Photoacoustic flow cytometry

Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy.     

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted.     

In vivo photoacoustic and photothermal cytometry for monitoring multiple blood rheology parameters

Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo assessment of multiple hemorheological parameters over long periods of time has not been reported. Here, we review the capabilities of label-free photoacoustic (PA) and photothermal (PT) flow cytometry for dynamic monitoring of hemorhelogical parameters in vivo which we refer to as photoacoustic and photothermal blood rheology. Using phenomenological models, we analyze correlations between both PT and PA signal characteristics in the dynamic modes and following determinants of blood rheology: red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, hematocrit, and likely shear rate. We present ex vivo and in vivo experimental verifications involving high-speed PT imaging of RBCs, identification of sickle cells in a mouse model of human sickle cell disease and in vivo monitoring of complex hemorheological changes (e.g., RBC deformability, hematocrit and RBC aggregation). The multi-parameter platform that integrates PT, PA, and conventional optical techniques has potential for translation to clinical applications using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency.     

An evaluation of the genotoxic properties of some chosen dyes using the micronucleus test in vivo

The frequency of micronucleated polychromatic erythrocytes and the polychromatic to normochromatic erythrocyte ratio was studied in BalbC mice treated with four azo dyes: Direct Blue 74, Direct Blue 296, Direct Blue 297 and Direct Green 98 at two (40and 80% LD₅₀/kg body weight) concentrations. None of the studied compounds revealed a genotoxic activity in the micronucleus test. However, it was found that two dyes, Direct Blue 297 at doses 40% and 80% LD₅₀ and Direct Green 98 at dose 80% LD₅₀, cause a significant decrease in the ratio of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at the doses specified above they can affect the proliferation of the blood cells.     

Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

Photoacoustic and fluorescent effects in multilayer plasmon‐dye interfaces

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near‐infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold‐ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low‐toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near‐infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.      

Noninvasive in vivo monitoring of methemoglobin formation and reduction with broadband diffuse optical spectroscopy.

We present noninvasive, quantitative in vivo measurements of methemoglobin formation and reduction in a rabbit model using broadband diffuse optical spectroscopy (DOS). Broadband DOS combines multifrequency frequency-domain photon migration (FDPM) with time-independent near infrared (NIR) spectroscopy to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1,000 nm. Tissue concentrations (denoted by brackets) of methemoglobin ([MetHb]), deoxyhemoglobin ([Hb-R]), and oxyhemoglobin ([HbO2]) were determined from absorption spectra acquired in "real time" during nitrite infusions in nine pathogen-free New Zealand White rabbits. As little as 30 nM [MetHb] changes were detected for levels of [MetHb] that ranged from 0.80 to 5.72 microM, representing 2.2 to 14.9% of the total hemoglobin content (%MetHb). These values agreed well with on-site ex vivo cooximetry data (r2= 0.902, P < 0.0001, n = 4). The reduction of MetHb to functional hemoglobins was also carried out with intravenous injections of methylene blue (MB). As little as 10 nM changes in [MB] were detectable at levels of up to 150 nM in tissue. Our results demonstrate, for the first time, the ability of broadband DOS to noninvasively quantify real-time changes in [MetHb] and four additional chromophore concentrations ([Hb-R], [HbO2], [H2O], and [MB]) despite significant overlapping spectral features. These techniques are expected to be useful in evaluating dynamics of drug delivery and therapeutic efficacy in blood chemistry, human, and preclinical animal models.     

DNA interaction, cytotoxicity, and radiosensitization with PtCl4(Nile Blue)2 and PtCl4(Neutral Red)2

Complexes of the positively charged, nuclear staining, quinone-imine dyes Nile Blue and Neutral Red with negatively charged tetrachloroplatinum (II) have been prepared in an effort to form neutral drugs which could gain ready access to the cellular nucleus and deliver significant quantities of the reactive tetrachloroplatinum anion to the vicinity of the DNA. Elemental analysis showed that both the Nile Blue and Neutral Red complexes with tetrachloroplatinum (II) comprised 2 mol of dye and 1 mol of tetrachloroplatinum, forming Pt(Nile Blue)2 and Pt(Neutral Red)2. Exposure of superhelical pBR322 DNA to the complexes or the dyes for 24 h followed by agarose gel electrophoresis showed that Neutral Red and Pt(Neutral Red)2 had little effect on DNA conformation, but that both Nile Blue and Pt(Nile Blue)2 could produce single-strand DNA breaks in a dose-dependent fashion. Studies in exponentially growing asynchronous, hypoxic, and normally oxygenated EMT6 cells at normal pH (7.40) and pH 6.45 demonstrated that neither dye was highly toxic, but that both complexes were capable of producing significant cytotoxicity. Both complexes killed normally oxygenated cells more efficiently than hypoxic cells, but Pt(Neutral Red)2 was more cytotoxic at pH 6.45, while Pt(Nile Blue)2 killed significantly more cells at normal pH. Both complexes decreased the survival of hypoxic EMT6 cells as indicated by the slope of the radiation survival curve [dose modifying factor (DMF) 2.90 for Pt(Nile Blue)2 and 1.45 for Pt(Neutral Red)2]. Studies with the FSaIIC murine tumor showed that both complexes were active radiosensitizing agents in vivo [DMF 1.76 for Pt(Nile Blue)2 and 1.25 for Pt(Neutral Red)2]. These results indicate that these new platinum complexes have characteristics which may make them and similar complexes effective radiosensitizing agents in humans.     

Technical and physiological background of plasma volume measurement with indocyanine green: a clarification of misunderstandings.

The indocyanine green (ICG) dilution technique (DT) is frequently used for plasma volume (PV) measurement. However, because of inadequate knowledge about the properties of this dye, lack of accuracy has been attributed to the method. The aim of this report is to provide physiological background information about the ICG-DT to avoid some profound misunderstandings. When performing tracer dilution, one has to consider the tracer's distribution space before interpreting the result. For ICG, the distribution space is the total PV, i.e., circulating + noncirculating PV, fixed within the endothelial glycocalyx. The distribution space of red blood cells and large molecules, in contrast, is only the circulating part of PV. Therefore, it is erroneous to compare directly PV derived from different tracer dilution methods. The transcapillary escape rate of ICG should not relevantly influence measured PV if the method is performed properly, i.e., if a short time window of measurement is subjected to monoexponential extrapolation. A major problem of PV measurement in general is that the target itself is very inconstant. Thus, checking for constancy of ICG-DT with two consecutive measurements is unreliable. Nevertheless, the ICG-DT is a useful tool for determining PV, provided it is well understood by the investigator to enable correct interpretation of the results.     

Choice of Activity-Intensity Classification Thresholds Impacts upon Accelerometer-Assessed Physical Activity-Health Relationships in Children

Background

It is unknown whether using different published thresholds (PTs) for classifying physical activity (PA) impacts upon activity-health relationships. This study explored whether relationships between PA (sedentary [SED], light PA [LPA], moderate PA [MPA], moderate-to-vigorous PA, vigorous PA [VPA]) and health markers differed in children when classified using three different PTs.

Methods

104 children (63 girls) aged 10–14 years wore an RT3 triaxial accelerometer for seven days and measures of adiposity and cardiometabolic risk markers were taken.

Results

Significant associations (p< .05) in boys were found between LPA and body mass index z-score and waist circumference z-score for the Rowlands et al PT only (β =  .459 and.401, respectively) and body fat% (BF%) for the Chu et al PT only (β = .322) and in girls with BF% for the Rowlands et al PT only (β =  .303) and systolic BP and blood glucose for the Vanhelst et al PT only (β = −.298 and −.283, respectively). MPA was significantly (p<.05) associated with BF%, diastolic BP, and cardiorespiratory fitness (CRF) for the Chu et al PT only in girls (β = −.436, −.529, and .446, respectively). SED was significantly (p<.05) associated with triglycerides (β = .492) for the Rowlands et al PT only in boys and VPA with CRF (p<.05) for the Rowlands et al and Vanhelst et al PTs only in girls (β = .416 and .352, respectively).

Conclusions

The choice of PT impacted upon activity-health relationships. A consensus on appropriate accelerometer thresholds for quantifying PA intensity and sedentary behaviour is needed in order to make accurate evidence-based recommendations for health promotion.     

一株高效广谱染料降解细菌的分离鉴定及脱色特性研究

通过梯度驯化,从印染废水长期污染土壤中分离筛选出能以4种不同结构类型的染料(刚果红、美蓝、孔雀绿和活性艳蓝KN-R)为唯一碳源的菌株XSMR,根据其形态学特征和生理生化鉴定及16S rDNA序列分析,初步鉴定为无色杆菌属(Achromobacter sp.)的菌株。菌株XSMR对4种染料均具有强的脱色降解能力,且对染料脱色的同时,自身能够生长繁殖,培养24h菌体干重超过不加染料的对照。在振荡培养条件下对该菌株的脱色反应条件进行研究,结果表明,当刚果红、美蓝、孔雀绿及活性艳蓝KN-R的初始浓度分别小于200mg/L、200mg/L、150mg/L及150mg/L时,在pH7.5、温度35℃、接种量4%(V/V)条件下,接种菌株XSMR脱色14h对4种染料的脱色率均可达到98%以上。通过对降解产物的紫外-可见光谱分析,进一步证明了菌株XSMR能彻底降解染料。菌株XSMR对染料脱色的机理包括生物降解和菌株吸附两方面。     

Nanotechnology‐based molecular photoacoustic and photothermal flow cytometry platform for in‐vivo detection and killing of circulating cancer stem cells

In‐vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem‐like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet‐induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor‐initiating cells in circulation before they form metastasis. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In Vivo Magnetic Enrichment,Photoacoustic Diagnosis,and Photothermal Purging of Infected Blood Using Multifunctional Gold and Magnetic Nanoparticles

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic.     

Cytotoxicity, radiosensitization, and DNA interaction of platinum complexes of thiazin and xanthene dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosorption of dyes using dead macro fungi: effect of dye structure, ionic strength and pH

Biosorbents prepared from dead macro fungi, namely Fomes fomentarius and Phellinus igniarius, were applied for the uptake of Methylene Blue (MB) and Rhodamine B (RB). Equilibrium isotherm data could be well described by the Langmuir and Freundlich models. Methylene Blue was found to be more adsorbable than Rhodamine B. Langmuir monolayer coverage was determined as 204.38-232.73 mg/g and 25.12-36.82 mg/g for MB and RB, respectively. Molecular structure and ionic radius of dyes were found to be responsible for differences in their uptakes. Results showed that sorption of MB increased while that of RB decreased as pH of respective dye solutions changed from 3 to 11. An increase in ionic strength also exhibited an adverse effect on dye sorption capacity. Ionic strength and pH affected the sorption of MB more as compared to the sorption of RB. The presence of carboxylic (-ve) and amino (+ve) groups in RB could explain the lower sorption of RB compared to MB.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bioelectrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 μW cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 μW cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxidase bioanodes and/or biofuel cells.     

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G相似文献   

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic–PT switching of conventional and gold‐coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple‐modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near‐infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super‐resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)