Temporal modulation of optical energy for enhanced photothermal hemostasis of intraoperative bleeding during laser treatment

Intraoperative bleeding during laser treatment of benign prostate hyperplasia (BPH) often impedes cystoscopic vision, necessitating the use of conventional hemostatic devices. This study proposes an optical technique to improve the efficacy of photothermal hemostasis of bleeders during laser prostatectomy by temporally modulating a 532 nm laser beam. A perfused porcine kidney model is established to quantitatively investigate various optical pulse patterns and irradiation modes. Thermal simulations demonstrate a high success rate of complete hemostasis achieved by the modulated 532 nm pulse pattern. In comparison to the irradiation modes typically employed for hemostasis, the modulated 532 nm mode exhibits a short coagulation time and minimal thermal injury. ex vivo and in vivo cystoscopic observations validate the clinical feasibility of the proposed optical energy modulation method to regulate intraoperative bleeding.     

In‐vitro investigations on laser‐induced smoke generation mimicking the laparoscopic laser surgery purposes

Intraoperative smoke‐generation limits the quality of vision during laparoscopic/endoscopic laser‐assisted surgeries. The current study aimed at the evaluation of factors affecting this phenomenon. As a first step, a suitable experimental setup and a test tissue model were established for this investigation. The experimental setup is composed of a specific sample container, a laser therapy component suitable for the ablation of model tissue at different treatment wavelengths (λ = 980 nm, 1350 nm, 1470 nm), a suction unit providing continuous smoke extraction, and a detection unit for smoke quantification via detection of light (λ = 633 nm) scattered from smoke particles. The ablation rate (AR) was calculated by dividing the ablated volume by the ablation time (60 sec). The laser‐induced scattering signal intensity of the smoke (SI) was determined from time‐charts of the signal intensity as a measure for vision, in addition a delay‐time t_delay could be derived defining the onset of SI after the laser was switched on. The ratio SI/AR is used as a measure for smoke generation in relation to the ablation rate. Additionally the light transmission of the tissue samples was used to estimate their optical properties. In this set‐up, smoke generation using λ = 980 nm as ablation laser wavelength was detected after a delay‐time t_delay = (121.6 ± 24.8) sec which is significantly longer compared to the wavelengths λ = 1350 nm with t_delay = (89.8 ± 19.3) sec and λ = 1470 nm with t_delay = (24.7 ± 5.4) sec. Thus, the delay

Experimental set‐up consisting of sample container, laser therapy component, suction unit and scattered‐light detection compartment. time is wavelength‐dependent. The SI/AR ratio was significantly different (p < 0.001) for 1470 nm irradiation compared to 980 nm irradiation [SI/AR(1470) = (11.8 ± 2.6) · 10³ vs. SI/AR(980) = (8.6 ± 2.0) · 10³]. The ablation crater for 980 nm irradiation was comparable with 1470 nm irradiation, but the coagulation rim was thicker in the 980 nm case. In conclusion, it could be shown experimentally that smoke‐generation depends on the wavelength used for laser ablation.     

Feasibility of photoacoustic evaluations on dual‐thermal treatment of ex vivo bladder tumors

A variety of thermal therapeutic methods have been investigated to treat bladder tumors but often cause bowel injury and bladder wall perforation due to high treatment dosage and limited clinical margins. The objective of the current study is to develop a dual‐thermal modality to deeply coagulate the bladder tumors at low thermal dosage and to evaluate therapeutic outcomes with high contrast photoacoustic imaging (PAI). High intensity focused ultrasound (HIFU) is combined with 532 nm laser light to enhance therapeutic depth during thermal treatments on artificial tumor‐injected bladder tissue ex vivo. PAI is employed to identify the margins of the tumors pre‐ and post‐treatments. The dual‐thermal modality achieves 3‐ and 1.8‐fold higher transient temperature changes and 2.2‐ and 1.5‐fold deeper tissue denaturation than laser and HIFU, respectively. PAI vividly identifies the position of the injected tumor and entails approximately 7.9 times higher image contrast from the coagulated tumor as that from the untreated tumor. Spectroscopic analysis exhibits that both 740 nm and 760 nm attains the maximum photoacoustic amplitudes from the treated areas. The proposed PAI‐guided dual‐thermal treatments (laser and HIFU) treatments can be a feasible therapeutic modality to treat bladder tumors in a controlled and efficient manner.

     

Dose‐dependent study of effects of 532‐nm continuous wave laser on rat skin: A mechanistic insight

Visible lasers emitting in the green spectral region are being routinely employed in various medical and defense fields namely treatment of pigmented lesions, tattoo inks, port wine stains, dazzling the target or mob dispersal. Despite their increasing applications, lasers also tend to pose occupational hazards to operators, ancillary personnel, individuals undergoing laser therapies. This study was aimed at investigating the effects of different doses of 532‐nm continuous wave laser on rat skin. The present study demonstrated that higher fluences of 532‐nm continuous wave (CW) laser induces significant tissue damage through induction of tumor necrosis factor‐α, cyclooxygenase‐2, tumor protein (p53), PARP 1, caspase3 which in turn leads to tissue damage and cell death. Furthermore, level of heat shock proteins, pAkt were found up‐regulated as a cope up response to laser‐induced stress. On the basis of the findings, irradiation with 532‐nm CW laser up to 2.5 J/cm² was found within the safe exposure limits. Thus, it is probably the first attempt to demonstrate the tissue damage induced by 532‐nm CW laser on skin, which may help in choosing safe laser dose for certain skin‐based applications and evolving methods to ameliorate laser‐inflicted injuries.     

Concentric photothermal coagulation with basket‐integrated optical device for treatment of tracheal stenosis

A basket‐integrated optical device is developed to consistently treat tubular tissue by centering an optical diffuser in the lumen. Four nitinol arms in conjunction with the optical diffusing applicator are deployed to induce homogeneous circumferential light emission and concentric photothermal coagulation on tracheal tissue. A 1470‐nm laser light is employed for the tissue testing at various irradiation conditions and evaluated in terms of thermal gradient and temperature evolution. Preliminary experiments on liver tissue demonstrate the concentric development of the radial thermal coagulation in the tissue (eccentric ratio = ~5.5%). The interstitial tissue temperature increases with the total amount of energy delivery (around 65°C). Ex vivo trachea testing yields up to 16.5% tissue shrinkage due to dehydration as well as uniform ablation of the cilia and goblet cells in a mucosa layer under 7‐W irradiation for 10 s. The proposed optical device may be a feasible therapeutic method to entail the circumferential coagulation in the tubular tissues in a reliable manner.      

Which wavelength is optimal for transcranial low‐level laser stimulation?

One of the challenges in transcranial low‐level laser therapy (LLLT) is to optimally choose illumination parameters, such as wavelength. However, there is sparse study on the wavelengths comparison especially on human transcranial LLLT. Here, we employed Monte Carlo modeling and visible human phantom to compute the penetrated photon fluence distribution within cerebral cortex. By comparing the fluence distribution, penetration depth and the intensity of laser‐tissue‐interaction within brain among all candidate wavelengths, we found that 660, 810 nm performed much better than 980, 1064 nm with much stronger, deeper and wider photon penetration into cerebral tissue; 660 nm was shown to be the best and slightly better than 810 nm. Our computational finding was in a surprising accordance with previous LLLT‐neurobehavioral studies on mice. This study not only offered quantitative comparison among wavelengths in the effect of LLLT light penetration effectiveness but also anticipated a delightful possibility of online, precise and visible optimization of LLLT illumination parameters.      

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Real‐time functional optical‐resolution photoacoustic microscopy using high‐speed alternating illumination at 532 and 1064 nm

Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps.      

In situ diode laser fenestration: An ex‐vivo evaluation of irradiation effects on human aortic tissue

The in situ laser fenestration is an interesting option for the endovascular treatment of short‐necked aneurysms with an intraoperative modification of a standard endograft. According to literature evidence, diode laser emitting in the near‐infrared wavelength (810 nm) can be successfully used to fenestrate the endograft fabric. This paper describes a three‐dimensional navigation system for the accurate targeting of the fenestration site, then reports results of an ex vivo study to assess whether the laser operative conditions, which ensure the fabric fenestration, are harmless for the biological tissue surrounding the endoprosthesis. Two hundred twenty‐five samples of human aorta, including healthy specimens and abdominal aortic aneurysm samples, were irradiated ex vivo using a 810 nm diode laser. Energy and pulse duration were varied. Irradiated tissues were fixed in formaldehyde, sectioned and subjected to histological examination. Only 7.5% of the irradiated samples exhibited a thermal damage, which was always confined to the contact point between the laser fiber tip and the aortic wall. These experiments suggest that the diode laser can be safely used for the proposed surgical application.     

Wavelength‐Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS) for noninvasive early cancer detection and tissue hypoxia monitoring

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO₂). Unlike conventional photoacoustic spectroscopy, WM‐DPAS measures simultaneously two signals induced from square‐wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy‐ and oxy‐hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two‐wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM‐DPAS to detect very small changes in total hemoglobin concentration (C_Hb) and oxygenation levels, thereby identifying pre‐malignant tumors before they are anatomically apparent. The non‐invasive nature also makes WM‐DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM‐DPAS can be tuned for different applications such as quick cancer screening and accurate StO₂ quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM‐DPAS theory has been validated with sheep blood phantom measurements.

Sensitivity comparison between conventional single‐ended signal and differential signal.     

Laser‐induced plasmon‐mediated treatment of retinoblastoma in viscous vitreous phantom

Retinoblastoma (RB) is a rare form of cancer of the retina most prevalent in young children. We successfully show that laser‐induced cell disruption, mediated by gold plasmonic nanoparticle (NP), is a potential and efficient therapy to kill the cancerous cells. The proof of concept is demonstrated in vitro on cultured Y79 RB cancer cells with a nanosecond laser at 527 nm, for both attached cells at the bottom of a Petri dish and for floating, clustered cells in a viscous vitreous phantom comprised of hyaluronan. We report a cellular death of 82% after irradiation in classic culture medium and a cellular death of 98% in vitreous phantom, for similar number of NPs in each sample. It is found that the NPs efficiently penetrate the floating Y79 clusters cells in the vitreous phantom, leading to a cellular death of over 85% even within the centre of the aggregates. The proposed treatment technique is based on a similar nanosecond laser used to eliminate floaters in the vitreous, but with much lower (100‐1000 times) fluences of 20 J cm^?2.      

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.      

Ground‐ and excited‐state proton transfer and antioxidant activity of 3‐hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited‐state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3‐hydroxyflavone (3‐HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3‐HF against lipid peroxidation in this membrane system. When 3‐HF molecules are partitioned into EYPC liposomes, a weak long‐wavelength absorption band with λ ～410 nm appears in addition to the principal absorption at ～λ = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (λ～460 nm) of the ground‐state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (λ = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground‐state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3‐HF binding, suggesting that 3‐HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes. Copyright © 2008 John Wiley & Sons, Ltd.     

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Endoluminal application of glass‐capped diffuser for ex vivo endovenous photocoagulation

Endovenous laser ablation (EVLA) has frequently been used to treat varicose veins for 20 years. In spite of 90?95% occlusion rates, clinical complications such as burn and ecchymosis still occur due to excessive thermal injury to perivenous tissue. In the current study, a glass‐capped diffusing applicator is designed to validate the feasibility of EVLA as an effective therapeutic device by applying circumferential light distribution. The proposed device is evaluated with a flat fiber as a reference in terms of temperature elevation, fiber degradation, and degree of coagulative necrosis after 532 nm‐assisted EVLA at 100 J/cm. The diffusing fiber generates a 40% lower maximum temperature with a 90% lower transient temperature change in blood, compared to the flat fiber. Due to low irradiance (13.5 kW/cm²) and wide light distribution, the diffuser tip experiences no significant thermal degradation while severe carbonization occurs at the flat fiber tip. Ex vivo tissue tests verify that the diffusing fiber induces circumferential and consistent tissue denaturation to the vein wall (107.8 ± 7.8 µm) along with 19% vessel shrinkage. The proposed glass‐capped diffusing applicator can be a feasible therapeutic device for EVLA with minimal complications by entailing low maximum temperatures and uniform tissue denaturation in the venous tissue.

     

Second harmonic imaging of plants tissues and cell implosion using two‐photon process in ZnO nanoparticles

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto‐fluorescence above 750 nm and minimal auto‐fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near‐infrared femtosecond laser source ranging from 750‐980 nm. For laser energies exceeding the two‐photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two‐photon emission becomes the dominant signal. The heat generated due to two‐photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710–750 nm. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade‐offs between the spatiotemporal resolution, field‐of‐view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual‐wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large‐scale cerebral vascular networks. The system offers 3‐dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 × 8 mm², thus covering the entire cortical vasculature in mice. The large‐scale OA imaging capacity is complemented by simultaneously acquired pulse‐echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.      

On the role of interference in laser‐based mid‐infrared widefield microspectroscopy

A laser's high degree of coherence leads to interferences, which—in the absence of precautions—can cause severe image distortions such as fringes and speckles and which thereby strongly hamper a meaningful interpretation of hyperspectral images in laser‐based widefield microspectroscopy. While images and spectra of homogenous samples may already suffer from interferences, any structured object such as a tissue thin section will add to these distortions due to wavelength‐ and, in particular, sample‐dependent phase shifts (structure sizes, absorption coefficients, refractive indices). This effect is devastating for the universal applicability of laser‐based microspectroscopy especially in the mid‐infrared (MIR), where cell sizes are of the same dimension as the wavelength of the illumination source. Here, we show that the impact of interferences is strongly mitigated by reducing the time‐averaged spatiotemporal coherence properties of the illumination using a moving plus a stationary scatterer. In this case, the illumination path provides a pseudothermal radiation source and spatially resolved spectra can be obtained at the quality of the reference method, that is, Fourier‐transform infrared microspectroscopy, without compromising spectral or spatial resolution.      

Front Cover: Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks (J. Biophotonics 9/2018)

We present a hybrid dual‐wavelength optoacoustic and ultrasound bio‐microscope capable of rapid transcranial visualization of morphology and oxygenation status of large‐scale cerebral vascular networks. Imaging of entire cortical vasculature in mice is achieved with single capillary resolution and complemented by simultaneously acquired pulse‐echo ultrasound microscopy scans of the mouse skull. The new approach holds potential to facilitate studies into neurological and vascular abnormalities of the brain. Further details can be found in the article by Johannes Rebling, Héctor Estrada, Sven Gottschalk, et al. ( e201800057 ).

     

Non‐Invasive Multi‐Dimensional Two‐Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non‐invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two‐photon microscopy to non‐invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence‐based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients.

Two photon based autofluorescence features of immune cells enables non‐invasive differentiation.