Fluorescence Intensity Distribution Analysis Research Articles

Elevated Aβ aggregates in feces from Alzheimer’s disease patients: a proof-of-concept study

BackgroundMisfolding and aggregation of amyloid β (Aβ), along with neurofibrillary tangles consisting of aggregated Tau species, are pathological hallmarks of Alzheimer’s disease (AD) onset and progression. In this study, we hypothesized the clearance of Aβ aggregates from the brain and body into the gut.MethodsTo investigate this, we used surface-based fluorescence intensity distribution analysis (sFIDA) to determine the Aβ aggregate concentrations in feces from 26 AD patients and 31 healthy controls (HC).ResultsAβ aggregates were detectable in human feces and their concentrations were elevated in AD patients compared to HC (specificity 90.3%, sensitivity 53.8%).ConclusionThus, fecal Aβ aggregates constitute a non-invasive biomarker candidate for diagnosing AD. Whether digestion-resistant Aβ aggregates in feces are secreted via the liver and bile or directly from the enteric neuronal system remains to be elucidated.

IAPP - oligomerisation levels in plasma of people with type 2 diabetes

Islet amyloid polypeptide (IAPP) is co-secreted with insulin from pancreatic ß-cells. Its oligomerisation is regarded as disease driving force in type 2 diabetes (T2D) pathology. Up to now, IAPP oligomers have been detected in affected tissues. IAPP oligomer concentrations in blood have not been analysed so far. Using the IAPP single-oligomer-sensitive and monomer-insensitive surface-based fluorescence intensity distribution analysis (sFIDA) technology, levels of IAPP oligomers in blood plasma from healthy controls and people with T2D in different disease stages where determined. Subsequently, the level of IAPP oligomerisation was introduced as the ratio between the IAPP oligomers determined with sFIDA and the total IAPP concentration determined with ELISA. Highest oligomerisation levels were detected in plasma of people with T2D without late complication and without insulin therapy. Their levels stand out significantly from the control group. Healthy controls presented with the lowest oligomerisation levels in plasma. In people with T2D without complications, IAPP oligomerisation levels correlated with disease duration. The results clearly demonstrate that IAPP oligomerisation in insulin-naïve patients correlates with duration of T2D. Although a correlation per se does not identify, which is cause and what is consequence, this result supports the hypothesis that IAPP aggregation is the driving factor of T2D development and progression. The alternative and conventional hypothesis explains development of T2D with increasing insulin resistance causing exhaustion of pancreatic ß-cells due to over-secretion of insulin, and thus IAPP, too, resulting in subsequent IAPP aggregation and fibril deposition in the pancreas. Further experiments and comparative analyses with primary tissues are warranted.

Aβ oligomers peak in early stages of Alzheimer's disease preceding tau pathology.

Soluble amyloid beta (Aβ) oligomers have been suggested as initiating Aβ related neuropathologic change in Alzheimer's disease (AD) but their quantitative distribution and chronological sequence within the AD continuum remain unclear. A total of 526 participants in early clinical stages of AD and controls from a longitudinal cohort were neurobiologically classified for amyloid and tau pathology applying the AT(N) system. Aβ and tau oligomers in the quantified cerebrospinal fluid (CSF) were measured using surface-based fluorescence intensity distribution analysis (sFIDA) technology. Across groups, highest Aβ oligomer levels were found in A+ with subjective cognitive decline and mild cognitive impairment. Aβ oligomers were significantly higher in A+T- compared to A-T- and A+T+. APOEε4 allele carriers showed significantly higher Aβ oligomer levels. No differences in tau oligomers were detected. The accumulation of Aβ oligomers in the CSF peaks early within the AD continuum, preceding tau pathology. Disease-modifying treatments targeting Aβ oligomers might have the highest therapeutic effect in these disease stages. Using surface-based fluorescence intensity distribution analysis (sFIDA) technology, we quantified Aβ oligomers in cerebrospinal fluid (CSF) samples of the DZNE-Longitudinal Cognitive Impairment and Dementia (DELCODE) cohortAβ oligomers were significantly elevated in mild cognitive impairment (MCI)Amyloid-positive subjects in the subjective cognitive decline (SCD) group increased compared to the amyloid-negative control groupInterestingly, levels of Aβ oligomers decrease at advanced stages of the disease (A+T+), which might be explained by altered clearing mechanisms.

Disrupted-in-schizophrenia 1 protein aggregates in cerebrospinal fluid are elevated in patients with first-episode psychosis.

The disrupted-in-schizophrenia 1 (DISC1) protein is a key regulator at the intersection of major signaling pathways relevant for adaptive behavior. It is prone to posttranslational changes such as misassembly and aggregation but the significance of such transformations for human mental illness has remained unclear. We aimed to demonstrate the occurrence of DISC1 protein aggregates in patients with first-episode psychosis (FEP). Cerebrospinal fluid samples of patients with FEP (n = 50) and matched healthy controls (HCs; n = 47) were measured by the highly sensitive surface-based fluorescence intensity distribution analysis technology that enables single aggregate detection. We demonstrate that DISC1 protein aggregates are increased in cerebrospinal fluid samples of patients with FEP versus HCs. The concentration was in the low femtomolar range. No correlations were found with specific symptom levels, but the difference was particularly significant in the subset of patients with the diagnoses schizophrenia, unspecified (DSM-IV 295.9) or schizoaffective disorder (DSM-IV 295.70) at 18-month follow-up. DISC1 protein aggregate levels did not significantly change within the 18-month observation interval and were on average higher for individuals carrying the major DISC1 rs821577 allele, before correction. The occurrence of protein aggregates in vivo in patients with psychotic disorders has not been previously reported. It underscores the significance of posttranslational modifications of proteins both as pathogenetic mechanisms and as potential diagnostic markers in these disorders.

Development and Implementation of an Internal Quality Control Sample to Standardize Oligomer-Based Diagnostics of Alzheimer's Disease.

Protein misfolding and aggregation are pathological hallmarks of various neurodegenerative diseases. In Alzheimer's disease (AD), soluble and toxic amyloid-β (Aβ) oligomers are biomarker candidates for diagnostics and drug development. However, accurate quantification of Aβ oligomers in bodily fluids is challenging because extreme sensitivity and specificity are required. We previously introduced surface-based fluorescence intensity distribution analysis (sFIDA) with single-particle sensitivity. In this report, a preparation protocol for a synthetic Aβ oligomer sample was developed. This sample was used for internal quality control (IQC) to improve standardization, quality assurance, and routine application of oligomer-based diagnostic methods. We established an aggregation protocol for Aβ1-42, characterized the oligomers by atomic force microscopy (AFM), and assessed their application in sFIDA. Globular-shaped oligomers with a median size of 2.67 nm were detected by AFM, and sFIDA analysis of the Aβ1-42 oligomers yielded a femtomolar detection limit with high assay selectivity and dilution linearity over 5 log units. Lastly, we implemented a Shewhart chart for monitoring IQC performance over time, which is another important step toward quality assurance of oligomer-based diagnostic methods.

Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool

Misfolded and aggregated α-synuclein is a neuropathological hallmark of Parkinson’s disease (PD). Thus, α-synuclein aggregates are regarded as a biomarker for the development of diagnostic assays. Quantification of α-synuclein aggregates in body fluids is challenging, and requires highly sensitive and specific assays. Recent studies suggest that α-synuclein aggregates may be shed into stool. We used surface-based fluorescence intensity distribution analysis (sFIDA) to detect and quantify single particles of α-synuclein aggregates in stool of 94 PD patients, 72 isolated rapid eye movement sleep behavior disorder (iRBD) patients, and 51 healthy controls. We measured significantly elevated concentrations of α-synuclein aggregates in stool of iRBD patients versus those of controls (p = 0.024) or PD patients (p < 0.001). Our results show that α-synuclein aggregates are excreted in stool and can be measured using the sFIDA assay, which could support the diagnosis of prodromal synucleinopathies.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson’s disease (PD), dementia with Lewy bodies (DLB), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo

SummaryThe elimination of amyloid beta (Aβ) oligomers is a promising strategy for therapeutic drug development of Alzheimer’s disease (AD). AD mouse models that develop Aβ pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aβ oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aβ oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.

Binding of Metal-Ion-Induced Tau Oligomers to Lipid Surfaces Is Enhanced by GSK-3β-Mediated Phosphorylation

While fibrillar deposits of hyperphosphorylated protein tau are a key hallmark of several neurodegenerative diseases such as Alzheimer's disease, small oligomers have been speculated to be the key toxic aggregate species. Trivalent metal ions were shown to promote tau oligomer formation in vitro. However, little is known about potential intercellular spreading mechanisms or toxic modes of action of such oligomers. We investigated interactions of tau monomers and Fe3+/Al3+-induced oligomers with small unilamellar vesicles derived from 1-palmitoyl-2-oleoyl-phosphatidylcholine (neutral, liquid-crystalline phase) and dipalmitoyl-phosphatidylcholine (neutral, gel-phase). We further evaluated the influence of glycogen synthase kinase 3β (GSK-3β)-mediated tau phosphorylation applying the single-particle fluorescence spectroscopy techniques fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and scanning for intensely fluorescent targets. In these experiments, no binding to neutral lipid surfaces was observed for tau monomers. In contrast, metal-ion-induced tau oligomers showed a gain of function in binding to neutral lipid surfaces. Of note, tau phosphorylation by GSK-3β increased both oligomer formation and membrane affinity of the resulting oligomers. In conclusion, our data imply a pathological gain of function of metal-ion-induced oligomers of hyperphosphorylated tau, enabling membrane binding irrespective of surface charge even at nanomolar protein concentrations.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid β (Aβ) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aβ oligomers in body fluids are hampered by the low analyte concentration and presence of Aβ monomers. The surface-based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aβ. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α-synuclein oligomers, and significant improvement of essential assay parameters.

SFIDA: PLATFORM TECHNOLOGY FOR DIAGNOSTICS OF PROTEIN MISFOLDING DISEASES

Protein misfolding is a common hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases. Mounting evidence argues that especially smaller aggregates, so called oligomers, are the pathological culprits in these proteinopathies. Therefore the pathological oligomers might be the most direct and reliable biomarker of the respective disorder. To quantify oligomers in body fluids like blood plasma and cerebrospinal fluid (CSF), however, two major technical challenges have to be addressed: i) the extremely low concentration of oligomers and ii) the ubiquitous presence of an excess of monomers. We have developed a method designated sFIDA (surface-based fluorescence intensity distribution analysis) for quantitating single oligomers of amyloid beta, alpha-synuclein and prion protein, respectively. In sFIDA, protein aggregates are fixed on a capture-coated glass surface and loaded with at least two antibodies carrying two different fluorescence dyes. Single protein aggregates are detected by high-resolution imaging like laser scanning and total internal reflection fluorescence microscopy. The use of the same epitope for capturing and probing renders the assay insensitive against monomeric proteins. By sFIDA analysis of CSF samples, a small cohort of AD-affected patients could be distinguished from an age-matched control group. Here we present recent advancements of the sFIDA technology including application of standard molecules for calibration and quantification, assay automatization as well as novel data obtained on patient samples. The sFIDA assay will be employed for biomarker-based monitoring of therapeutic success in drug trials and further adapted for differential diagnostics of other protein misfolding diseases.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms.The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD.Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes.The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

SFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids

ObjectivesAlzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. We have previously introduced a platform technology designated ‘sFIDA’ for the quantitation of amyloid β peptide (Aβ) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay. Design and methodsIn sFIDA (surface-based fluorescence intensity distribution analysis) Aβ species are immobilized by a capture antibody to a glass surface. Aβ aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aβ aggregates, we used Aβ-conjugated silica nanoparticles (Aβ-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer. ResultsThe automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range. ConclusionsAutomation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics.

Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay.

Early diagnostics at the preclinical stage of Alzheimer's disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer's Disease.

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Fluorescence cumulants analysis with non-ideal observation profiles

One of the challenges of fluorescence fluctuation fpectroscopy (FFS) is an adequate approximation of a brightness profile. The key feature of fluorescence intensity distribution analysis (FIDA) is a polynomial approximation of a brightness profile. A broad range of brightness profile shapes can be well described by this approximation. A different approach consisting of the introduction of additional fitting parameters, defined as a relative difference between integrals of the actual brightness profile and its Gaussian approximation, is used in photon counting histogram (PCH) analysis. It is sufficient to introduce only one additional fitting parameter (first-order correction) to get an adequate fit to the experimental data in many practical applications.In the current study, we apply these approaches to the theory of time integrated fluorescence cumulants analysis. We demonstrate that developed corrections improve results of FFS analysis applied to simulated and experimental data. The use of different brightness profile approximations and normalizations in PCH and FIDA leads to different estimates of brightness and the number of molecules, even though they represent the same physical quantities. Based on the developed theory, we derive equations that relate brightness and the number of molecules in PCH and FIDA.

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection.

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.

Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.

Distribution Characterization with Fluorescence Cumulant Analysis

Fluorescence correlation spectroscopy (FCS) is a powerful tool for biophysical research, capable of monitoring concentrations and molecular interactions on a femtoliter scale. However, it is difficult in practice to resolve multiple molecular species using FCS unless there is a large difference in molecular weight. Several techniques have been developed to combat this problem, including photon counting histograms (PCH), fluorescence intensity distribution analysis (FIDA), and fluorescence cumulant analysis (FCA). These are capable of resolving multiple components on the basis of differences in molecular brightness, but have typically focused on studying samples with a small number of species. Fluorescence cumulant analysis can be extended to determine the parameters of brightness distributions containing many components, yielding a set of simple algebraic relations between the factorial cumulants of a measured fluorescence intensity series and the moments of the brightness distribution. Using PCH in conjunction with FCA, the best-fit distribution may be distinguished from several candidate models. We present simulations and experiments testing the applicability of this analysis. This technique may be useful in studies where the molecules of interest form a variety of multimers or aggregates.

Detection of Mobile Single Fluorescent Particles by Confocal Microscopy: A Comparison with Fluorescence Correlation Techniques

To improve our understanding of proteins interactions with cellular membranes, we have developed a method based on confocal microscopy that enable us to correlate the number of proteins bound to a lipid vesicle with the vesicle properties (membrane integrity, membrane potential, vesicle size¼). This method consists in the simultaneous recording of confocal images in two different channels, and in the coincidental detection of single mobile particles in these channels. To demonstrate the usefulness of this method, we have used a model system consisting of small unilamellar liposomes (∼ 100 nm in diameter) rendered fluorescent by the addition of a small percentage of fluorophore and interacting with fluorescently labeled proteins.To optimize image acquisition parameters for the detection of single diffusing liposomes, we systematically varied the fluorophore to vesicle ratio, the fluorescence excitation intensity, and the confocal pixel size and dwell time. We analyzed the images using different pixel binning values and different detection threshold. We then determined the range of parameters for which we were able to detect particles with independent positions. We also verified that the particle concentration and average particle fluorescence emission extracted from the images using this single particle detection method were consistent with results obtained by fluorescence correlation spectroscopy (FCS), raster-scanning image correlation spectroscopy (RICS) and fluorescence intensity distribution analysis (FIDA). Using vesicles labeled with two different fluorophores, we measured the probability that a vesicle detected in one channel would also be detected in the second channel, and compared our results with those obtained by fluorescence cross-correlation spectroscopy (XCS).Our method should be useful for studies of protein or peptide insertion and pore formation into lipid membranes.