Novel cell culture system for monitoring cells during continuous and variable negative-pressure wound therapy.

BACKGROUND: Although the clinical efficacy of negative-pressure wound therapy (NPWT) is well known, many of its molecular biological mechanisms remain unresolved, mainly due to the difficulty and paucity of relevant in vitro studies. We attempted to develop an in vitro cell culture system capable of real-time monitoring of cells during NPWT treatment. MATERIALS AND METHODS: A novel negative-pressure cell culture system was developed by combining an inverted microscope, a stage-top incubator, a sealed metal chamber for cell culture, and an NPWT treatment device. Human keratinocytes, PSVK-1, were divided into ambient pressure (AP), continuous negative-pressure (NPc), and intermittent negative-pressure (NPi) groups and cultured for 24 h with scratch assay using our real-time monitoring system and device. Pressure inside the device, medium evaporation rate, and the residual wound area were compared across the groups. RESULTS: Pressure in the device was maintained at almost the same value as set in all groups. Medium evaporation rate was significantly higher in the NPi group than in the other two groups; however, it had negligible effect on cell culture. Residual wound area after 9 h evaluated by the scratch assay was significantly smaller in the NPc and NPi groups than in the AP group. CONCLUSION: We developed a negative-pressure cell culture device that enables negative-pressure cell culture under conditions similar to those used in clinical practice and is able to monitor cells under NPWT. Further experiments using this device would provide high-quality molecular biological evidence for NPWT.

Negative-Pressure Wound Therapy: What We Know and What We Need to Know.

Negative-pressure wound therapy (NPWT) promotes wound healing by applying negative pressure to the wound surface. A quarter of a century after its introduction, NPWT has been used in various clinical conditions, although molecular biological evidence is insufficient due to delay in basic research. Here, we have summarized the history of NPWT, its mechanism of action, what is currently known about it, and what is expected to be known in the future. Particularly, attention has shifted from the four main mechanisms of NPWT to the accompanying secondary effects, such as effects on various cells, bacteria, and surgical wounds. This chapter will help the reader to understand the current status and shortcomings of NPWT-related research, which could aid in the development of basic research and, eventually, clinical use with stronger scientific evidence.

Artificially Created Reentry Circuit by Laser Irradiation Causes Atrial Tachycardia to Persist in Murine Atria.

BACKGROUND: There is a gradual progression from paroxysmal to persistent atrial fibrillation (AF) in humans. To elucidate the mechanism involved, the creation of an artificial atrial substrate to persist AF in mice was attempted.Methods and Results:This study used wild type (WT) mice, but it is difficult to induce AF in them. A novel antegrade perfusion method from the left ventricle (LV) to enlarge both atria for artificial atrial modification was proposed in this study. Short duration AF was induced by burst pacing under this method. Optical mapping analysis revealed non-sustained focal type and meandering spiral reentrants after short duration AF. A tiny artificial substrate (~1.2 mm in diameter) was added in by laser irradiation to create a critical atrial arrhythmogenic substrate. Burst pacing was performed in a non-laser group (n=8), a circular-shape laser group (n=8), and a wedge-shaped dent laser group (n=8). We defined AF and atrial tachycardia (AT) as atrial arrhythmia (AA). Long-lasting AA was defined as lasting for ≥30 min. Long-lasting AA was observed in 0/8, 0/8, and 6/8 (75%) mice in each group. Optical mapping analysis revealed that the mechanism was AT with a stationary rotor around the irradiated margin. CONCLUSIONS: Regrettably, this study failed to reproduce persistent AF, but succeeded in creating an arrhythmic substrate that causes sustained AT in WT mice.

Ability of photocurable gelatin to prevent stricture recurrence after urethral dilation in rabbits.

OBJECTIVES: To evaluate the ability of photocurable gelatin to prevent stricture recurrence after urethral dilation in a rabbit urethral stricture model. METHODS: We created urethral strictures in the bulbar urethras of 10 male Japanese white rabbits using electrocoagulation. After 1 month, the rabbits were randomly divided into Group A (n = 5; urethral stricture dilation and the local application of photocurable gelatin using a ruthenium photoinitiator and irradiation with a light-emitting diode light [λ = 455 nm, 50 mW/cm2 ] for 1 min) and Group B (n = 5; dilation only). Urethral stricture status was evaluated 1-2 months later by retrograde urethrography and urethroscopy. The lumen ratio (urethral width at the stricture site to the normal urethral width on retrograde urethrography) was calculated. Urethral patency was considered to be improved when the urethral lumen could accommodate a 10-Fr urethroscope without resistance. Urethral specimens were harvested for histopathological examination. RESULTS: The mean lumen ratio did not differ significantly between Groups A and B before dilation (25.8% vs 23.4%; P = 0.40), but differed significantly after dilation (65.5% vs 27.3%, respectively; P = 0.03). Urethral patency improved in all rabbits in Group A (100%) versus one rabbit in Group B (20%; P = 0.02). The mean circumference of the regenerated urethral epithelium at the stricture site was larger in Group A than in Group B (14 mm vs 6.6 mm; P = 0.06). CONCLUSIONS: Photocurable gelatin can reduce urethral stricture recurrence after dilation in a rabbit model.

Chromatic-aberration-free multispectral optical-resolution photoacoustic microscopy using reflective optics and a supercontinuum light source.

A supercontinuum (SC) light source enables multispectral photoacoustic imaging at excitation wavelengths in the visible-to-near-infrared range. However, for such a broad optical wavelength range, chromatic aberration is non-negligible. We developed a multispectral optical-resolution photoacoustic microscopy (MS-OR-PAM) setup with a nanosecond pulsed SC light source and a reflective objective lens to avoid chromatic aberration. Chromatic aberrations generated by reflective and conventional objective lenses were compared, and the images acquired using the reflective objective were not affected by chromatic aberration. Hence, MS-OR-PAM with the reflective objective was used to distinguish red blood cells from melanoma cells via spectral subtraction processing.

Insulin-like growth factor 1 sustained-release collagen on urethral catheter prevents stricture after urethral injury in a rabbit model.

OBJECTIVES: To evaluate the preventive effect of an insulin-like growth factor 1 sustained-release collagen urethral catheter on urethral stricture after urethral injury in a rabbit model. METHODS: We made urethral catheters coated either with insulin-like growth factor 1 impregnated collagen or with only collagen, and we divided 19 male Japanese white rabbits into three groups according to the kind of catheter inserted immediately after the rabbit's urethra was injured by electrocoagulation. Group 1 (n = 7) had a catheter coated with insulin-like growth factor 1 impregnated collagen inserted; group 2 (n = 7) had a catheter coated with only collagen inserted; and group 3 (n = 5) had an uncoated catheter inserted. A total of 14 days later, the injured urethras were evaluated by urethrography and urethroscopy, and were also histologically examined. RESULTS: Urethrography showed that the ratio of the urethral lumen diameter in injured urethra to that in normal urethra was the largest in group 1 (P < 0.0001). In addition, five of the seven rabbits in group 1 (71.4%) had a urethral lumen large enough for passage of a urethroscope, a fraction larger than the corresponding fractions in groups 2 (57.1%) and 3 (20%). On histological analysis, the injured area not covered with regenerated urethral epithelium tended to be smaller in group 1 than the other two groups, but the mean difference was not significant (P = 0.19). CONCLUSIONS: An insulin-like growth factor 1 sustained-release collagen urethral catheter significantly improves wound healing and prevents urethral stricture after urethral injury.

Measurement of blood-oxygen saturation using a photoacoustic technique in the rabbit hypoxemia model.

The golden standard method to obtain accurate blood oxygen saturation is blood gas analysis that needs invasive procedure of blood sampling. Photoacoustic technique enables us to measure real-time blood oxygen saturation without invasive procedure. The aim of this study is to use the photoacoustic technique, an optical method, for accurately determining oxygen saturation in vivo. We measured induced photoacoustic signals of arterial blood in the rabbit model of stable hypoxemia after irradiation at 750 and 800 nm. Oxygen saturation was calculated from the photoacoustic signals using two calibration curves. Calibration curve 1 is a conventional curve derived from the absorbance coefficient of hemoglobin, whereas calibration curve 2 is derived from the photoacoustic signals obtained from the original blood vessel model. Simultaneously, blood-gas analysis was performed to obtain the reference standard of oxygen saturation. Regression analysis and Bland-Altman analysis were performed to assess the accuracy of oxygen saturation obtained using the two methods. The oxygen saturation calculated using calibration curves 1 and 2 showed strong correlations with the reference standard in regression analysis (R = 0.965, 0.964, respectively). The Bland-Altman analysis revealed better agreement and precision with calibration curve 2, whereas there was significant underestimation of values obtained using calibration curve 1. Photoacoustic measurement of oxygen saturation using calibration curve 2 provided an accurate estimate of oxygen saturation, which was similar to that obtained using a portable blood-gas analyzer.

Spectral-differential-based unmixing for multispectral photoacoustic imaging.

We propose the use of a spectral differential method (SDM) to emphasize the spectral peaks of multispectral photoacoustic images. Because contrast agent signals have spectral peaks at the contrast agent absorption peak, the SDM can selectively emphasize contrast agent signals. Unlike the conventional spectral fitting method (SFM), the SDM does not require reference background spectra and, consequently, does not suffer from separation error caused by the deviation of reference spectra from the measured spectra. We performed multispectral photoacoustic imaging of tissue-mimicking phantoms and subcutaneous tumors of mice injected with small organic molecule-based contrast agents. Contrast agent images obtained by the SDM were clearer than those obtained by SFM.

A pilot study of photoacoustic imaging system for improved real-time visualization of neurovascular bundle during radical prostatectomy.

BACKGROUND: Photoacoustic imaging, a noninvasive imaging based on optical excitation and ultrasonic detection, enables one to visualize the distribution of hemoglobin and acquire a map of microvessels without using contrast agents. We examined whether it helps visualize periprostatic microvessels and improves visualization of the neurovascular bundle. METHODS: We developed a photoacoustic imaging (PAI) system with a hand-held probe combining optical illumination and a conventional linear array ultrasound probe. In experiments with a phantom model, it was able to visualize vessels with diameters as small as 300 µm within a depth of 10 mm. We also developed a TRUS type probe for our photoacoustic imaging system and used it to intraoperatively monitor periprostatic tissues in seven patients with clinically organ-confined prostate cancer who were undergoing non-nerve-sparing retropubic radical prostatectomy. Images of periprostatic tissues from resected prostatectomy specimens were also obtained using the linear photoacoustic probe, and the consistency of the microvessel distribution and co-existence of nerve fibers was examined by double immunostaining of paraffin-embedded sections with anti-CD31 and anti-S-100 antibodies. RESULTS: Intraoperative monitoring of periprostatic tissues with the TRUS photoacoustic probe showed substantial signals on the posterolateral surface of the prostate and clearly demonstrated the location and extent of the neurovascular bundle better than does TRUS alone. Photoacoustic images of the periprostatic tissues in resected specimens also showed substantial signals that were especially strong on the posterolateral surface of the prostate. Nerve fibers were closely co-localized with periprostatic microvessels and the pattern of their distribution was consistent with that of PAI signals. CONCLUSIONS: The intraoperative photoacoustic imaging located the microvascular complex in the neurovascular bundle. Moreover, the neurovascular bundle was easier to identify by PAI than by TRUS alone, suggesting that PAI could be helpful in nerve-sparing radical prostatectomy.

Appropriate timing of blood sampling for blood gas analysis in the ventilated rabbit.

BACKGROUND: Arterial and venous blood gas analyses (BGAs) are essential to evaluate devices that measure biological oxygenation. The appropriate timing of blood sampling for BGA after respiratory rate (RR) change in animal experiments has not been reported. This study investigated the appropriate timing of blood sampling for BGA in ventilated rabbits and whether venous samples are an alternative to arterial samples. MATERIALS AND METHODS: Under general anesthesia, 14 rabbits (body weight, 3.02 ± 0.09 kg) were ventilated and their RR was changed (40/min, 30/min, and 20/min). Blood was sampled through cervical arterial and venous catheters. Experiment 1: in seven rabbits, arterial BGA was measured at 0, 0.5, 1, 2, 3, 5, 10, 15, and 20 min after the RR change. Experiment 2: in seven different rabbits, simultaneous arterial and venous BGA were measured at 0, 2, 5, 10, 15, and 20 min after the RR change. RESULTS: Oxygen partial pressure (PO2) and saturation (SO2) of the arterial blood stabilized 0.5 min after the RR changed. In venous BGA, no index stabilized during observation. The arterial and venous values of the carbon dioxide partial pressure (PCO2) and pH had significant correlations (arterial PCO2 = 0.9316 × venous PCO2-4.4425 [r = 0.9178]; arterial pH = 1.0835 × venous pH-0.5795 [r = 0.9453]). CONCLUSIONS: In ventilated rabbits, arterial PO2 and SO2 stabilized in 0.5 min. No venous value stabilized after the RR change. Only the PCO2 and pH of venous samples may be an alternative to arterial samples under the defined formula.

Improved angiogenesis and healing in crush syndrome by fibroblast growth factor-2-containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles.

BACKGROUND: We produced fibroblast growth factor (FGF)-2-containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles (FGF-2 + F/P NPs). The purpose of this study was to evaluate the effectiveness of the local administration of FGF-2 + F/P NPs on repairing crush syndrome (CS)-injured lesions after compression release using a nonlethal and reproducible CS injury rat model. MATERIALS AND METHODS: The hind limbs of the anesthetized rats were compressed for 6 h using 3.6 kg blocks, as previously described. The effects of administering FGF-2 + F/P NPs (group A), F/P NPs alone (group B), FGF-2 alone (group C), and saline (control; group D) were examined. Motor function, surface blood flow in the hind limbs, and the wet/dry weight ratio in the tibialis anterior muscle were examined for 1-28 d after the compression release. Histologic analyses were also performed. RESULTS: At the middle and late stages (3-28 d after the compression release), group A had higher scores in the motor function, improved blood flow, increased number of blood vessels, and faster recovered muscle tissue, compared with the other groups. There was no significant difference in enhanced edema in the tibialis anterior muscle among all groups. CONCLUSIONS: The local administration of FGF-2 + F/P NPs to a CS-injured lesion was effective in repairing damaged muscle tissue after compression release.

Improved survival rate by temperature control at compression sites in rat model of crush syndrome.

BACKGROUND: Crush syndrome (CS) has been reported in disasters, terrorist incidents, and accidents, and the clinical and pathologic picture has gradually been clarified. Few lethal and reproducible animal models of CS with use of a quantitative load are available. A new model is needed to investigate pathologic and therapeutic aspects of this injury. MATERIALS AND METHODS: Using a device built from commercially available components, both hindlimbs of anesthetized rats were respectively compressed for 6 h using 3.6-kg blocks. The effects of trunk warming alone without compressed hindlimbs (Group A), non-warming at room temperature (Group B), whole-body warming including compressed hindlimbs (Group C), or warming of compressed hindlimbs alone (Group D) during compression were examined. Survival rates were compared and hematological and histologic analyses were performed at specific time points after compression release. RESULTS: Limb or whole-body warming significantly worsened the survival of rats. We found a much lower survival rate of 0%-10% in animals, in which the hindlimbs were warmed during compression (Groups C and D) at 12 h after compression release, compared with 90%-100% in animals without warming of the hindlimbs (Groups A and B). Groups C and D showed significantly enhanced hyperkalemia at ≥4 h after compression release and all blood samples from dead cases showed hyperkalemia (>10 mEq/L). CONCLUSIONS: We developed a new lethal and reproducible rat CS model with a quantitative load. This study found that warming of compressed limbs worsened the survival rate and significantly enhanced hyperkalemia, apparently leading to cardiac arrest.

Real-time fetal monitoring using photoacoustic measurement of placental oxygen saturation in a rabbit hypoxia model.

INTRODUCTION: Ensuring adequate fetal oxygenation is an essential aim of fetal monitoring. The purpose of this study was to establish a basic technique for real-time measurement of blood oxygen saturation of the placenta by photoacoustic (PA) technique as a new fetal monitoring method. METHODS: The hypoxia model established in our previous study was applied to 7 pregnant rabbits. Three phases were induced: normal phase, hypoxia phase, and recovery phase. Three methods were simultaneously used for real-time fetal monitoring: fetal heat rate (FHR) monitoring, oxygen saturation (SO2) measurement by near-infrared spectroscopy (SNO2), and placenta SO2 measured by PA technique (SplO2). The maternal hypoxia was assessed by skin SO2 measured by PA technique (SsO2), and arterial blood SO2 by blood gas analysis (SaO2). RESULTS: The average of SplO2 in normal phase was 52.6 ± 13.9 %. The averages of SNO2, SSO2, and SplO2 in the seven rabbits changed in parallel from the normal phase to hypoxia phase. In the recovery phase, the SplO2 rose in parallel with recovery of SaO2. There was lag in increase of the FHR compared to the change in the other values. In the detailed analysis of PA signals from the labyrinth and decidua, a unique change in oxygen saturation was seen in one case. DISCUSSION: Results of this study showed that sensitivity of our novel PA technique in detecting tissue hypoxia was similar to near-infrared spectroscopy (NIRS). As an advantage, unlike NIRS, monitoring with PA technique was unaffected by ischemia and surface changes in oxygen saturation because of its higher spatial resolution. We conclude that PA technique provides more accurate information about fetal blood placenta than NIRS. Ultrasound imaging, combined with oxygen saturation monitoring by PA technique, would improve fetal monitoring and fetal diagnosis in the future.

Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

Significance: We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. Aim: In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). Approach: A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. Results: The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. Conclusions: The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Quantification of effective attenuation coefficients using continuous wavelet transform of photoacoustic signals.

A method for quantifying the effective attenuation coefficients of optical absorbers by using the continuous wavelet transform (CWT) to calculate the time-resolved frequency spectra of photoacoustic signals is proposed. Because the coefficients can be quantified according to the relative intensity of the frequency content of the signals, it is unnecessary to determine the fluences. A computational simulation reveals that the time-resolved frequency spectra exhibit better correlation with the coefficients than do power spectra calculated using a Fourier transformation. The CWT-based method was experimentally verified, and the coefficients were quantified with mean square error of 2.0 cm(-1).

Regulation of miRNA expression by low-level laser therapy (LLLT) and photodynamic therapy (PDT).

Applications of laser therapy, including low-level laser therapy (LLLT), phototherapy and photodynamic therapy (PDT), have been proven to be beneficial and relatively less invasive therapeutic modalities for numerous diseases and disease conditions. Using specific types of laser irradiation, specific cellular activities can be induced. Because multiple cellular signaling cascades are simultaneously activated in cells exposed to lasers, understanding the molecular responses within cells will aid in the development of laser therapies. In order to understand in detail the molecular mechanisms of LLLT and PDT-related responses, it will be useful to characterize the specific expression of miRNAs and proteins. Such analyses will provide an important source for new applications of laser therapy, as well as for the development of individualized treatments. Although several miRNAs should be up- or down-regulated upon stimulation by LLLT, phototherapy and PDT, very few published studies address the effect of laser therapy on miRNA expression. In this review, we focus on LLLT, phototherapy and PDT as representative laser therapies and discuss the effects of these therapies on miRNA expression.

Optimal blue light irradiation conditions for the treatment of acne vulgaris in a mouse model.

BACKGROUND: Although blue light is one of the therapeutic approaches used to treat acne vulgaris (AV), there is no consensus on its effectiveness. As a result, it is not recommended in the major acne vulgaris treatment guidelines. OBJECTIVE: The goal of this study was to look into the mechanism, safety, and efficacy of blue light therapy. We achieved this by examining the pathological response, inflammation, and depth of light penetration in a mouse model of cystic AV. METHODS: The aims of the study were addressed by exposing the mice to light with a wavelength of 415 nm under four different irradiation conditions. The exposure was done for five consecutive days followed by a no irradiation period of 72 h. RESULTS: Blue light treatment was most effective when irradiation was performed at 100 mW/cm2 for 20 min for five consecutive days. Inflammatory responses emerged 72 h after the final irradiation dose was administered. These responses were not associated with apoptosis as cleaved caspase-3 staining revealed no significant increases in apoptosis in the skin under any of the tested conditions. Blue light reached the superficial layer of the acne cyst at 5% of the total irradiation power and was attenuated by half for every 50 µm of progress through the cyst. CONCLUSION: In conclusion, blue light could control severe dermatologic inflammatory responses; therefore, it can be used to irradiate AV with high inflammation levels on a daily basis until improvement is observed. In addition, porphyrin, a metabolite of Cutibacterium acnes, and reactive oxygen species generated by the surrounding skin tissue may have essential roles in AV treatment.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential.

BACKGROUND: Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span. METHODS: We transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo. RESULTS: Our results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components. CONCLUSIONS: Focal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Blue Laser Irradiation Decreases the ATP Level in Mouse Skin and Increases the Production of Superoxide Anion and Hypochlorous Acid in Mouse Fibroblasts.

Photobiomodulation studies have reported that blue light irradiation induces the production of reactive oxygen species. We investigated the effect of blue laser (405 nm) irradiation on the ATP levels in mouse skin and determined the types of reactive oxygen species and reactive nitrogen species using cultured mouse fibroblasts. Blue laser irradiation caused a decrease in the ATP level in the mouse skin and triggered the generation of superoxide anion and hypochlorous acid, whereas nitric oxide and peroxynitrite were not detected. Moreover, blue laser irradiation resulted in reduced cell viability. It is believed that the decrease in the skin ATP level due to blue light irradiation results from the increased levels of oxidative stress due to the generation of reactive oxygen species. This method of systematically measuring the levels of reactive oxygen species and reactive nitrogen species may be useful for understanding the effects of irradiation conditions.

Spectroscopic photoacoustic microscopic imaging during single spatial scan using broadband excitation light pulses with wavelength-dependent time delay.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.