Nonlinear photoacoustic microscopy via a loss modulation technique: from detection to imaging

In order to achieve high-resolution deep-tissue imaging, multi-photon fluorescence microscopy and photoacoustic tomography had been proposed in the past two decades. However, combining the advantages of these two imaging systems to achieve optical-spatial resolution with an ultrasonic-penetration depth is still a field with challenges. In this paper, we investigate the detection of the two-photon photoacoustic ultrasound, and first demonstrate background-free two-photon photoacoustic imaging in a phantom sample. To generate the background-free two-photon photoacoustic signals, we used a high-repetition rate femtosecond laser to induce narrowband excitation. Combining a loss modulation technique, we successfully created a beating on the light intensity, which not only provides pure sinusoidal modulation, but also ensures the spectrum sensitivity and frequency selectivity. By using the lock-in detection, the power dependency experiment validates our methodology to frequency-select the source of the nonlinearity. This ensures our capability of measuring the background-free two-photon photoacoustic waves by detecting the 2nd order beating signal directly. Furthermore, by mixing the nanoparticles and fluorescence dyes as contrast agents, the two-photon photoacoustic signal was found to be enhanced and detected. In the end, we demonstrate subsurface two-photon photoacoustic bio-imaging based on the optical scanning mechanism inside phantom samples

In vivo sub-femtoliter resolution photoacoustic microscopy with higher frame rates

Microscopy based on non-fluorescent absorption dye staining is widely used in various fields of biomedicine for 400 years. Unlike its fluorescent counterpart, non-fluorescent absorption microscopy lacks proper methodologies to realize its in vivo applications with a sub-femtoliter 3D resolution. Regardless of the most advanced high-resolution photoacoustic microscopy, sub-femtoliter spatial resolution is still unattainable, and the imaging speed is relatively slow. In this paper, based on the two-photon photoacoustic mechanism, we demonstrated a in vivo label free laser-scanning photoacoustic imaging modality featuring high frame rates and sub-femtoliter 3D resolution simultaneously, which stands as a perfect solution to 3D high resolution non-fluorescent absorption microscopy. Furthermore, we first demonstrated in vivo label-free two-photon acoustic microscopy on the observation of non-fluorescent melanin distribution within mouse skin

The squeeze film effect on micro-electromechanical resonators

The air squeeze film damping effect on the dynamic responses of clamped micro- electromechanical resonators is investigated in this study. A dynamic model for a clamped micro-electromechanical resonator with the damping consideration is derived using Lagrange’s equation. The corresponding resonator eigen solutions are formulated and solved by employing the assumed-mode method. The effect of different parameters; i.e. the resonator size, ambient temperature and pressure on the squeeze film damping characteristics were simulated and investigated. The results indicate that the squeeze film damping effect may significantly affect the dynamic responses of micro-scale electromechanical resonator

Glycogen synthase kinase 3α and 3β have distinct functions during cardiogenesis of zebrafish embryo

<p>Abstract</p> <p>Background</p> <p>Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3α (51 kDa) and GSK3β (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3α and GSK3β, making it difficult to identify an inhibitor that can be selective against GSK3α or GSK3β. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3α or GSK3β and uncover the isoform-specific roles that GSK3 plays during cardiogenesis.</p> <p>Results</p> <p>We blocked <it>gsk3α </it>and <it>gsk3β </it>translations by injection of morpholino antisense oligonucleotides (MO). Both <it>gsk3α</it>- and <it>gsk3β</it>-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the <it>gsk3α</it>- and <it>gsk3β</it>-MO-induced heart defects, we found that the reduced number of cardiomyocytes in <it>gsk3α </it>morphants during the heart-ring stage was due to apoptosis. On the contrary, <it>gsk3β </it>morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in <it>gsk3β </it>morphants. <it>bmp4 </it>expression in <it>gsk3β </it>morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in <it>gsk3β </it>morphants were similar to those observed in <it>axin1 </it>and <it>apc</it>^{<it>mcr </it>}mutants, suggesting that GSK3β might play a role in cardiac valve development through the Wnt/β-catenin pathway. Finally, the phenotypes of <it>gsk3α </it>mutant embryos cannot be rescued by <it>gsk3β </it>mRNA, and vice versa, demonstrating that GSK3α and GSK3β are not functionally redundant.</p> <p>Conclusion</p> <p>We conclude that (1) GSK3α, but not GSK3β, is necessary in cardiomyocyte survival; (2) the GSK3β plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3α and GSK3β play distinct roles during zebrafish cardiogenesis.</p

Rapid virtual H&E histology of breast tissue specimens using a compact fluorescence nonlinear microscope

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to artifacts on the tissue surface from electrocautery, surgical ink or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin fixed, paraffin embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in-situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections

Autophagy Induction by HIV-Tat and Methamphetamine in Primary Midbrain Neuronal Cells of Tree Shrews via the mTOR Signaling and ATG5/ATG7 Pathway

Background: Addictive stimulant drugs, such as methamphetamine (METH), increase the risk of exposure to the human immunodeficiency virus-1 (HIV-1) infection and thus predispose individuals to the development of HIV-associated neurocognitive disorders (HANDs). Previous studies have indicated that HIV-Tat (the transactivator of transcription) and METH can synergistically induce autophagy in SH-SY5Y neuroblastoma cells and that autophagy plays a pivotal role in the neuronal dysfunction in HANDs. However, the underlying mechanism of METH-and HIV-Tat-induced neuronal autophagy remains unclear.Methods: We cultured primary midbrain neuronal cells of tree shrews and treated them with METH and HIV-Tat to study the role of METH and HIV-Tat in inducing autophagy. We evaluated the effects of the single or combined treatment of METH and HIV-Tat on the protein expressions of the autophagy-related genes, including Beclin-1 and LC3B, ATG5, and ATG7 in METH and HIV-Tat-induced autophagy. In addition, the presence of autophagosomes in the METH and/or HIV-Tat treatment was revealed using transmission electron microscopy.Results: The results indicated that METH increased the protein levels of LC3B and Beclin-1, and these effects were significantly enhanced by HIV-Tat. Moreover, the results suggested that ATG5 and ATG7 were involved in the METH and HIV-Tat-induced autophagy. In addition, it was found that mTOR inhibition via pharmacological intervention could trigger autophagy and promote METH and HIV-Tat-induced autophagy.Discussion: Overall, this study contributes to the knowledge of the molecular underpinnings of METH and HIV-Tat-induced autophagy in primary midbrain neuronal cells. Our findings may facilitate the development of therapeutic strategies for METH-and HIV-Tat-induced autophagy in HANDs

Chronic Kidney Disease Stage Is a Modulator on the Association between High-Sensitivity C-Reactive Protein and Coronary Vasospastic Angina

The prevalence of coronary vasospasm and also the factors associated with coronary vasospasm in CKD is still unclear. In this cross-sectional study of 859 consecutive CKD patients with angina pectoris received coronary catheterization, we evaluated the factors associated with coronary vasospasm. Patients with vasospasm were older and had higher peripheral blood white cell counts, higher peripheral blood monocyte cell counts, higher haemoglobin levels, higher hs-CRP levels, and lower levels of serum creatinine than patients without vasospasm. The results of multivariate logistic regression analysis revealed that peripheral blood monocyte count and hs-CRP level were independently associated with coronary vasospasm in patients with stage 1 CKD. Only peripheral blood monocyte count but not hs-CRP was independently associated with coronary vasospasm in patients with stages 2 and 3 of CKD. In conclusion, peripheral blood monocyte count is independently associated with coronary vasospasm in patients with stage 1–3 CKD, whereas hs-CRP is only independently associated with coronary vasospasm in patients with stage 1 CKD

F-box protein-32 down-regulates small-conductance calcium-activated potassium channel 2 in diabetic mouse atria

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation