds to a shorter distance between donor and acceptor. The color bars were shown on the upper-left of each picture. (e) Quantification of FRET efficiency and donor/acceptor distance in cells expressing mPlum-IFP 1.4 fusion protein or co-transfected mPlum and IFP 1.4 constructs (mPlum+IFP). *: p < 0.05.  
Effects of biliverdin (BV) on mPlum-IFP 1.4 fusion protein. (a) A dose dependent experiment demonstrated that BV increased the brightness of IFP 1.4 expressed in 293T cells detected using the in vivo imaging system (IVIS) 50 system (ex: 580 to 610 nm; em: 695 to 770 nm). Photon flux was quantified and compared across different concentrations of BV. **: p < 0.01 and ***: p < 0.005. (b) Comparison of the brightness of cells transfected with IFP 1.4, mPlum, and mPlum-IFP 1.4 fusion construct and exposed to the excitation wavelength at DsRed channel (ex: 580 to 610 nm) or Cy5.5 channel (615 to 665 nm) with and without BV (25 μM). (c) Quantification of photon flux in (b). *: p < 0.05.  
Imaging of mPlum-IFP 1.4 fusion protein expression in a xenograft tumor model. (a) Imaging of the tumor-bearing nude mice with and without BV individual excitation at DsRed channel or Cy5.5 channel (ex: 580 to 610 nm and 615 to 665 nm), and collected using the same Cy5.5 channel (em: 695 to 770 nm) via the IVIS 50 system. For each nude mouse, IFP 1.4 transfected 293T cells were subcutaneously implanted in the left thigh, and mPlum-IFP 1.4 fusion protein construct transfected cells were implanted in the right thigh. Control is the nude mouse without injection of cells. The regions of interests (ROIs) were indicated by circles. (b) Semi-quantification of the fluorescent signals by selecting ROIs in different conditions with duplicated experiments. *: p < 0.001. FP: mPlum-IFP 1.4 fusion protein.  
Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Förster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovativefluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo.
Block diagram of laser system for delivering 0.6-ms pulses at 1.54 ␮ m to skin. 
Block diagram of laser system for delivering Q -switched, 31-ns pulses at 1.54 ␮ m to skin. 
Thresholds for second- and third-degree burns versus spot size. 
Takata Model temperature rises for second-degree burns for various spot diameters. 
Pigskin showing Q -switched, 1.54- ␮ m superthreshold lesions 2 weeks postexposure. 
Er:glass lasers have been in operation with both long pulses (hundreds of microseconds) and Q-switched pulses (50 to 100 ns) for more than 35 yr. The ocular hazards of this laser were reported early, and it was determined that damage to the eye from the 1.54-microm wavelength occurred mainly in the cornea where light from this wavelength is highly absorbed. Research on skin hazards has been reported only in the past few years because of limited pulse energies from these lasers. Currently, however, with pulse energies in the hundreds of joules, these lasers may be hazardous to the skin in addition to being eye hazards. We report our minimum visible lesion (MVL) threshold measurements for two different pulse durations and three different spot sizes for the 1.54-microm wavelength using porcine skin as an in vivo model. We also compare our measurements to results from our model, based on the heat transfer equation and the rate process equation. Our MVL-ED50 thresholds for the long pulse (600 micros) at 24 h postexposure were measured to be 20, 8.1, and 7.4 J cm(-2) for spot diameters of 0.7, 1.0, and 5 mm, respectively. Q-switched laser pulses of 31 ns had lower ED50 (estimated dose for a 50% probability of laser-induced damage) thresholds of 6.1 J cm(-2) for a 5-mm-diam, top-hat spatial profile laser pulse.
We demonstrate two short-coherence-length, rare-earth-doped fiber optical sources for performing optical coherence tomography (OCT) in human tissue. The first source is a stretched-pulse, mode-locked Er-doped fiber laser with a center wavelength of 1.55 μm, a power of 100 mW, and a bandwidth of 80 nm. The second is a Tm-doped silica fiber fluorescent source emitting up to 7 mW of power at 1.81 μm with a bandwidth of 80 nm. The OCT imaging depth of penetration in in vitro human aorta is compared using these sources and conventional 1.3-μm sources. © 1998 Society of Photo-Optical Instrumentation Engineers.
Optical properties of cornea and sclera of the eye and their alterations under the effect of 1.56-μm laser radiation are studied. The laser settings corresponded to the laser treatment regimens used (1) to correct the shape of the cornea and change the refraction of the eye and (2) to improve the hydraulic permeability of the sclera in glaucoma cases. A fiber-optical system to investigate the dynamics of the reflected and transmitted scattered laser radiation and a setup with a double integrating sphere to determine the optical properties of the ocular tissues on the basis of the Monte-Carlo simulation of the propagation of light was used. When the radiation characteristics corresponded to the treatment regimens for correcting the shape of the cornea, no noticeable changes were detected in its optical properties. When irradiating the sclera in conditions corresponding to the treatment regimens for improving its hydraulic permeability, the optical characteristics of the tissue showed definite changes. The results obtained as to the dynamics of the optical signals during the course of laser irradiation of the cornea and sclera create prerequisites for designing test systems to be used with novel medical laser techniques for correcting visual abnormalities.
Two photon fluorescence images of cortical vasculature in a mouse brain. (a) 235 x-y frames from 60 μm above the cortical surface to 1110 μm below are taken at a depth increment of 5 μm. The depth increments in the stack are 20 μm from 1110 to 1490 μm and 30 μm from 1490 to 1670 μm. These increments are adjusted to keep the total imaging time at a practical limit while covering all the available depths. 3D reconstruction is made in IMAGE J software using the volume viewer plug-in. Expanded 3D stacks are shown for the deepest sections (> 1130 μm). (b) Four normalized x-y frames from the z-stack at various depths. 8, 16, 32, and 224 frames (at 1 frame/s acquisition speed) were averaged for the sections at imaging depths of 1110, 1310, 1470, and 1610 μm, respectively. Scale bars are 50 μm for both (a) and (b).  
Fluorescence intensity as a function of imaging depth for the stack shown in Fig. 1. Fluorescence signal strength at a particular depth is represented by the average value of the brightest 1% of the pixels in the x-y image at that depth.
Intensity line profiles across lateral blood vessels are used to characterize the SBR and resolution. (a) A sample x-y frame at 1010 μm depth, where a line profile (b) is taken along the dashed line. The scale bar is 50 μm. (b) The FWHM of the profiles presents an upper bound for the lateral resolution. Background is calculated by averaging the values between − 15 and − 5 μm and between 5 and 15 μm. (c) The SBR as a function of depth. Journal of Biomedical Optics October 2011 r Vol. 16(10) 106014-2  
Two photon fluorescence images of mouse cortical vasculature at approximately 2 mm posterior and lateral to bregma, where the cortical thickness is approximately 800 μm. (a) 161 x-y frames from 80 μm above the cortical surface to 725 μm below were taken at a depth increment of 5 μm. For the top 635-μm imaging depth, the excitation powers were adjusted so that approximately the same SNR can be achieved at a constant frame rate of 1 frame/s. At the imaging depths beyond 635 μm, the full output power of the OPO (120 mW at the brain surface) was used. Imaging integration times were then increased in accordance with the imaging depths to maintain reasonably constant SNRs throughout the imaging depth of 1.2 mm. The depth increments in the stack were 10 μm from 725 to 905 μm and 25 μm from 905 to 1230 μm. 3D reconstruction was made in IMAGE J software using the volume viewer plug-in. (b) Four normalized x-y frames from the z-stack at various depths. 16 frames (at 1 frame/s acquisition speed) were averaged for the sections at imaging depths of 805 and 905 μm. Thirty frames were averaged for the sections at imaging depths of 1005 and 1105 μm. Scale bars are 50 μm for both (a) and (b). (c) Fluorescence intensity as a function of imaging depth for the stack shown in (a). Fluorescence signal strength at a particular depth is represented by the average value of the brightest 1% of the pixels in the x-y image at that depth. For the first 800-μm imaging depth (approximately the cortical thickness), a constant fluorescence signal attenuation length of approximately 330 μm is observed. However, beyond 800 μm an abrupt change of the slope is present since the tissue beneath the gray matter is significantly more scattering. A rapid decrease of SBR and SNR is observed in x-y images at this region, as shown in (b).  
Deep tissue in vivo two-photon fluorescence imaging of cortical vasculature in a mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.6 mm in mouse cortex is achieved in vivo, approximately reaching the fundamental depth limit in scattering tissue.
In this paper we present the absorption coefficient mu(a) and the isotropic scattering coefficient mu(s)(') for 22 human skin samples measured using a double integrating sphere apparatus in the wavelength range of 1000-2200 nm. These in vitro results show that values for mua) follow 70% of the absorption coefficient of water and values for mu(s)(') range from 3 to 16 cm(-1). From the measured optical properties, it was found that a 2% Intralipid solution provides a suitable skin tissue phantom.
Photodynamic therapy (PDT) efficacy depends on the amount of light distribution within the tissue. However, conventional PDT does not consider the laser irradiation dose during PDT. The optical properties of biological tissues (absorption coefficient μ(a), reduced scattering coefficient μ's), anisotropy factor g, refractive index, etc.) help us to recognize light propagation through the tissue. The goal of this paper is to acquire the knowledge of the light propagation within tissue during and after PDT with the optical property of PDT-performed mouse tumor tissue. The optical properties of mouse tumor tissues were evaluated using a double integrating sphere setup and the algorithm based on the inverse Monte Carlo method in the wavelength range from 350 to 1000 nm. During PDT, the μ(a) and μ's were not changed after 1 and 5 min of irradiation. After PDT, the μ's in the wavelength range from 600 to 1000 nm increased with the passage of time. For seven days after PDT, the μ's increased by 1.7 to 2.0 times, which results in the optical penetration depth decreased by 1.4 to 1.8 times. To ensure an effective procedure, the adjustment of laser parameters for the decreasing penetration depth is recommended for the re-irradiation of PDT.
Terahertz corneal hydration sensing has shown promise in ophthalmology applications and was recently shown to be capable of detecting water concentration changes of about two parts in a thousand in ex vivo corneal tissues. This technology may be effective in patient monitoring during refractive surgery and for early diagnosis and treatment monitoring in diseases of the cornea. In this work, Fuchs dystrophy, cornea transplant rejection, and keratoconus are discussed, and a hydration sensitivity of about one part in a hundred is predicted to be needed to successfully distinguish between diseased and healthy tissues in these applications. Stratified models of corneal tissue reflectivity are developed and validated using ex vivo spectroscopy of harvested porcine corneas that are hydrated using polyethylene glycol solutions. Simulation of the cornea's depth-dependent hydration profile, from 0.01 to 100 THz, identifies a peak in intrinsic reflectivity contrast for sensing at 100 GHz. A 100 GHz hydration sensing system is evaluated alongside the current standard ultrasound pachymetry technique to measure corneal hydration in vivo in four rabbits. A hydration sensitivity, of three parts per thousand or better, was measured in all four rabbits under study. This work presents the first in vivo demonstration of remote corneal hydration sensing.
(a) The schematic of the system setup and (b) its measured sensitivity rolling-off curve versus imaging depth. The dashed line indicates the −6 dB level. CIR: circulator, OC: optical coupler, CL: collimating lens, FL: focusing lens, M: mirror, OL: objective lens, DG: diffraction grating, PC: polarization controller.  
Extended depth SDOCT versus a typical SSOCT (in air)
Typical OCT cross-sectional image acquired from corneo-scleral limbus at 120 kHz A-scan rate and 1050 nm wavelength. Insert is an enlarged view of the region marked with dashed square. CC: collector channel, TM: trabecular meshwork, SC: Schlemm's canal, SS: sclera spur, CB: ciliary body.  
3-D imaging of the whole anterior segment of the human eye with the EID-SDOCT at 120 kHz A-scan rate and 1050 nm wavelength. (a) 3-D rendering of the full anterior segment (2048 pixels × 2048 lines × 200 frames covering 12 × 18 × 18 mm 3 ), (b) typical cross-sectional image obtained by averaging three consecutive B-frames extracted from the volumetric dataset in (a). (c) shows the zoomed images from (b) for the subregions of (from left to right) the corneoscleral limbus, cornea and anterior and posterior parts of the crystalline lens. AR: angle recess, EP: epithelium, S: stroma, CP: lens capsule and CR: lens cortex.  
We demonstrate a 1050-nm spectral domain optical coherence tomography (OCT) system with a 12 mm imaging depth in air, a 120 kHz A-scan rate and a 10 μm axial resolution for anterior-segment imaging of human eye, in which a new prototype InGaAs linescan camera with 2048 active-pixel photodiodes is employed to record OCT spectral interferograms in parallel. Combined with the full-range complex technique, we show that the system delivers comparable imaging performance to that of a swept-source OCT with similar system specifications.
(a) Representative B-scan OCT/OMAG images resulting from 800 nm and (b) 1 μm (right) systems, indicating the microstructural images at the top and the blood flow images at the bottom. White bar = 500 μm. 
Depth resolved retinal blood vessel networks imaged by the 1-μm system from: (a) R1 layer (beyond 425 μm above the RPE layer); (b) R2 layer (between 300 and 425 μm above the RPE layer); (c) R3 layer (between 50 and 300 μm above the RPE layer). (d) Depth encoded color map to appreciate the vasculature located at different depths with red for (a), green for (b) and blue for (c). White bar = 500 μm 
Depth resolved choroidal blood perfusion maps from: (a) C1 layer; (b) C2 layer; and (c) C3 layer. (d) 3D visualization of choroidal vasculature. White bar = 500 μm. 
The light source at similar to 1-mu m wavelength is attractive for enhanced imaging depth in retinal optical coherence tomography (OCT). In this paper, we report on a 1050-nm spectral domain OCT system, combined with optical microangiography that operates at a 92-kHz line scan rate for multifunctional imaging of the human eye, delivering the volumetric imaging of microstructure and microvasculature within retina and choroid. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3582159]
Frequency domain optical coherence tomography (FD-OCT), based on an all-reflective high-speed InGaAs spectrometer, operating in the 1050 nm wavelength region for retinal diagnostics, enables high-speed, volumetric imaging of retinal pathologies with greater penetration into choroidal tissue is compared to conventional 800 nm three-dimensional (3-D) ophthalmic FD-OCT systems. Furthermore, the lower scattering at this wavelength significantly improves imaging performance in cataract patients, thereby widening the clinical applicability of ophthalmic OCT. The clinical performance of two spectrometer-based ophthalmic 3-D OCT systems compared in respect to their clinical performance, one operating at 800 nm with 150 nm bandwidth (approximately 3 microm effective axial resolution) and the other at 1050 nm with 70 nm bandwidth (approximately 7 microm effective axial resolution). Results achieved with 3-D OCT at 1050 nm reveal, for the first time, decisive improvements in image quality for patients with retinal pathologies and clinically significant cataract.
Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was recognized as regions of motion by creating differential phase-variance (DPV) tomograms: multiple B-scans of individual slices through the retina were collected and the variance of the phase differences was calculated. DPV captured the small vessels and the meshwork of capillaries associated with the inner retina in en-face images over 4 mm(2). The swept-source laser at 1060 nm offered the needed phase sensitivity to perform DPV and generated en-face images that capture motion in the inner choroidal layer exceeding the capabilities of previous spectrometer-based instruments. In comparison with the power Doppler phase-shift method, DPV provided better visualization of the foveal avascular zone in en-face images.
Set-up of FDML Source, SOA: semiconductor optical amplifier, FC: fiber coupler, DL: delay line, ISO: optical isolator, TF: tunable filter, PC: polarization controller, WFG: waveform generator, PD: photodetector.  
Spectral shaping of forward and backward sweep of the FDML source: (a) Instantaneous power of forward and backward sweep at constant SOA current (gray trace) and at modulated SOA current (black trace); (b) modulated SOA current for spectral shaping; (c) resulting point spread functions with constant or modulated SOA current.  
Polarization sensitive optical coherence tomography set-up used for all images acquired, PC: polarization control, POL: polarizer, DC: dispersion compensation, PBS: polarizing beam splitter, L1: lense f ¼ 80 mm, L2: lense f ¼ 50 mm. For further details see description in the text.
3-D data set of the central retinal region of a healthy human volunteer acquired in 0.65 s. (200 B-scans, 1000 A-scans per B-scan, scanning angle ∼32 deg ×32 deg): (a) En face intensity image (log scale), yellow line: B-scans shown in (c), (e), (g); red line: B-scans shown in (d), (f), (h); (b) En face retardation image (scale bar 0 deg to 60 deg); (c) and (d) Intensity B-scan single frames (log scale); (e) and (f) Retardation B-scan single frames (scale bar: 0 deg to 90 deg); (g) and (h) Axis orientation B-scan single frames (scale bar: 0 deg to 180 deg). B-scan dimensions: 32 deg (horizontal) ×1 mm (vertical; geometric distance).
Averaged 2-D data sets of the fovea of a healthy human volunteer. (50 frames averaged): (a) intensity image (log scale); (b) retardation image (scale bar 0 deg to 90 deg); (c) fast axis orientation (scale bar 0 deg to 180 deg); (d) degree of polarization uniformity B-scan (scale bar 0-1); (e) and (f) Segmentation of choroid based on PS-OCT data; (e) Intensity B-scans (red line: RPE, green line: CSI); (f) Retardation B-scans (top white line: RPE, bottom white line: CSI; scale bar 0 deg to 90 deg). B-scan dimensions: 32 deg (horizontal) ×1 mm (vertical; geometric distance).  
We present a novel, high-speed, polarization-sensitive, optical coherence tomography set-up for retinal imaging operating at a central wavelength of 1060 nm which was tested for in vivo imaging in healthy human volunteers. We use the system in combination with a Fourier domain mode locked laser with active spectral shaping which enables the use of forward and backward sweep in order to double the imaging speed without a buffering stage. With this approach and with a custom designed data acquisition system, we show polarization-sensitive imaging with an A-scan rate of 350 kHz. The acquired three-dimensional data sets of healthy human volunteers show different polarization characteristics in the eye, such as depolarization in the retinal pigment epithelium and birefringence in retinal nerve fiber layer and sclera. The increased speed allows imaging of large volumes with reduced motion artifacts. Moreover, averaging several two-dimensional frames allows the generation of high-definition B-scans without the use of an eye-tracking system. The increased penetration depth of the system, which is caused by the longer probing beam wavelength, is beneficial for imaging choroidal and scleral structures and allows automated segmentation of these layers based on their polarization characteristics.
To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.
Nonlinear optical phenomena, such as two-photon fluorescence (2PF) and second harmonic generation (SHG), in combination with voltage sensitive dyes, can be used to acquire high-resolution spatio temporal maps of electrical activity in excitable cells and tissue. Developments in 1064-nm fiber laser technology have simplified the generation of high-intensity, long-wavelength, femtosecond light pulses, capable of penetrating deep into tissue. To merge these two advances requires the design and synthesis of new dyes that are optimized for longer wavelengths and that produce fast and sensitive responses to membrane potential changes. In this work, we have systematically screened a series of new dyes with varying chromophores and sidechains that anchor them in cell membranes. We discovered several dyes that could potentially be used for in vivo measurements of cellular electrical activity because of their rapid and sensitive responses to membrane potential. Some of these dyes show optimal activity for SHG; others for 2PF. This regulated approach to dye screening also allows significant insight into the molecular mechanisms behind both SHG and 2PF. In particular, the differing patterns of sensitivity and kinetics for these two nonlinear optical modalities indicate that their voltage sensitivity originates from differing mechanisms.
Raman spectra of whole blood and oxy-hemoglobin (Hb) were measured under the same conditions with visible (514.5 nm) and near-infrared (NIR; 720 and 1064 nm) excitation, and the obtained spectra were compared in detail. The Raman spectrum of blood excited with visible light is dominated by very intense bands due to carotenoids, so that it was difficult to obtain information about Hb from the spectrum. The Raman spectra of whole blood and oxy-Hb excited with 720 nm light are very close to each other; both spectra are essentially Raman spectra of the heme chromophore that is preresonant with Q bands. Qualitative spectral analysis including band assignment and investigation of nature of resonance effect were carried out for the Raman spectra with 720 nm excitation. The spectra of whole blood and oxy-Hb excited with 1064 nm light contain contributions from nonresonance Raman spectra of the heme chromophore and Raman spectra of proteins. The 1064 nm excited spectra of blood and oxy-Hb are similar to each other but different in some features. For example, bands due to protein appear stronger in the spectrum of whole blood than in that of oxy-Hb which does not contain protein except globin part. The comparison between the 514.5, 720, and 1064 nm excited Raman spectra reveal that the excitation wavelength of 720 nm is more practical than that of visible light and 1064 nm in the Raman analysis of Hb, such as oxygenation, specially in situ measurement.
Lasers have shown great advantages in enhancing transdermal drug delivery. However, the physical or physiological mechanisms are not clear, which limits the application in clinical medicine. Here, 1064 nm-Nd:YAG lasers with long-pulsed (LP, 15 J/cm2) and Q-switched (QS, 0.5 J/cm2) output modes inducing short- and long-term effects on the stratum corneum (SC) of skin are investigated. Infrared thermography is applied to monitor the dynamical temperature distribution of the skin surface, while histopathological analysis and two-photon fluorescence microscopy are employed to examine changes in the microstructure of skin and molecular constitution of SC, respectively. Results have shown that the LP laser irradiation increases skin temperature evidently and loosens keratin, making corneocytes fragile or exfoliative, whereas the QS laser irradiation disrupts the keratin or corneocytes completely, perforating some micropores on the SC. It can be concluded that the mechanisms of enhancing transdermal delivery caused by lasers depends on the output modes. The LP laser irradiation produces thermal effects on skin, which loosens the SC, while the QS laser induces mechanical effects on skin, which punches micropores on the SC. Moreover, the laser-induced enhancing effects on transdermal glycerol delivery can last for one week to wait for the recovery of SC.
In the search for improved imaging modalities for detection and diagnosis of breast cancer, a high negative prediction value is also important. Photoacoustic (optoacoustic) imaging is a relatively new technique that has high potential for visualizing breast malignancies, but little is known about the photoacoustic appearance of benign lesions. In this work, we investigate the visibility of benign breast cysts in forward-mode photoacoustic mammography using 1064-nm light, as currently applied in the Twente photoacoustic mammoscope. Results from (Monte Carlo and k-wave) simulations and phantom measurements were used to interpret results from patient measurements. There was a strong agreement among the results from simulations, phantom, and patient measurements. Depending on the absorption contrast between cyst and breast tissue, cysts were visible as either one or two confined high-contrast areas representing the front and the back of the cyst, respectively. This edge enhancement is most likely the consequence of the local sudden change in the absorbed energy density and Grüneisen coefficients. Although the current forward-mode single-wavelength photoacoustic mammoscope cannot always unambiguously discriminate cysts from malignancies, this study reveals specific features of cysts compared to malignancies, which can be exploited for discrimination of the two abnormalities in future modifications of the imager.
The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.
An in vivo exposure to 197 W/cm(2) 1090-nm infrared radiation (IRR) requires a minimum 8 s for cataract induction. The present study aims to determine the ocular temperature evolution and the associated heat flow at the same exposure conditions. Two groups of 12 rats were unilaterally exposed within the dilated pupil with a close to collimated beam between lens and retina. Temperature was recorded with thermocouples. Within 5 min after exposure, the lens light scattering was measured. In one group, the temperature rise in the exposed eye, expressed as a confidence interval (0.95), was 11 +/- 3 degrees C at the limbus, 16 +/- 6 degrees C in the vitreous behind lens, and 16 +/- 7 degrees C on the sclera next to the optic nerve, respectively. In the other group, the temperature rise in the exposed eye was 9 +/- 1 degrees C at the limbus and 26 +/- 11 degrees C on the sclera next to the optic nerve, respectively. The difference of forward light scattering between exposed and contralateral not exposed eye was 0.01 +/- 0.09 tEDC. An exposure to 197 W/cm(2) 1090-nm IRR for 8 s induces a temperature increase of 10 degrees C at the limbus and 26 degrees C close to the retina. IRR cataract is probably of thermal origin. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.
The damage mechanism for near-infrared radiation (IRR) induced cataract is unclear. Both a photochemical and a thermal mechanism were suggested. The current paper aims to elucidate a photochemical effect based on investigation of irradiance-exposure time reciprocity. Groups of 20 rats were unilaterally exposed to 96-W/cm(2) IRR at 1090 nm within the dilated pupil accumulating 57, 103, 198, and 344 kJ/cm(2), respectively. Temperature was recorded at the limbus of the exposed eye. Seven days after exposure, the lenses were macroscopically imaged and light scattering was quantitatively measured. The average maximum temperature increases for exposure times of 10, 18, 33, and 60 min were expressed as 7.0 +/- 1.1, 6.8 +/- 1.1, 7.6 +/- 1.3, and 7.4 +/- 1.1 degrees C [CI (0.95)] at the limbus of the exposed eye. The difference of light scattering in the lenses between exposed and contralateral not-exposed eyes was 0.00 +/- 0.02, 0.01 +/- 0.03, -0.01 +/- 0.02, and -0.01 +/- 0.03 transformed equivalent diazepam concentration (tEDC), respectively, and no apparent morphological changes in the lens were observed. An exposure to 96-W/cm(2) 1090-nm IRR projected on the cornea within the dilated pupil accumulating radiant exposures up to 344 kJ/cm(2) does not induce cataract if the temperature rise at the limbus is <8 degrees C. This is consistent with a thermal damage mechanism for IRR-induced cataract. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.
The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 micros, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.
The intrinsic optical parameters-absorption coefficient mua, scattering coefficient mus, anisotropy factor g, and effective scattering coefficient mus'--are determined for human red blood cells of hematocrit 42.1% dependent on the shear rate in the wavelength range 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulation are carried out for different wall shear rates between 0 and 1000 s(-1). Randomly oriented cells show maximal mua, mus, and mus' values. Cell alignment and elongation, as well as the Fahraeus effect at increasing shear rates, lead to an asymptotical decrease of these values. The anisotropy factor shows this behavior only below 600 nm, dependent on absorption; above 600 nm, g is almost independent of shear rate. The decrease of mus' is inversely correlated with the hemoglobin absorption. Compared to randomly oriented cells, aggregation reduces all parameters by a different degree, depending on the hemoglobin absorption. It is possible to evaluate the influence of collective scattering phenomena, the absorption within the cell, and the cell shape.
The feasibility of in vivo measurements in the range of 1000 to 1100 nm and the potential benefits of operation in that wavelength range for diagnostic applications are investigated. To this purpose, an existing system for time-resolved diffuse spectroscopy is modified to enable in vivo studies to be carried out continuously from 600 to 1100 nm. The optical characterization of collagen powder is extended to 1100 nm and an accurate measurement of the absorption properties of lipid is carried out over the entire spectral range. Finally, the first in vivo absorption and scattering spectra of breast tissue are measured from 10 healthy volunteers between 600 and 1100 nm and tissue composition is evaluated in terms of blood parameters and water, lipid, and collagen content using a spectrally constrained global fitting procedure.
The absorption coefficient mu(a), scattering coefficient mu(s), and anisotropy factor g of diluted and undiluted human blood (hematocrit 0.84 and 42.1%) are determined under flow conditions in the wavelength range 250 to 1100 nm, covering the absorption bands of hemoglobin. These values are obtained by high precision integrating sphere measurements in combination with an optimized inverse Monte Carlo simulation (IMCS). With a new algorithm, appropriate effective phase functions could be evaluated for both blood concentrations using the IMCS. The best results are obtained using the Reynolds-McCormick phase function with the variation factor alpha = 1.2 for hematocrit 0.84%, and alpha = 1.7 for hematocrit 42.1%. The obtained data are compared with the parameters given by the Mie theory. The use of IMCS in combination with selected appropriate effective phase functions make it possible to take into account the nonspherical shape of erythrocytes, the phenomenon of coupled absorption and scattering, and multiple scattering and interference phenomena. It is therefore possible for the first time to obtain reasonable results for the optical behavior of human blood, even at high hematocrit and in high hemoglobin absorption areas. Moreover, the limitations of the Mie theory describing the optical properties of blood can be shown.
Schematic of a photoacoustic microscope equipped with a BaðNO 3 Þ 2 crystal-based Raman laser. M1, M2, M3, and M4: 45 deg 1064 nm reflective mirror. PBS: polarizing beam splitter; HWP: half wave plate; QWP: quarter wave plate; M5: resonator end mirror; M6: output coupler; M7: silver mirror; PC: computer.
Characteristics of the BaðNO 3 Þ 2 crystal-based Raman laser. (a) Spectral profile of the Raman laser output. (b) The 1st Stokes energy as a function of the pump energy incident on the Raman crystal. Red solid line is a linear fit. (c) Conversion efficiency with respect to the pump intensity incident on the Raman crystal. (d) Pulse energy of Raman laser as a function of time.
PA imaging of intramuscular fat performed with the Raman laser. (a) En face maximum intensity projection PA image of intramuscular fat sample with 1197 nm excitation. (b) En face maximum intensity projection PA image of intramuscular fat sample with 1064 nm excitation. (c) Histological evaluation of the same intramuscular fat sample. (d) Three-dimensional PA image of a separate intramuscular fat sample. Pulse energy: 60 J. Image size: 120 × 120 pixels.
Photoacoustic imaging employing molecular overtone vibration as a contrast mechanism opens a new avenue for bond-selective imaging of deep tissues. Broad use of this modality is, however, hampered by the extremely low conversion efficiency of optical parametric oscillators at the overtone transition wavelengths. To overcome such a barrier, we demonstrate the construction and use of a compact, barium nitrite crystal-based Raman laser for photoacoustic imaging of C-H overtone vibrations. Using a 5-ns Nd∶YAG laser as the pumping source, up to 21.4 mJ pulse energy at 1197 nm was generated, corresponding to a conversion efficiency of 34.8%. Using the 1197 nm pulses, three-dimensional photoacoustic imaging of intramuscular fat was demonstrated.
Photoacoustic microscopy has achieved submicron lateral resolution, but its axial resolution is much lower. Here an axial resolution of 7.6 μm, the highest axial resolution validated by experimental data, has been achieved by using a commercial 125 MHz ultrasonic transducer for signal detection followed by the Wiener deconvolution for signal processing. Limited by the working distance, the high-frequency ultrasonic transducer can penetrate 1.2 mm into biological tissue from the ultrasound detection side. At this depth, the signal-to-noise ratio decreases by 11 dB, and the axial resolution degrades by 36%. The new system was demonstrated in imaging melanoma cells ex vivo and mouse ears in vivo.
In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.
Optoacoustic (photoacoustic) imaging has already showcased the capacity to offer high-resolution small animal visualization in vivo in a variety of cancer, cardiovascular, or neuroimaging applications. In particular, multispectral optoacoustic tomography (MSOT) has shown imaging along the spectral and the time dimensions, enabling sensing of multiple molecules over time and, more recently, in real time. Furthermore, cross-sectional imaging of at least 20 mm diameter has been showcased in vivo in animals and humans using 64-element curved transducers placed along a single curved line. Herein, we investigated the imaging improvements gained by utilizing a larger number of detectors and inquired whether more detectors will result in measurable image quality improvements. For this reason, we implemented MSOT using 64-, 128-, and 256-element transducers and imaged the same phantoms and animals under similar conditions. Further, corroborated by numerical simulation analysis, our findings quantify the improvements in resolution and overall image quality for the increasing number of detectors used pointing to significant improvements in image quality for the 256 detector array, over 64 or 128 detectors.
We report the application of optical coherence tomography (OCT) to generate images of the remaining dentin and pulp chamber of in vitro human teeth. Bidimensional images of remaining dentin and of the pulp chamber were obtained parallel to the long axis of the teeth, by two OCT systems operating around 1280 and 850 nm, and compared to tomography images using the i-CAT(R) Cone Beam Volumetric Tomography system as the gold standard. The results demonstrated the efficacy of the OCT technique; furthermore, the wavelength close to 1280 nm presented greater penetration depth in the dentine than 850 nm, as expected from scattering and absorption coefficients. The OCT technique has great potential to be used on clinical practice, preventing accidental exposure of the pulp and promoting preventive restoration treatment.
The increased sensitivity of spectral domain optical coherence tomography (OCT) has driven the development of a new generation of technologies in OCT, including rapidly tunable, broad bandwidth swept laser sources and spectral domain OCT interferometer topologies. In this work, the operation of a turnkey 1300-nm swept laser source is demonstrated. This source has a fiber ring cavity with a semiconductor optical amplifier gain medium. Intracavity mode selection is achieved with an in-fiber tunable fiber Fabry-Perot filter. A novel optoelectronic technique that allows for even sampling of the swept source OCT signal in k space also is described. A differential swept source OCT system is presented, and images of in vivo human cornea and skin are presented. Lastly, the effects of analog-to-digital converter aliasing on image quality in swept source OCT are discussed.
(a) System setup where the cameras are externally triggered by the trigger-sequences shown in (b). (c) Measured system-sensitivity for each spectrometer. SLD: super luminescent diode, OC: optical circulator, PC: polarization controller, and M: mirror.  
Cross-sectional structure and phase-difference images obtained from the flow-phantom experiments. (a) and (b) SDOCT structural images resulting from the first and second spectrometers, respectively. (c) Structural image from the dual-spectrometer system. (d), (e) and (f) Corresponding PRODT phase difference maps evaluated from the system employing the first, second, and dual-spectrometers, respectively . (g) Phase-difference plot along the dashed line (see middle images ), passing through the center of the flow-tube. The solid curves are the parabolic fitting to the measured data.  
We propose a useful method to boost the imaging speed for spectral domain optical coherence tomography (SDOCT) by multiplying a number of high-speed spectrometers used in the system with selective precise control of data-recording and data-reading phases for spectral cameras employed in each spectrometer. To demonstrate the proposed method, we use two spectrometers built in a 1310 nm-band SDOCT system, each equipped with a high-speed InGaAs line-scan camera capable of 92-kHz line-scan rate, to achieve an unprecedented imaging speed at 184,000 lines/s. We validate the multiplied imaging speed by measuring Doppler-induced phase shift in the spectrograms using a flow phantom.
A fundamental understanding of how near-IR light propagates through sound and carious dental hard tissues is essential for the development of clinically useful optical diagnostic systems, since image contrast is based on changes in the optical properties of these tissues on demineralization. During the caries (decay) process, micropores are formed in the lesion due to partial dissolution of the individual mineral crystals. Such small pores behave as scattering centers, strongly scattering visible and near-IR light. The optical properties of enamel can be quantitatively described by the absorption and scattering coefficients, and the scattering phase function. Our aim is to measure the optical scattering behavior of natural and artificial enamel caries. Near-IR attenuation measurements and angular-resolved goniometer measurements coupled with Monte Carlo simulations are used to determine changes in the scattering coefficient and the scattering anisotropy on demineralization at 1310 nm. An ultra-high resolution digital microradiography system is used to quantify the lesion severity by measurement of the relative mineral loss for comparison with optical scattering measurements. The scattering coefficient increases exponentially with increasing mineral loss. Natural and artificial demineralization increases the scattering coefficient more than two orders of magnitude at 1310 nm, and the scattering is highly forward directed.
The high transparency of dental enamel in the near-infrared (NIR) at 1310 nm can be exploited for imaging dental caries without the use of ionizing radiation. The objective of this study is to determine whether the lesion contrast derived from NIR imaging in both transmission and reflectance can be used to estimate lesion severity. Two NIR imaging detector technologies are investigated: a new Ge-enhanced complementary metal-oxide-semiconductor (CMOS)-based NIR imaging camera, and an InGaAs focal plane array (FPA). Natural occlusal caries lesions are imaged with both cameras at 1310 nm, and the image contrast between sound and carious regions is calculated. After NIR imaging, teeth are sectioned and examined using polarized light microscopy (PLM) and transverse microradiography (TMR) to determine lesion severity. Lesions are then classified into four categories according to lesion severity. Lesion contrast increases significantly with lesion severity for both cameras (p<0.05). The Ge-enhanced CMOS camera equipped with the larger array and smaller pixels yields higher contrast values compared with the smaller InGaAs FPA (p<0.01). Results demonstrate that NIR lesion contrast can be used to estimate lesion severity.
Top-cited authors
Caglar Elbuken
  • University of Oulu
Carolyn L Ren
  • University of Waterloo
J.P. Huissoon
  • University of Waterloo
Konstantin Maslov
  • Washington University in St. Louis
Ruikang Wang
  • University of Washington Seattle