In this work, we theoretically and experimentally deal with photoacoustic resolution enhancement by means of saturated modulation quenching. It is shown that experimental systems for resolution enhancement with saturated modulation quenching in fluorescence microscopy are not necessarily suited for photoacoustic modulation quenching. Here, we show that modulation quenching is not limited to fluorescent dyes but can be also applied to metallic nanoparticles. For modulation quenching in photoacoustic microscopy it is sufficient that the signal saturates with increasing excitation intensity.
KEYWORDS: Quantum dots, Cadmium, In vivo imaging, Confocal microscopy, In vitro testing, Toxicology, Tissues, Transmission electron microscopy, Luminescence
Cadmium still represents a stigma for many research- and/or industrial applications. Some deleterious effects are attributed to Cadmium. In the present work, highly fluorescent Cadmium sulfide quantum dots are investigated by e.g. physical-chemical characterization. Most important however is their application as fluorescent probes for bio-imaging in living cells and tissues. This work presents their toxicological evaluation by means of in vivo studies. Bio-imaging experiments are performed without any pre-treatment. The toxicological studies performed, strongly indicate that the use of Cadmium based nanoparticles as fluorescent probes may be nonhazardous and not induce side effects for cells/tissues.
QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.
Biopsies are conventionally performed in two dimensions. Histological slices in general present some micrometers in thickness, allowing that some molecular domains stay out of the resulting image. Thus the histopathological assay potentially is based on an incomplete set of information. The use of quantum dots as fluorescente probes allows the investigation of labelling pattern and biomarkers expression, along the three-dimensions of fresh histological slices, leading to more precise results. Present work show and discuss pattern and fluorescence intensity emission at the visible region obtained as a function of tissue thickness in histological (thickness(z)=7.6μm) breast cancer samples labeled with compact (7-10 nm) water soluble quantum dots. Series of 154 three-dimensional (3D) images were recorded from each tissue sample by laser scanning confocal microscopy, using 488 nm excitation.. In order to compare the results obtained, all the acquisition parameters were maintained constant. Results point to the possibility of more accurate histological diagnostics, once they clearly show distinct labeling patterns across sample thickness.
In the last few years, quantum confinement effects in semiconductor nanocrystals (quantum dots - QDs) have attracted a
significant amount of interest due to their new optical properties and also because of their potential applications in
biological systems. In this work, cadmium sulphide (CdS) nanoparticles were synthesized in aqueous medium and
passivated with Cd(OH)2. Polyphosphate ions were used in order to avoid particle aggregation. After the passivation
step, CdS/Cd(OH)2 quantum dots were coated with silica. Silica coating has been extensively investigated concerning its
properties in biocompatibilizing QDs to biological systems. Silica coated core-shell CdS/Cd(OH)2 water soluble QDs
optical properties were studied by absorption, excitation and emission spectroscopies, while their morphological
characterization was carried out by transmission electron microscopy.
ZnSe quantum dots are wide-band gap materials suitable for UV-blue-emitting applications like laser diode or
biolabeling probes. Initially, an organometallic approach have been proposed for preparing these quantum dots with high
quantum yield (QY), crystallinity and monodispersity. Although, the chemicals used in this procedures are toxic,
expensive, pyrophoric and even explosive. Aiming to develop a more convenient and applicable methodology we
propose an alternative methods to synthesize blue-emitting ZnSe/ZnS nanocrystals in aqueous media. This work presents
a new methodology using chemometric analysis for improving the quantum yield of these blue probes under aqueous
colloidal synthesis. The structural characterization performed by X-Ray diffraction and transmission electronic
microscopy shows that this material is in the strong confinement regime with average diameter of 2.5 nm with zinc blend
crystalline structure. The optical properties of the colloids demonstrate a large Stoke´s shift between the absorption band
(360 nm) and the emission peak (420 nm) with a half maximum at full width of 30 nm and a maximum quantum yield of
43%.
Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell
antibodies. For this reason, simple, cheap and reproducible routes of QDs´s syntheses are the main goal of many
researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow
cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and
applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous
route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can
target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as
biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with
permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non
permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In
this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully
functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for
A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method
shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient
and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of
equipments.
This works deals with the use of quantum chemical calculations in order to investigate the interaction between
cadmium telluride quantum dot (CdTe) and the following functionalizers: mecapto acetic acid (MAA),
mercaptopropionic acid (MPA) and cysteine (CYS). This interaction between the quantum dot and each
functionalizer was quantified by an energy value resulting from theoretical calculations. Interesting features relating
this theoretical energy and the experimental results related to stability of these systems were found. The passivation
of the CdTe core generating a CdS shell was also considered in the calculations.
Semiconductor colloidal quantum dots have been, for the past two decades, incorporated in a wide range of
applications from catalysis and optical sensors to biolabels. For this reason, simple, cheap and reproducible routes of
synthesis are the main goal of many research groups around the world. They seek the production of a very stable and
extremely quantum efficient nanocrystal that can afford rough changes in the external environment. Silica capping is
becoming a very common tool in the quest for a stable quantum dot, because of its strong and stable structure, this
material provides a great insulator to the nanocrystal from the outside. The nanocrystal surface is not chemically
favorable to the deposition of the bare silica shell, what demands a bifunctional molecule that provides the linkage
between the core and the shell. In this work we present a comparison between several silanization methods of thiol
capped CdSe and CdTe quantum dots, showing some simplifications of the routes and an application of the quantum dots
produced as fluorescent cell markers in acquisition of confocal microscopy images.
Hybrid nanomaterials consisted of II-VI semiconductor quantum dots covered by layers of functionalizing organic
compounds, may be prepared to develop good biolabeling and consequent bioimaging applications. In this study we
present some well succeeded results related to the preparation, functionalization, and to the use of II-VI semiconductor
quantum dots, prepared via colloidal synthesis in aqueous medium, as highly luminescent labels for bioimaging. The
resulting systems possess the combined properties of both forming elements: the optical properties of the quantum dots
and the biological functionalities provided by the organic capping layers, which leads to specific association to biological
systems.These nanostructured materials nanoparticles presented good optical properties and excellent resistance to
photodegradation. Their conjugation to biological samples was evaluated by their fluorescence intensities and patterns,
by using conventional fluorescence and laser scanning confocal microscopies. The resulting images showed very good
quality concerning morphological features of the analyzed cells and tissues.
In this study we use CdS/Cd(OH)2 quantum dots functionalized with concanavalin-A (Con-A) lectin, specific to glucose/mannose residues, to investigate cell alterations regarding carbohydrate profile in human mammary tissues diagnosed as fibroadenoma (benign tumor). These particles were functionalized with glutaraldehyde and Con-A and incubated with tissue sections of normal and to Fibroadenoma, a benign type of mammary tumor. The tissue sections were deparafinized, hydrated in graded alcohol and treated with a solution of Evans Blue in order to avoid autofluorescence. The fluorescence intensity of QD-Con-A stained tissues showed different patterns, which reflect the carbohydrate expression of glucose/mannose in fibroadenoma when compared to the detection of the normal carbohydrate expression. The pattern of unspecific labeling of the tissues with glutaraldehyde functionalized CdS/Cd(OH)2 quantum dots is compared to the targeting driven by the Con-A lectin. The preliminary findings reported here support the use of CdS/Cd(OH)2 quantum dots as specific probes of cellular alterations and their use in diagnostics.
In this work we present the preparation, characterization and conjugation of colloidal core shell CdS-Cd(OH)2 quantum dots to health and cancer glial rats living cells in culture media. The particles were obtained via colloidal synthesis in aqueous medium, with final pH=7.3-7.4. Laser Scan Confocal Microscopy (LSCM) and Fluorescence Microscopy were used to evaluate fluorescence intensities and patterns of health and cancer (glioblastoma) glial cells labeled with the quantum dots in different time intervals. Health and cancer glial cells clearly differ in their fluorescence intensities and patterns. These different fluorescence intensities and patterns may be associated to differences concerning cellular membrane and metabolic features of health and cancer cells. The results obtained indicate the potential of the methodology for fast and precise cancer diagnostics.
Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well known features which make them very useful tools for biological labeling purposes. Low photo-bleaching rates, high chemical stability, active surface allowing conjugation to living cells, explains the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with high fluorescent core-shell CdTe-CdS (diameter = 3-7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules, incubated with living yeast cells, in order to investigate their glucose up-take activity.
In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λexc=795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.
In this work we developed a setup consisting of an Optical Tweezers equipped with linear and non-linear micro-spectroscopy system to add the capabilities of manipulation and analysing captured objects. Our setup includes a homemade confocal spectrometer using a monochromator equipped with a liquid nitrogen cooled CCD. The spectroscopic laser system included a cw and a femtosecond Ti:sapphire lasers that allowed us to perform Raman, hyper-Raman, hyper-Rayleigh and two photon Excited (TPE) luminescence in particles trapped with an Nd:YAG cw laser. We obtained Raman spectra of a single trapped polystyrene microsphere and a single trapped red blood cell to evaluate the performance of our system. We also observed hyper-Rayleigh and hyper-Raman peaks for SrTiO3 with 60s integration time only. This was possible because the repetition rate of the femtosecond Ti:sapphire lasers, on the order of 80 MHz, are much higher than the few kHz typical picosecond laser repetition rate used before in hyper- Raman experiment, which required acquisition times of order of few hours. We used this system to perform scanning microscopy and to acquire TPE luminescence spectra of captured single stained microsphere and cells conjugated with quantum dots of CdS and CdTe and hyper-Rayleigh spectra of a noncaptured ZnSe microparticle. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.
We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.
Semiconductor nanocrystals composed by few hundred to a few thousand atoms also known as quantum dots have received substantial attention due to their size tunable narrow-emission spectra and several other advantages over organic molecules as fluorescent labels for biological applications, including resistence to photodegradation, improved brightness and only one laser excitation that enable the monitoring of several processes simultaneously. In this work we have synthesized and characterized thiol-capped CdTe and bioconjugated them to macrophages. We have mapped the fluoroscence intensity along the macrophage's body in our set up consisting of an optical tweezer plus a non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited luminescence.
We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.
In this work we used our set up consisting of an optical tweezers plus non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited (TPE) luminescence of captured single cells conjugated with quantum dots of CdS and CdTe. The CdS nanocrystals are obtained by our group via colloidal synthesis in aqueous medium with final pH = 7 using sodium polyphosphate as the stabilizing agent. In a second step the surface of CdS particles is functionalized with linking agents such as Glutaraldehyde. The CdTe quantum dots are functionalized in the its proper synthesis using mercaptoacetic acid (AMA). We used a femtosecond Ti:sapphire laser to excite the hyper Rayleigh or TPE luminescence in particles trapped with an Nd:YAG cw laser and a 30 cm monochromator equipped with a cooled back illuminated CCD to select the spectral region for imaging. With this system we obtained hyper Rayleigh and TPE luminescence images of macrophages and other samples. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.
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