Marcel Ameloot | Universiteit Hasselt (original) (raw)

Papers by Marcel Ameloot

ABSTRACTExtracellular vesicles (EV) are biological nanoparticles that play an important role in c... more ABSTRACTExtracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, we used multicolor single-molecule burst analysis microscopy to detect multiple biomarkers present on single EV. We classified the recorded signals and applied the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, we applied the method to assess both the purity and the inflammatory status of EV, and compared cell culture and plasma-derived EV isolated via different purification methods. We then applied this methodology to identify intercellular adhesion molecule-1 (...

Materials, 2019

Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agent... more Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agents. In this paper, we report the synthesis of PCDTBT (poly([9-(1’-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophene-diyl)) nanoparticles of varying sizes using the mini-emulsion and emulsion/solvent evaporation approach. The effect of the size of the particles on the optical properties is investigated using UV-Vis absorption and fluorescence emission spectroscopy. It is shown that PCDTBT nanoparticles have a fluorescence emission maximum around 710 nm, within the biological near-infrared “optical window”. The photoluminescence quantum yield shows a characteristic trend as a function of size. The particles are not cytotoxic and are taken up successfully by human lung cancer carcinoma A549 cells. Irrespective of the size, all particles show excellent fluorescent brightness for bioimaging. The fidelity of the particles as fluorescent probes to...

IUPAC Standards Online, 2016

The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular... more The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular concentrations of Ca2+ and K+, respectively. To investigate the complex forming reaction between Fura-2 and Ca2+, and between PBFI and K+ in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surfaces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution: (1) for Fura-2: k01 equals 1.2 X 109 s-1, k21 equals 1.0 X 1011 M-1x-1, k02 equals 5.5 X 108s-1, k12 equals 2.2 X 107s-1 (2) for PBFI: k01 equals 1.1 X 109s-1, k21 equals 2.7 X 108M-1s-1, k02 equals 1.8 X 109s-1, k12 equals 1.4 X 109s-1 k01 and k02 denote the deactivation rate constants of the free and bound forms of the indicator, respectively k21 represents the bimolecular rate constant of binding of the cation by the indicator whereas k12 is the rate constant of dissociation of the ca...

Biophysical Journal, 2019

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracti... more Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three-and fivefold. For D ¼ 1 mm 2 s À1 , a typical value for intracellular measurements, $25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (<<50 Â 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E ¼ 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM-mM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical leastsquares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.

Journal of biophotonics, 2018

Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human ... more Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label-free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump-probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump-probe approach allows label-free optical detection and unambig...

IUPAC Standards Online, 2016

The Journal of Physical Chemistry, 1995

In this report we investigate how, even in the absence of any a priori information, one can disti... more In this report we investigate how, even in the absence of any a priori information, one can distinguish between competing irreversible intramolecular two-state excited-state processes. Only two different kinetic models are possible for irreversible intramolecular two-state excited-state processes: (i) one with a unidirectional excited-state process and (ii) one without an excited-state process. The distinction between these models is based on the collection of a fluorescence decay surface with at least three quencher concentrations and the use of standard global biexponential and global compartmental analyses. Standard global biexponential analysis provides estimates for the preexponential factors and decay times which, together with the steady-state fluorescence spectrum, allow the construction of decay-associated emission spectra. Two series of global compartmental analyses have to be performed whereby (i) k01 is kept constant at various preset values while k12 is held fixed at zero and whereby (ii) k 0 2 is scanned while k21 is held constant at zero. kol denotes the rate constant of deactivation of excited species i*, klz represents the rate constant of transformation of excited species 2* into 1*, and k21 describes the opposite process. If negative preexponentials-indicative of a unidirectional excited-state process-are obtained in the global biexponential analysis, the statistical goodnessof-fit criteria of the repetitive global compartmental analyses allows one to determine the direction of the unidirectional excited-state process. When only positive preexponentials are obtained in the global biexponential analysis, the decay-associated emission spectra should be compared to the species-associated emission spectra. If both sets of spectra coincide, no excited-state process occurs. Otherwise, two solutions in which one rate constant of interconversion is zero are mathematically possible. It is impossible to distinguish between these two nontrivial alternative solutions.

Time-Resolved Laser Spectroscopy in Biochemistry II, 1990

fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms... more fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms of reaction rates and species associated spectra [Beechem et al., Chem. Phys. Letters 120 (1985) 466]. The identifiability of two-state excited state reactions has been investigated for properly normalized decay curves assuming that the ratio of the ground state absorbances is known [Ameloot et al, Chem. Phys. Letters 129 [1986] 21 1]. It is demonstrated in this paper that the condition of proper normalization is not always required. In addition, it is shown that the ratio of the absorbances of the species in the ground state can be obtained from fluorescence decay surfaces. The required experimental design is indicated.

Standardization and Quality Assurance in Fluorescence Measurements I

In this chapter we describe how artifacts can be avoided in the two most commonly used time-resol... more In this chapter we describe how artifacts can be avoided in the two most commonly used time-resolved fluorometries, namely the single-photon timing and the multifrequency phase-modulation techniques. The most frequently encountered artifacts (inner filter effect, autofluorescence, polarization effects, color effect, photobleaching, deoxygenation, pulse pile-up, and linearity of the time response in the time-to-amplitude converter) are described in detail and remedies are presented to avoid these pitfalls. An extensive list of fluorescence lifetime standards is presented, which allows the spectroscopist to calibrate and test time-resolved instruments for systematic errors.

Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994

ABSTRACT

Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994

ABSTRACTExtracellular vesicles (EV) are biological nanoparticles that play an important role in c... more ABSTRACTExtracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, we used multicolor single-molecule burst analysis microscopy to detect multiple biomarkers present on single EV. We classified the recorded signals and applied the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, we applied the method to assess both the purity and the inflammatory status of EV, and compared cell culture and plasma-derived EV isolated via different purification methods. We then applied this methodology to identify intercellular adhesion molecule-1 (...

Materials, 2019

Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agent... more Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agents. In this paper, we report the synthesis of PCDTBT (poly([9-(1’-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophene-diyl)) nanoparticles of varying sizes using the mini-emulsion and emulsion/solvent evaporation approach. The effect of the size of the particles on the optical properties is investigated using UV-Vis absorption and fluorescence emission spectroscopy. It is shown that PCDTBT nanoparticles have a fluorescence emission maximum around 710 nm, within the biological near-infrared “optical window”. The photoluminescence quantum yield shows a characteristic trend as a function of size. The particles are not cytotoxic and are taken up successfully by human lung cancer carcinoma A549 cells. Irrespective of the size, all particles show excellent fluorescent brightness for bioimaging. The fidelity of the particles as fluorescent probes to...

IUPAC Standards Online, 2016

The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular... more The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular concentrations of Ca2+ and K+, respectively. To investigate the complex forming reaction between Fura-2 and Ca2+, and between PBFI and K+ in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surfaces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution: (1) for Fura-2: k01 equals 1.2 X 109 s-1, k21 equals 1.0 X 1011 M-1x-1, k02 equals 5.5 X 108s-1, k12 equals 2.2 X 107s-1 (2) for PBFI: k01 equals 1.1 X 109s-1, k21 equals 2.7 X 108M-1s-1, k02 equals 1.8 X 109s-1, k12 equals 1.4 X 109s-1 k01 and k02 denote the deactivation rate constants of the free and bound forms of the indicator, respectively k21 represents the bimolecular rate constant of binding of the cation by the indicator whereas k12 is the rate constant of dissociation of the ca...

Biophysical Journal, 2019

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracti... more Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three-and fivefold. For D ¼ 1 mm 2 s À1 , a typical value for intracellular measurements, $25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (<<50 Â 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E ¼ 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM-mM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical leastsquares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.

Journal of biophotonics, 2018

Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human ... more Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label-free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump-probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump-probe approach allows label-free optical detection and unambig...

IUPAC Standards Online, 2016

The Journal of Physical Chemistry, 1995

In this report we investigate how, even in the absence of any a priori information, one can disti... more In this report we investigate how, even in the absence of any a priori information, one can distinguish between competing irreversible intramolecular two-state excited-state processes. Only two different kinetic models are possible for irreversible intramolecular two-state excited-state processes: (i) one with a unidirectional excited-state process and (ii) one without an excited-state process. The distinction between these models is based on the collection of a fluorescence decay surface with at least three quencher concentrations and the use of standard global biexponential and global compartmental analyses. Standard global biexponential analysis provides estimates for the preexponential factors and decay times which, together with the steady-state fluorescence spectrum, allow the construction of decay-associated emission spectra. Two series of global compartmental analyses have to be performed whereby (i) k01 is kept constant at various preset values while k12 is held fixed at zero and whereby (ii) k 0 2 is scanned while k21 is held constant at zero. kol denotes the rate constant of deactivation of excited species i*, klz represents the rate constant of transformation of excited species 2* into 1*, and k21 describes the opposite process. If negative preexponentials-indicative of a unidirectional excited-state process-are obtained in the global biexponential analysis, the statistical goodnessof-fit criteria of the repetitive global compartmental analyses allows one to determine the direction of the unidirectional excited-state process. When only positive preexponentials are obtained in the global biexponential analysis, the decay-associated emission spectra should be compared to the species-associated emission spectra. If both sets of spectra coincide, no excited-state process occurs. Otherwise, two solutions in which one rate constant of interconversion is zero are mathematically possible. It is impossible to distinguish between these two nontrivial alternative solutions.

Time-Resolved Laser Spectroscopy in Biochemistry II, 1990

fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms... more fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms of reaction rates and species associated spectra [Beechem et al., Chem. Phys. Letters 120 (1985) 466]. The identifiability of two-state excited state reactions has been investigated for properly normalized decay curves assuming that the ratio of the ground state absorbances is known [Ameloot et al, Chem. Phys. Letters 129 [1986] 21 1]. It is demonstrated in this paper that the condition of proper normalization is not always required. In addition, it is shown that the ratio of the absorbances of the species in the ground state can be obtained from fluorescence decay surfaces. The required experimental design is indicated.

Standardization and Quality Assurance in Fluorescence Measurements I

In this chapter we describe how artifacts can be avoided in the two most commonly used time-resol... more In this chapter we describe how artifacts can be avoided in the two most commonly used time-resolved fluorometries, namely the single-photon timing and the multifrequency phase-modulation techniques. The most frequently encountered artifacts (inner filter effect, autofluorescence, polarization effects, color effect, photobleaching, deoxygenation, pulse pile-up, and linearity of the time response in the time-to-amplitude converter) are described in detail and remedies are presented to avoid these pitfalls. An extensive list of fluorescence lifetime standards is presented, which allows the spectroscopist to calibrate and test time-resolved instruments for systematic errors.

Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994

ABSTRACT

Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994