A Review of Endogenous and Exogenous Contrast Agents Used in Photoacoustic Tomography with Different Sensing Configurations.

Optical-based sensing approaches have long been an indispensable way to detect molecules in biological tissues for various biomedical research and applications. The advancement in optical microscopy is one of the main drivers for discoveries and innovations in both life science and biomedical imaging. However, the shallow imaging depth due to the use of ballistic photons fundamentally limits optical imaging approaches' translational potential to a clinical setting. Photoacoustic (PA) tomography (PAT) is a rapidly growing hybrid imaging modality that is capable of acoustically detecting optical contrast. PAT uniquely enjoys high-resolution deep-tissue imaging owing to the utilization of diffused photons. The exploration of endogenous contrast agents and the development of exogenous contrast agents further improve the molecular specificity for PAT. PAT's versatile design and non-invasive nature have proven its great potential as a biomedical imaging tool for a multitude of biomedical applications. In this review, representative endogenous and exogenous PA contrast agents will be introduced alongside common PAT system configurations, including the latest advances of all-optical acoustic sensing techniques.

Conduction and Excess Charge in Silicate Glass/Air Interfaces.

The glass/air interface shows electrical properties that are unexpected for a widely used electrical insulator. The mobility of interfacial charge carriers under 80% relative humidity (RH) is 4.81 × 10-5 m2 s-1 V-1, 3 orders of magnitude higher than the electrophoretic mobility of simple ions in water and less than 2 orders of magnitude lower than the electron mobility in copper metal. This allows the glass/air interface to reach the same potential as a biased contacting metal quickly. The interfacial surface resistance R increases by more than 5 orders of magnitude when the RH decreases from 80 to 2%, following an S-shaped curve with small hysteresis. Moreover, the biased surfaces store charge, as shown by Kelvin potential measurements. Applying an electric field parallel to the surface produces RH-dependent results: under low humidity, the interface behaves as expected for an ideal two-dimensional parallel-plate capacitor, while under high RH, it acquires and maintains excess negative charge, which is lost under low RH. The glass surface morphology and potential distribution change on the glass/air interface under high RH and applied potential, including the extensive elimination of nonglass contaminating particles and potential levelling. All these surprising results are explained by using a protonic-charge-transfer mechanism: mobile protons dissociated from silanol groups migrate rapidly along a field-oriented adsorbed water layer, while the matrix-bound silicate anions remain immobile. Glass may thus be classified as the ionic analogue of a topological insulator but based on structural features and charge-transfer mechanisms different from the chalcogenides that have been receiving great attention in the literature.

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape.

Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of seven Mycobacterium tuberculosis gene regions (katG, inhA, ahpC, rpoB, gyrA, rrs, and eis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INH(r)) specimens was the katG 315ACC mutation (87%). However, in the Eastern Cape, INH(r) specimens had a lower frequency of katG mutations (44%) and higher frequencies of inhA (47%) and ahpC (10%) promoter mutations. The most common mutation among rifampin-resistant (RIF(r)) specimens was the rpoB 531TTG mutation (80%). The mutation was common in RIF(r) specimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was the gyrA 94GGC mutation (44%). The rrs 1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KAN(r)) specimens, respectively. The eis promoter mutation -12T was found in 26% of KAN(r) and 4% of KAN-susceptible (KAN(s)) specimens. Inclusion of the ahpC and eis promoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered at ClinicalTrials.gov under registration number NCT02170441.).

Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug-Resistant Tuberculosis in South Africa.

We show that the interpretation of molecular epidemiological data for extensively drug-resistant tuberculosis (XDR-TB) is dependent on the number of different markers used to define transmission. Using spoligotyping, IS6110 DNA fingerprinting, and DNA sequence data, we show that XDR-TB in South Africa (2006 to 2008) was predominantly driven by the acquisition of second-line drug resistance.

Deep learning-assisted smartphone-based quantitative microscopy for label-free peripheral blood smear analysis.

Hematologists evaluate alterations in blood cell enumeration and morphology to confirm peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose using smartphone-based autofluorescence microscopy (Smart-AM) for imaging label-free blood smears at subcellular resolution with automatic hematological analysis. Smart-AM enables rapid and label-free visualization of morphological features of normal and abnormal blood cells (including leukocytes, erythrocytes, and thrombocytes). Moreover, assisted with deep-learning algorithms, this technique can automatically detect and classify different leukocytes with high accuracy, and transform the autofluorescence images into virtual Giemsa-stained images which show clear cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.

Tri-modality in vivo imaging for tumor detection with combined ultrasound, photoacoustic, and photoacoustic elastography.

A comprehensive understanding of a tumor is required for accurate diagnosis and effective treatment. However, currently, there is no single imaging modality that can provide sufficient information. Photoacoustic (PA) imaging is a hybrid imaging technique with high spatial resolution and detection sensitivity, which can be combined with ultrasound (US) imaging to provide both optical and acoustic contrast. Elastography can noninvasively map the elasticity distribution of biological tissue, which reflects pathological conditions. In this study, we incorporated PA elastography into a commercial US/PA imaging system to develop a tri-modality imaging system, which has been tested for tumor detection using four mice with different physiological conditions. The results show that this tri-modality imaging system can provide complementary information on acoustic, optical, and mechanical properties. The enabled visualization and dimension estimation of tumors can lead to a more comprehensive tissue characterization for diagnosis and treatment.

Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission.

Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P < 0.01). Moreover, these putative compensatory mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P < 0.001). Our study supports a role for rpoC mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Rapid slide-free and non-destructive histological imaging using wide-field optical-sectioning microscopy.

Histopathology based on formalin-fixed and paraffin-embedded tissues has long been the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to guide surgeons intraoperatively. In this report, we propose a practical and low-cost histological imaging method with wide-field optical-sectioning microscopy (i.e., High-and-Low-frequency (HiLo) microscopy). HiLo can achieve rapid and non-destructive imaging of freshly-excised tissues at an extremely high acquisition speed of 5 cm2/min with a spatial resolution of 1.3 µm (lateral) and 5.8 µm (axial), showing great potential as an SMA tool that can provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that HiLo enables rapid extraction of diagnostic features for different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing surface images of rough specimens with large field-of-views and cellular features that are comparable to the clinical standard. Our results show promising clinical translations of HiLo microscopy to improve the current standard of care.

Three-dimensional label-free histological imaging of whole organs by microtomy-assisted autofluorescence tomography.

Three-dimensional (3D) histology is vitally important to characterize disease-induced tissue heterogeneity at the individual cell level. However, it remains challenging for both high-throughput 3D imaging and volumetric reconstruction. Here we propose a label-free, cost-effective, and ready-to-use 3D histological imaging technique, termed microtomy-assisted autofluorescence tomography with ultraviolet excitation (MATE). With the combination of block-face imaging and serial microtome sectioning, MATE can achieve rapid and label-free imaging of paraffin-embedded whole organs at an acquisition speed of 1 cm3 per 4 h with a voxel resolution of 1.2 × 1.2 × 10 µm3. We demonstrate that MATE enables simultaneous visualization of cell nuclei, fiber tracts, and blood vessels in mouse/human brains without tissue staining or clearing. Moreover, diagnostic features, including nuclear size and packing density, can be quantitatively extracted with high accuracy. MATE is augmented to the current slide-based 2D histology, holding great promise to facilitate histopathological interpretation at the organelle level.

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning.

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 µm, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining.

Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.

Low-cost high-resolution photoacoustic microscopy of blood oxygenation with two laser diodes.

Optical-resolution photoacoustic microscopy (OR-PAM) has been widely used for imaging blood vessel and oxygen saturation of hemoglobin (sO2), providing high-resolution functional images of living animals in vivo. However, most of them require one or multiple bulky and costly pulsed lasers, hindering their applicability in preclinical and clinical settings. In this paper, we demonstrate a reflection-mode low-cost high-resolution OR-PAM system by using two cost-effective and compact laser diodes (LDs), achieving microvasculature and sO2 imaging with a high lateral resolution of ∼6 µm. The cost of the excitation sources has dramatically reduced by ∼20-40 times compared to that of the pulsed lasers used in state-of-the-art OR-PAM systems. A blood phantom study was performed to show a determination coefficient R 2 of 0.96 in linear regression analysis. Experimental results of in vivo mouse ear imaging show that the proposed dual-wavelength LD-based PAM system can provide high-resolution functional images at a low cost.

Ultraviolet photoacoustic microscopy with tissue clearing for high-contrast histological imaging.

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

High-speed high-resolution laser diode-based photoacoustic microscopy for in vivo microvasculature imaging.

Laser diodes (LDs) have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy (PAM). However, the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously. In this paper, we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD, operating at a pulsed mode, with a repetition rate of 30 kHz, as an excitation source. A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio. By optimizing the optical system, a high lateral resolution of 4.8 µm has been achieved. In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

Efficacy and Safety of Repeated Micropulse Transscleral Diode Cyclophotocoagulation in Advanced Glaucoma.

PRECIS: Repeat micropulse transscleral cyclophotocoagulation (MPTCP) has some benefit in lowering intraocular pressure (IOP). There was a small risk of loss of vision, prolonged hypotony, and phthisis bulbi. AIM: This study aimed to determine the efficacy and safety of repeated MPTCP for an Asian population with refractory glaucoma. METHODS: This is a retrospective case series of 43 eyes (43 patients) with severe glaucoma which underwent repeated MPTCP. Baseline parameters were taken from the visit just before the second MPTCP session. Success was defined as IOP of 6 to 21 mm Hg or ≥20% reduction in IOP without an increase in glaucoma medication from baseline, without further glaucoma reoperation, and ≤3 total MPTCP episodes. The IOP, number of IOP-lowering medications, and best-corrected visual acuity were documented preoperatively and postoperatively. Postoperative complications were also analyzed. RESULTS: The mean age±SD was 57.4±18.2 years with a mean follow-up duration of 28.9±27.5 months. Neovascular glaucoma was the most common type of glaucoma [18 eyes (41.9%)]. The success rates at postoperative years 1, 2, and 3, and the latest follow-up were 36.4%, 42.9%, 32.0%, and 39.5%, respectively. The median survival time of repeat MPTCP was 4.6 months. Compared with the preoperative mean IOP (35.2±11.0 mm Hg), the mean IOP at postoperative years 1, 2, and 3, and latest follow-up, was 27.8±13.7 mm Hg (P=0.004), 27.4±12.4 (P=0.003), 31.8±13.2 (P=0.35), and 27.1±13.8 mm Hg (P=0.002), respectively. The mean number of IOP-lowering medications was reduced from 3.3±0.9 preoperatively to 2.8±1.3 at the final follow-up (P=0.007). Postoperative complications included prolonged hypotony [3 eyes (7.0%)] and phthisis bulbi [2 eyes (4.7%)]. CONCLUSION: Repeated MPTCP is at best moderately effective in lowering IOP for eyes with advanced glaucoma.

Clinical Efficacy and Safety Outcomes of Micropulse Transscleral Diode Cyclophotocoagulation in Patients With Advanced Glaucoma.

PRECIS: Micropulse transscleral cyclophotocoagulation (MPTCP) is only moderately effective in lowering intraocular pressure (IOP) and is useful as an adjunct procedure to other glaucoma surgeries. There was a small risk of loss of vision, prolonged hypotony, and phthisis bulbi. AIM: The aim of this study was to determine the efficacy and safety of a single MPTCP treatment for an Asian population with advanced glaucoma. METHODS: This is a retrospective single-center study of 207 eyes (207 patients) with advanced glaucoma which underwent first-time MPTCP between January 1, 2008, and March 31, 2018. Success was defined as IOP of 6 to 21 mm Hg or ≥20% reduction in IOP without an increase in glaucoma medication from baseline, and without glaucoma reoperation. The IOP, best-corrected visual acuity, and number of glaucoma medications were also analyzed. RESULTS: The mean (SD) age was 64.9±16.9 years. The mean follow-up duration was 18.7±16.2 months. The rate of success at postoperative years 1 and 2 follow-up was 44.1% and 32.6%, respectively. The median survival time of MPTCP was 9.0 months and 85 (40.9%) eyes received reoperation. The mean IOP decreased from 31.5±12.0 mm Hg preoperatively to 22.1±10.3 and 23.8±11.8 mm Hg at postoperative years 1 and 2, respectively (P<0.0001). The mean number of glaucoma medications was reduced from 3.3±1.0 preoperatively to 2.6±1.1 and 2.4±1.1 at postoperative years 1 and 2, respectively (P<0.0001). Significant complications included prolonged hypotony [1 eye (0.5%)], phthisis bulbi [7 eyes (3.4%)], and best-corrected visual acuity reduction [29 eyes (13.9%)]. CONCLUSION: Single first-time MPTCP for advanced glaucoma eyes was moderately effective in lowering IOP but >50% failed by 1 year.

Spread of a low-fitness drug-resistant Mycobacterium tuberculosis strain in a setting of high human immunodeficiency virus prevalence.

The fitness cost associated with the evolution of resistance to rifampin in Mycobacterium tuberculosis may be different in clinical isolates compared to in vitro-generated mutants. An atypical Beijing strain (attenuated phenotype) demonstrated the ability to spread despite acquiring resistance to rifampin. Transmission was linked to human immunodeficiency virus coinfection (P = 0.029), raising concern for the spread of drug resistance in vulnerable populations.