Biomedical subjects

Santiago Costantino

Publications and source records attributed to Santiago Costantino.

7 recordsLinked to original sources

PubMed2025

Focus on numbers - characterizing protein accumulation at DNA double-strand breaks.

Unrepaired DNA double-strand breaks can lead to cell death or genomic rearrangements. The DNA damage response (DDR) is a complex signaling cascade in which a plethora of factors act to finely tune repair pathway choice. Several DDR proteins have been shown to accumulate at sites of DNA lesions in characteristic dot-like structures known as DNA repair foci. Changes in foci brightness, commonly expressed in arbitrary intensity units, are often used as readout for DNA repair dynamics. However, due in part to technical challenges, the stoichiometry, absolute number of proteins recruited to DDR foci, and their impact on the resolution of the break remain incompletely characterized. Here, we combine spatial intensity distribution analysis (SpIDA) and a custom foci detection algorithm into an easy-to-use pipeline that, starting from confocal images, allows quantitative description of protein accumulation in DNA repair foci. Moreover, by quantifying foci based on their molecular count, SpIDA overcomes the limitations of ambiguous intensity units, enabling stoichiometric quantification between repair factors and providing a unifying means for experimental comparisons.

DNA Breaks, Double-Stranded↗

PubMed2006

A guide to accurate fluorescence microscopy colocalization measurements.

Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed on fluorescence microscopy images of fixed or live cells have been routinely applied for biophysical and cell biological studies. These approaches measure the fraction of interacting particles by analyzing dual color fluorescence images for colocalized pixels. Colocalization algorithms have proven to be effective, although the dynamic range and accuracy of these measurements has never been well established. Spatial image cross-correlation spectroscopy (ICCS), which cross-correlates spatial intensity fluctuations recorded in images from two detection channels simultaneously, has also recently been shown to be an effective measure of colocalization as well. Through simulations, imaging of fluorescent antibodies adsorbed on glass and cell measurements, we show that ICCS performs much better than standard colocalization algorithms at moderate to high densities of particles, which are often encountered in cellular systems. Furthermore, it was found that the density ratio between the two labeled species of interest plays a major role in the accuracy of the colocalization analysis. By applying a direct and systematic comparison between the standard, fluorescence microscopy colocalization algorithm and spatial ICCS, we show regimes where each approach is applicable, and more importantly, where they fail to yield accurate results.

Algorithms↗

PubMed2006

Measurement of monomer-oligomer distributions via fluorescence moment image analysis.

We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from individual images for specific ranges of densities and particle brightness ratios. Computer simulations and experiments with fluorescent microspheres and cells were performed to illustrate the detection limits and accuracy of this statistical approach. The simulation results show that the concentration of the dimer or oligomer population should be less than or equal to the monomeric concentration for the method to provide accurate results, and that the upper density detection limit of the population of monomers is one order-of-magnitude higher than the concentration of the oligomers. We implemented this technique to resolve two populations of fluorescent microspheres with different brightness ratios and we also applied the moment-analysis method to examine the distribution of aggregation states of PDGF-beta receptors in human fibroblast cells. The method was able to resolve a tetrameric population of the PDGF-beta receptors relative to the background distribution of nonspecifically bound fluorophore.

Cells, Cultured↗

PubMed2005

Two-photon fluorescent microlithography for live-cell imaging.

Fluorescent dyes added to UV-cure resins allow the rapid fabrication of fluorescent micropatterns on standard glass coverslips by two-photon optical lithography. We use this lithographic method to tailor fiduciary markers, focal references, and calibration tools, for fluorescence and laser scanning microscopy. Fluorescent microlithography provides spatial landmarks to quantify molecular transport, cell growth and migration, and to compensate for focal drift during time-lapse imaging. We show that the fluorescent patterned microstructures are biocompatible with cultures of mammalian cell lines and hippocampal neurons. Furthermore, the high-relief topology of the lithographed substrates is utilized as a mold for poly(dimethylsiloxane) stamps to create protein patterns by microcontact printing, representing an alternative to the current etching techniques. We present two different applications of such protein patterns for localizing cell adhesion and guidance of neurite outgrowth.

Animals↗

PubMed2005

Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy.

We present an extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS). Using simulations of laser scanning microscopy image time series, we investigate the effect of spatiotemporal sampling, particle density, noise, sampling frequency, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS. We show that the recovery of transport coefficients is usually limited by spatial sampling, while the measurement of accurate number densities is restricted by background noise in an image series. We also demonstrate that photobleaching of the fluorophore causes a consistent overestimation of diffusion coefficients and flow rates, and a severe underestimation of number densities. We derive a bleaching correction equation that removes both of these biases when used to fit temporal autocorrelation functions, without increasing the number of fit parameters. Finally, we image the basal membrane of a CHO cell with EGFP/alpha-actinin, using two-photon microscopy, and analyze a subregion of this series using TICS and apply the bleaching correction. We show that the photobleaching correction can be determined simply by using the average image intensities from the time series, and we use the simulations to provide good estimates of the accuracy and precision of the number density and transport coefficients measured with TICS.

Algorithms↗

PubMed2005

Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy.

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 microm(-2) within a 121 x 98 pixel subregion of a single cell.

Algorithms↗

PubMed2005

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Actinin↗