Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

Enhanced contrast acoustic‐resolution photoacoustic microscopy using double‐stage delay‐multiply‐and‐sum beamformer for vasculature imaging

In acoustic‐resolution photoacoustic microscopy (AR‐PAM) systems, the lateral resolution in the focal zone of the ultrasound (US) transducer is determined by the numerical aperture (NA) of the transducer. To have a high lateral resolution, a large NA is used. However, the larger the NA, the smaller the depth of focus [DOF]. As a result, the lateral resolution is deteriorated at depths out of the focal region. The synthetic aperture focusing technique (SAFT) along with a beamformer can be used to improve the resolution outside the focal region. In this work, for image formation in AR‐PAM, we propose the double‐stage delay‐multiply‐and‐sum (DS_DMAS) algorithm to be combined with SAFT. The proposed method is evaluated experimentally using hair targets and in vivo vasculature imaging. It is shown that DS_DMAS provides a higher resolution and contrast compared to other methods. For the B‐mode images obtained using the hair phantom, the proposed method reduces the average noise level for all the depths by about 134%, 57% and 23%, compared to the original low‐ resolution, SAFT+DAS and SAFT+DMAS methods, respectively. All the results indicate that the proposed method can be an appropriate algorithm for image formation in AR‐PAM systems.      

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

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.      

Comparison of high frequency ultrasound and optical coherence tomography as modalities for high resolution and non invasive skin imaging.

High frequency ultrasound (HFUS) and optical coherence tomography (OCT) are techniques for high resolution imaging of tissues. The penetration depth of these modalities is limited, but it is sufficiently large enough for non invasive skin imaging. HFUS and OCT are based on the same concept. Waves (ultrasonic waves, respectively light waves) propagate along a narrow beam, are backscattered at tissue inhomogeneities and analyzed over time of flight to obtain spatially resolved morphological information. The objective of this paper is to compare HFUS and OCT in terms of resolution, dynamic range and contrast and to assess their value as tools for high resolution skin imaging. Measurements on phantoms and in vivo have been performed with a 100 MHz ultrasound system and an OCT-scanner working in the near infrared spectrum at 1300 nm wave-length. From the measurements, it can be concluded that OCT delivers an almost isotropic resolution (axial resolution about 5.8 microns, lateral resolution about 4.1 microns), whereas the resolution of the investigated HFUS system is more anisotropic (axial resolution about 9.3 microns, lateral resolution about 60 microns). HFUS and OCT show different penetration depths and a different contrast. Both techniques can, therefore, be combined advantageously in a multimodality approach to account for their individual characteristics.     

Miniature probe for dual‐modality photoacoustic microscopy and white‐light microscopy for image guidance: A prototype toward an endoscope

In this study, a novel photoacoustic microscopy (PAM) probe integrating white‐light microscopy (WLM) modality that provides guidance for PAM imaging and complementary information is implemented. One single core of an imaging fiber bundle is employed to deliver a pulsed laser for photoacoustic excitation for PAM mode, which provides high resolution with deep penetration. Meanwhile, for WLM mode, the imaging fiber bundle is used to transmit two‐dimensional superficial images. Lateral resolution of 7.2 μm for PAM is achieved. Since miniature components are used, the probe diameter is only 1.7 mm. Imaging of phantom and animals in vivo is conducted to show the imaging capability of the probe. The probe has several advantages by introducing the WLM mode, such as being able to conveniently identify regions of interest and align the focus for PAM mode. The prototype of an endoscope shows potential to facilitate clinical photoacoustic endoscopic applications.     

In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell–specific, fluorescent reporter mouse skin with induced ischemia‐reperfusion (I‐R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual‐wavelength micro electro mechanical system (MEMS) scanning–based optical resolution photoacoustic microscopy (OR‐PAM) system for continuous label‐free functional imaging of vascular reperfusion in an IR mouse model. This MEMS‐OR‐PAM system provides fast scanning speed for concurrent dual‐wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO₂) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS‐OR‐PAM system provides novel perspectives to understand the I‐R injuries. It solves the problem of dynamic label‐free functional monitoring of the vascular reperfusion at high spatial resolution.      

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.      

Two‐photon focal modulation microscopy for high‐resolution imaging in deep tissue

Two‐photon microscopy (2PM) is one of the most widely used tools for in vivo deep tissue imaging. However, the spatial resolution and penetration depth are still limited due to the strong scattering background. Here we demonstrate a two‐photon focal modulation microscopy. By utilizing the modulation and demodulation techniques, background rejection capability is enhanced, thus spatial resolution and imaging penetration depth are improved. Compared with 2PM, the transverse resolution is increased by 70%, while the axial resolution is increased to 2‐fold. Furthermore, when applied in conventional 2PM mode, it can achieve inertial‐free scanning in either transverse or axial direction with in principle unlimited scanning speed. Finally, we applied 2PFMM in thick scattering samples to further examine the imaging performance. The results show that the signal‐to‐background ratio of 2PFMM can be improved up to five times of 2PM at the depth of 500 μm. Fluorescent imaging in the mouse brain tissue. 3D Thy1‐GFP hippocampal neurons imaged by (A) 2PM compared with (B) 2PFMM; (C‐H) xy maximum‐intensity projection imaged by 2PM compared with 2PFMM. Scale bar 50 μm.      

Correcting the limited view in optical‐resolution photoacoustic microscopy

Optical‐resolution photoacoustic microscopy (OR‐PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection‐mode detection in OR‐PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited‐view phenomenon in OR‐PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof‐of‐concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection‐mode OR‐PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture‐cross phantom and in a mouse brain in vivo.      

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Quantitative analysis on in vivo tumor‐microvascular images from optical‐resolution photoacoustic microscopy

Optical‐resolution photoacoustic microscopy (OR‐PAM) has been shown to be an excellent imaging modality for monitoring and study of tumor microvasculature. However, previous studies focused mainly on the normal tissues and did not quantify the tumor microvasculature. In this study, we present an in vivo OR‐PAM imaging of the melanomas and hepatoma implanted in the mouse ear. We quantify the vessel growth by extracting the skeletons of both dense and thin branches of the tumor microvasculature obtained by Hessian matrix enhancement followed by improved two‐step multistencils fast marching method. Compared with the previous methods of using OR‐PAM for normal tissues, our method was more effective in extracting the binary vascular network in the tumor images and in obtaining the complete and continuous microvascular skeleton maps. Our demonstration of using OR‐PAM in improving microvasculature of tumors and quantification of tumor growth would push deep this technology for the early diagnosis and treatment of cancers.      

Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.     

Structured illumination microscopy with interleaved reconstruction (SIMILR)

Structured illumination microscopy (SIM) is the commonly used super‐resolution (SR) technique for imaging subcellular dynamics. However, due to its need for multiple illumination patterns, the frame rate is just a fraction of that of conventional microscopy and is thus too slow for fast dynamic studies. A new SR image reconstruction method that maximizes the use of each subframe of the acquisition series is proposed for improving the super‐resolved frame rate by N times for N illumination directions. The method requires no changes in raw data and is appropriate for many versions of SIM setup, including those implementing fast illumination pattern generation mechanism based on spatial light modulator or digital micromirror device. The performance of the proposed method is demonstrated through imaging the highly dynamic endoplasmic reticulum where continuous rapid growths or shape changes of tiny structures are observed.      

In vivo study of endometriosis in mice by photoacoustic microscopy

Endometriosis (EM) impacts the healthcare and the quality of life for women of reproductive age. However, there is no reliable noninvasive diagnosis method for either animal study or clinical use. In this work, a novel imaging method, photoacoustic microscopy (PAM) was employed to study the EM on the mouse model. Our results demonstrated the PAM noninvasively provided the high contrast and 3D imaging of subcutaneously implanted EM tissue in the nude mouse in vivo. The statistical study also indicated PAM had high sensitivity and specificity in the diagnosis of EM in this animal study. In addition, we also discussed the potential clinical application for PAM in the diagnosis of EM. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Indocyanine green-enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA-approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG-RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15-fold at 24 h post-injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.     

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix‐assisted laser desorption/ionization tandem mass spectrometry imaging

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high‐resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix‐assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high‐resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 μm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue‐specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full‐scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high‐throughput profiling of metabolites in plant tissues.     

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.