Use of nanosecond excitation pulses in fluorescence lifetime measurement via phasor analysis.

We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52-38 ns) and modulation frequencies (10-200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.

Intravascular polarization-sensitive optical coherence tomography based on polarization mode delay.

Intravascular polarization-sensitive optical coherence tomography (IV-PSOCT) provides depth-resolved tissue birefringence which can be used to evaluate the mechanical stability of a plaque. In our previous study, we reported a new strategy to construct polarization-sensitive optical coherence tomography in a microscope platform. Here, we demonstrated that this technology can be implemented in an endoscope platform, which has many clinical applications. A conventional intravascular OCT system can be modified for IV-PSOCT by introducing a 12-m polarization-maintaining fiber-based imaging probe. Its two polarization modes separately produce OCT images of polarization detection channels spatially distinguished by an image separation of 2.7 mm. We experimentally validated our IV-PSOCT with chicken tendon, chicken breast, and coronary artery as the image samples. We found that the birefringent properties can be successfully visualized by our IV-PSOCT.

Achieving a high photon count rate in digital time-correlated single photon counting using a hybrid photodetector.

We report an enhanced photon count rate in a digitally implemented time-correlated single-photon counting (TCSPC) system by utilizing a hybrid photodetector (HPD). In our digital TCSPC scheme, the photoelectronic responses from a single photon-sensitive photodetector are digitally analyzed through a high-speed analog-to-digital convertor (ADC). By virtue of the HPD which provides nearly a constant signal gain, the single-photon pulses can be effectively distinguished from pulses of simultaneously detected multiple photons by the pulse heights. Consequently, our digital TCSPC system can selectively collect single-photon signals even in the presence of intense multi-photon detections with its temporal accuracy not to be compromised. In our experiment of fluorescence lifetime measurement, the maximum count rate of single photons nearly reached the theoretical limit given by the Poisson statistics. This demonstrated that the digital TCSPC combined with the HPD provides an ultimate solution for the TCSPC implementation for high photon count rates.

Ultrahigh-sensitive optical coherence elastography.

The phase stability of an optical coherence elastography (OCE) system is the key determining factor for achieving a precise elasticity measurement, and it can be affected by the signal-to-noise ratio (SNR), timing jitters in the signal acquisition process, and fluctuations in the optical path difference (OPD) between the sample and reference arms. In this study, we developed an OCE system based on swept-source optical coherence tomography (SS-OCT) with a common-path configuration (SS-OCECP). Our system has a phase stability of 4.2 mrad without external stabilization or extensive post-processing, such as averaging. This phase stability allows us to detect a displacement as small as ~300 pm. A common-path interferometer was incorporated by integrating a 3-mm wedged window into the SS-OCT system to provide intrinsic compensation for polarization and dispersion mismatch, as well as to minimize phase fluctuations caused by the OPD variation. The wedged window generates two reference signals that produce two OCT images, allowing for averaging to improve the SNR. Furthermore, the electrical components are optimized to minimize the timing jitters and prevent edge collisions by adjusting the delays between the trigger, k-clock, and signal, utilizing a high-speed waveform digitizer, and incorporating a high-bandwidth balanced photodetector. We validated the SS-OCECP performance in a tissue-mimicking phantom and an in vivo rabbit model, and the results demonstrated a significantly improved phase stability compared to that of the conventional SS-OCE. To the best of our knowledge, we demonstrated the first SS-OCECP system, which possesses high-phase stability and can be utilized to significantly improve the sensitivity of elastography.

Digital implementation of TCSPC.

We present a digital instrumentation method of time-correlated single-photon counting (TCSPC). The pulsed signal of a single-photon sensitive photodetector is digitized by a high-speed analog-to-digital converter and digitally processed for determination of the photon detection times. We found that our digitally implemented TCSPC (dTCSPC) provides a smart way of discriminating valid photon pulses for the reliable measurement of fluorescence lifetimes and time-resolved spectroscopy.

Fiber-based polarization-sensitive optical coherence tomography of a minimalistic system configuration.

We present a very simple method of constructing a polarization-sensitive optical coherence tomography (PS-OCT) system. An ordinary fiber-based swept-source OCT system was reconfigured for PS-OCT by adding a long section of polarization-maintaining fiber in the sample arm. Two polarization modes of a large group-delay difference formed spatially distinguished polarization channels. The depth-encoded information on the polarization states was retrieved by an amplitude-based analysis. We found that our method provides an economic scheme of PS-OCT. It demonstrates that an ordinary OCT system can be easily reconfigured for PS-OCT imaging if it has sufficient margins in the imaging range.

Design of an Optical Probe to Monitor Vaginal Hemodynamics during Sexual Arousal.

An optical probe was developed to measure the change of oxy-hemoglobin (OHb), deoxy- hemoglobin (RHb), and total hemoglobin (THb) along with temperature from the vaginal wall of female rats. Apomorphine (APO, 80 µg/kg) was administered to elicit sexual arousal in female Sprague Dawley rats (SD, 180-200 g). The behavior changes caused by APO administration were checked before monitoring vaginal responses. The changes of oxy-, deoxy-, and total hemoglobin concentration and the temperature from the vaginal wall were monitored before, during, and after APO administration. Animals were under anesthesia during the measurement. After APO administration, the concentration of OHb (55 ± 29 µM/DPF), RHb (33 ± 25 µM/DPF), and THb (83 ± 59 µM/DPF) in the vaginal wall increased in a few min, while saline administration did not cause any significant change. In case of the vaginal temperature change, APO decreased the temperature slightly in the vaginal wall while saline administration did not show any temperature change in the vaginal wall. As the outcomes demonstrated, the developed probe can detect hemodynamic and temperature variation in the vaginal wall. The hemodynamic information acquired by the probe can be utilized to establish an objective and accurate standard of female sexual disorders.

Optically tunable fiber Bragg grating based on a photo-mechanical tuning mechanism.

We present an optically tunable fiber Bragg grating (FBG) based on a photo-mechanical tuning mechanism. Azobenzene containing polymer was utilized as a photo-mechanical tuning agent coated over a bare section of FBG. Controlled by ultraviolet (UV) irradiation, the polymer coating acts as a micro-actuator that deforms the fiber structure. Reversible tunability was obtained by the coating locally stretching the embedded FBG fiber by the intensity and the spatial distribution of the UV light. We found that our tunable FBG could linearly tune its center wavelength as large as 2.2 nm in a symmetric UV irradiation. We also found that a chirp of grating periods could be produced by an asymmetric irradiation, where the center of the UV irradiation profile is offset to that of the FBG device. The spectral width measured at 10 dB could be broadened from 0.4 to 1.2 nm by simply adjusting the relative position of the UV light source. The direction of the chirp, in either normal or anomalous dispersion, could be easily switched as well. Our observations demonstrated that our device provides a versatile means of tunable filters or dispersion generators, which is useful in various applications.

Effect of Air Injection Depth on Big-bubble Formation in Lamellar Keratoplasty: an Ex Vivo Study.

This study evaluated the effect of air injection depth in the big-bubble (BB) technique, which is used for corneal tissue preparation in lamellar keratoplasty. The BB technique was performed on ex vivo human corneoscleral buttons using a depth-sensing needle, based on optical coherence tomography (OCT) imaging technology. The needle tip, equipped with a miniaturized OCT depth-sensing probe, was inserted for air injection at a specified depth. Inside the corneal tissue, our needle obtained OCT line profiles, from which residual thickness below the needle tip was measured. Subjects were classified into Groups I, II, III, and IV based on injection depths of 75-80%, 80-85%, 85-90%, and >90% of the full corneal thickness, respectively. Both Type I and II BBs were produced when the mean residual thicknesses of air injection were 109.7 ± 38.0 µm and 52.4 ± 19.2 µm, respectively. Type II BB (4/5) was dominant in group IV. Bubble burst occurred in 1/16 cases of type I BB and 3/16 cases of type II BB, respectively. Injection depth was an important factor in determining the types of BBs produced. Deeper air injection could facilitate formation of Type II BBs, with an increased risk of bubble bursts.

Phase-stability optimization of swept-source optical coherence tomography.

Phase-resolved imaging of swept-source optical coherence tomography (SS-OCT) is subject to phase measurement instabilities involved with the sweep variation of a frequency-swept source. In general, optically generated timing references are utilized to track the variations imposed on OCT signals. But they might not be accurately synchronized due to relative time delays. In this research, we investigated the impact of the signal delays on the timing instabilities and the consequent deviations of the measured phases. We considered two types of timing signals utilized in a popular digitizer operation mode: a sweep trigger from a fiber Bragg grating (FBG) that initiates a series of signal sampling actions clocked by an auxiliary Mach-Zehnder interferometer (MZI) signal. We found that significant instabilities were brought by the relative delays through incoherent timing corrections and timing collisions between the timing references. The best-to-worst ratio of the measured phase errors was higher than 200 while only the signal delays varied. Noise-limited phase stability was achieved with a wide dynamic range of OCT signals above 50 dB in optimized delays. This demonstrated that delay optimization is very effective in phase stabilization of SS-OCT.

Characterization of spectral-domain OCT with autocorrelation interference response for axial resolution performance.

We present a class of novel system characterization methods for spectral-domain optical coherence tomography (SD-OCT) particularly on getting optimized axial resolution performance. Our schemes uniquely utilize the autocorrelation interference response, also known as the self-interference product, which is generated by the optical fields from the imaging sample in automatic interferences. In our methods, an autocorrelation-inducing calibration sample was prepared which was made by sandwiching glass plates. OCT images of the calibration sample were captured by an SD-OCT system under testing. And the image data were processed to find various system characteristics based on the unique properties of autocorrelation interferograms, free of dispersion- and polarization-involved modulations. First, we could analyze the sampling characteristic of the SD-OCT's spectrometer for spectral calibration that enables accurate linear-k resampling of detected spectral fringes. Second, we could obtain the systematic polarization properties for quantifying their impact on the achieved axial resolutions. We found that our methods based on the autocorrelation response provide an easy way of self-characterization and self-validation that is useful in optimizing and maintaining axial resolution performances. It was found very attractive that a variety of system characteristics can be obtained in a single-shot measurement without any increased system complexity.

Lamellar keratoplasty using position-guided surgical needle and M-mode optical coherence tomography.

Deep anterior lamellar keratoplasty (DALK) is an emerging surgical technique for the restoration of corneal clarity and vision acuity. The big-bubble technique in DALK surgery is the most essential procedure that includes the air injection through a thin syringe needle to separate the dysfunctional region of the cornea. Even though DALK is a well-known transplant method, it is still challenged to manipulate the needle inside the cornea under the surgical microscope, which varies its surgical yield. Here, we introduce the DALK protocol based on the position-guided needle and M-mode optical coherence tomography (OCT). Depth-resolved 26-gage needle was specially designed, fabricated by the stepwise transitional core fiber, and integrated with the swept source OCT system. Since our device is feasible to provide both the position information inside the cornea as well as air injection, it enables the accurate management of bubble formation during DALK. Our results show that real-time feedback of needle end position was intuitionally visualized and fast enough to adjust the location of the needle. Through our research, we realized that position-guided needle combined with M-mode OCT is a very efficient and promising surgical tool, which also to enhance the accuracy and stability of DALK.

VCSEL-based swept source for low-cost optical coherence tomography.

We present a novel wavelength-swept laser source for optical coherence tomography (OCT) which is based on the conventional laser diode technology of the vertical-cavity surface-emitting laser (VCSEL). In our self-heating sweep VCSEL (SS-VCSEL), a VCSEL device is simply driven by ramped pulses of currents in direct intensity modulation. The intrinsic property of VCSEL produces a frequency-swept output through the self-heating effect. By the injected current, the temperature of the active region is gradually increased in this effect. Consequently, it changes the wavelength of the laser output by itself. In this study, various characteristics of our SS-VCSEL were experimentally investigated for low-cost instrumentation of a swept source OCT system. A low-cost SS-VCSEL-based OCT system was demonstrated in this research that provided an axial resolution of 135 µm in air, sensitivity of -91 dB and a maximum imaging range longer than 10 cm when our source was operated at a sweep repetition rate of 5 kHz with an output power of 0.41 mW. Based on the experimental observations, we believe that our SS-VCSEL swept source can be an economic alternative in some of low-cost or long-range applications of OCT.

Ultra-thin and flexible endoscopy probe for optical coherence tomography based on stepwise transitional core fiber.

We present an ultra-thin fiber-body endoscopy probe for optical coherence tomography (OCT) which is based on a stepwise transitional core (STC) fiber. In a minimalistic design, our probe was made of spliced specialty fibers that could be directly used for beam probing optics without using a lens. In our probe, the OCT light delivered through a single-mode fiber was efficiently expanded to a large mode field of 24 µm diameter for a low beam divergence. The size of our probe was 85 µm in the probe's diameter while operated in a 160-µm thick protective tubing. Through theoretical and experimental analyses, our probe was found to exhibit various attractive features in terms of compactness, flexibility and reliability along with its excellent fabrication simplicity.

Dual-wavelength diffraction phase microscopy for simultaneous measurement of refractive index and thickness.

We present a quantitative phase microscopy scheme that simultaneously acquires two phase images at different wavelengths. The simultaneous dual-wavelength measurement was performed with a diffraction phase microscope (DPM) based on a transmission grating and a spatial filter that form a common-path imaging interferometer. With a combined laser source that generates two-color light continuously, a different diffraction order of the grating was utilized for each wavelength component so that the dual-wavelength interference pattern could be distinguished by the distinct fringe frequencies. Our dual-wavelength phase imaging allowed us to extract information on the physical thickness and the refractive index for a specimen immersed in a highly dispersive surrounding medium. We found that our dual-wavelength DPM (DW-DPM) provides an accurate measurement of the volume and the refractive index of a microscopy sample with good measurement stability that results from the common-path geometry.

Frequency-domain acquisition of fourth-order correlation by spectral intensity interferometry.

We report on the spectral intensity interferometer (SII) which is a frequency-domain variant of the fourth-order interferometry. In the SII, the power spectrum of the intensity is acquired for light fields of an interferometer. It produces a fringed spectral interferogram which can be acquired by means of an electric spectrum analyzer in keeping the relative time delay constant during the acquisition. Through both theoretical and experimental investigations, we have found that the SII interferogram provides the intensity correlation information without concern of field-sensitive disturbances which are vulnerable to minute variations of the optical paths. As an application example, a precision time-of-flight measurement was demonstrated by using a fiber-optic SII with an amplified spontaneous emission (ASE) light source. A large delay of 4.1-km long fiber was successfully analyzed from the fringe period. Its wavelength-dependent group delay or the group velocity dispersion (GVD) was also measured from the phase shift of the cosine fringe with a sub-picosecond delay precision.

Lens-free endoscopy probe for optical coherence tomography.

We present an ultrathin fiber-optic endoscopy probe for optical coherence tomography (OCT), which is made of a series of fused optical fibers instead of the conventional scheme based on an objective lens. The large-core fiber with a core diameter of 20 µm was utilized for the probe, while a single-mode fiber of core diameter 8.2 µm mainly delivered the OCT light. Those fibers were spliced with a bridge fiber of an intermediate core size. The guided light was stepwise converted to a beam of a large mode-field diameter to be radiated with a larger depth of focus. We obtained a 125 µm thick all-fiber endoscopy probe with a side-viewing capability implemented by an angled fiber end. Successful OCT imaging was demonstrated with a swept-source OCT system and showed the practical applicability of our lens-free endoscopy probe.

Mode-filtered large-core fiber for optical coherence tomography.

We have investigated the use of multimode fiber in optical coherence tomography (OCT) with a mode filter that selectively suppresses the power of the high-order modes (HOMs). A large-core fiber (LCF) that has a moderate number of guiding modes was found to be an attractive alternative to the conventional single-mode fiber for its large mode area and the consequentially wide Rayleigh range of the output beam if the HOMs of the LCF were efficiently filtered out by a mode filter installed in the middle. For this, a simple mode filtering scheme of a fiber-coil mode filter was developed in this study. The LCF was uniformly coiled by an optimal bend radius with a fiber winder, specially devised for making a low-loss mode filter. The feasibility of the mode-filtered LCF in OCT imaging was tested with a common-path OCT system. It has been successfully demonstrated that our mode-filtered LCF can provide a useful imaging or sensing probe without an objective lens that greatly simplifies the structure of the probing optics.

Mode-filtered large-core fiber for short-pulse delivery with reduced nonlinear effects.

We present a large-core fiber (LCF) with a reduced nonlinear property for a single-mode beam delivery of intense ultrashort pulses. A tapered-fiber mode filter was fabricated in an LCF with the core diameter decreased from 20 µm to 6 µm at the tapered waist region surrounded by index-matched liquid. By the tapered geometry, the high-order mode was rejected so that our mode-filtered LCF acted as a single-mode fiber despite the multimode property of the original LCF. It has been found that this fiber class is suitable for applications, such as an endoscopic multiphoton microscope, that demand a flexible short-distance (<4 m) delivery medium of ultrashort pulses.

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope.

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.