Descending aorta subject-specific one-dimensional model validated against in vivo data.

The aorta plays a major role in the cardiovascular system and its function and structure are primarily affected by aging, eating habits, life style and other cardiovascular risk factors, inducing increased stiffness which is associated with cardiovascular and cerebral morbi-mortality. Our objective was to develop and validate a robust subject-specific one-dimensional wave propagation numerical model of the descending aorta. This model with a cross-sectional area, velocity and pressure formulation is built using geometric and hemodynamic data measured on a specific person and is validated against in vivo data acquired on the same subject at three distinct anatomical locations along the thoracic aorta. We studied seven healthy volunteers, who underwent carotid applanation tonometry and aortic cardiovascular magnetic resonance (CMR). Responses of our model in terms of changes in central pressure waveform with arterial alterations were consistent with previously described physiological knowledge. Quantitative validation averaged over the three descending aortic locations and the seven subjects provided low rms errors (given in percentage of the maximal clinical value) between simulated and CMR data, i.e. area: 10±6%, velocity: 11±3%, flow rate: 9±3%. Finally, we also found low rms (5±2%) when comparing simulated pressure in the proximal aortic location against tonometric carotid pressure curves. In conclusion, this simple model performs similar to more complex models of the entire systemic arterial tree at a fraction of the cost, and could be of major usefulness in the non-invasive and local estimation of proximal biomechanical and hemodynamic indices.

Comparison of various methods for quantitative evaluation of myocardial infarct volume from magnetic resonance delayed enhancement data.

BACKGROUND: Late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) enables the estimation of myocardial infarct (MI) extent. Nevertheless, manual quantification is time consuming and subjective. We sought to assess MI volume with different quantitative methods in both acute (AMI) and chronic MI (CMI). METHODS: CMR was performed 50 ± 21 h after MI in 52 patients and was repeated 100 ± 21 days later in a subgroup of 34 patients. Then, necrosis volumes were quantified using: 1) manual delineation, 2) automated fuzzy c-means method, and 3) +2 to 6 SD thresholding approaches. Results were compared against peak values of serum Troponin I (TnI), creatine kinase (CK) and left ventricular (LV) functional parameters: LV ejection fraction (LVEF), indexed end-diastolic (EDVi), end-systolic volumes (ESVi) and the number of hypokinetic segments (NbHk). RESULTS: For CMI, quantitative evaluation of infarct size using manual, +2SD, +3 SD and fuzzy c-means provided equivalent results in terms of correlation coefficients for comparisons of MI volumes against LV function parameters (LVEF: r>0.79, p<0.0001; ESVi: r>0.82, p<0.0001, EDVi: r>0.67, p<0.0001, NbHk: r>0.54, p<0.0009). For AMI, +2SD and fuzzy c-means approaches provided higher correlations for comparisons of AMI volumes against biochemical markers (CK: r>0.79, p<0.0001,TnI: r>0.77, p<0.0001) and chronic LV function parameters (LVEF: r>0.82, p<0.0001, NbHk: r>0.59, p<0.0002). CONCLUSIONS: The fuzzy c-means and 2SD methods provided highest correlations with biochemical MI quantification as well as LV function parameters. The fuzzy c-means approach which does not require an arbitrary identification of the remote myocardium is fast and reproducible. It may be clinically useful in the evaluation of patients with MI.

Methodology for jointly assessing myocardial infarct extent and regional contraction in 3-D CMRI.

Automated extraction of quantitative parameters from cardiac magnetic resonance images is crucial for the management of patients with myocardial infarct. This paper proposes a postprocessing procedure to jointly analyze Cine and delayed-enhanced (DE) acquisitions, in order to provide an automatic quantification of myocardial contraction and enhancement parameters and a study of their relationship. For that purpose, the following processes are performed: 1) DE/Cine temporal synchronization and 3-D scan alignment, 2) 3-D DE/Cine rigid registration in a region about the heart, 3) myocardium segmentation on Cine-MRI and superimposition of the epicardial and endocardial contours on the DE images, 4) quantification of the myocardial infarct extent (MIE), 5) study of the regional contractile function using a new index, the amplitude to time ratio (ATR). The whole procedure was applied to ten patients with clinically proven myocardial infarction. The comparison between the MIE and the visually assessed regional function scores demonstrated that the MIE is highly related to the severity of the wall motion abnormality. In addition, it was shown that the newly developed regional myocardial contraction parameter (ATR) decreases significantly in delayed enhanced regions. This largely automated approach enables the combined study of regional MIE and left ventricular function.

An automatic respiratory gating method for the improvement of microcirculation evaluation: application to contrast-enhanced ultrasound studies of focal liver lesions.

Contrast-enhanced ultrasound (CEUS), with the recent development of both contrast-specific imaging modalities and microbubble-based contrast agents, allows noninvasive quantification of microcirculation in vivo. Nevertheless, functional parameters obtained by modeling contrast uptake kinetics could be impaired by respiratory motion. Accordingly, we developed an automatic respiratory gating method and tested it on 35 CEUS hepatic datasets with focal lesions. Each dataset included fundamental mode and cadence contrast pulse sequencing (CPS) mode sequences acquired simultaneously. The developed method consisted in (1) the estimation of the respiratory kinetics as a linear combination of the first components provided by a principal components analysis constrained by a prior knowledge on the respiratory rate in the frequency domain, (2) the automated generation of two respiratory-gated subsequences from the CPS mode sequence by detecting end-of-inspiration and end-of-expiration phases from the respiratory kinetics. The fundamental mode enabled a more reliable estimation of the respiratory kinetics than the CPS mode. The k-means algorithm was applied on both the original CPS mode sequences and the respiratory-gated subsequences resulting in clustering maps and associated mean kinetics. Our respiratory gating process allowed better superimposition of manually drawn lesion contours on k-means clustering maps as well as substantial improvement of the quality of contrast uptake kinetics. While the quality of maps and kinetics was satisfactory in only 11/35 datasets before gating, it was satisfactory in 34/35 datasets after gating. Moreover, noise amplitude estimated within the delineated lesions was reduced from 62 ± 21 to 40 ± 10 (p < 0.01) after gating. These findings were supported by the low residual horizontal (0.44 ± 0.29 mm) and vertical (0.15 ± 0.16 mm) shifts found during manual motion correction of each respiratory-gated subsequence. The developed technique could be used as a basis for accurate quantification of perfusion parameters for the evaluation and follow-up of patients under antiangiogenic therapies.

An automated four-point scale scoring of segmental wall motion in echocardiography using quantified parametric images.

The aim of this paper is to develop an automated method which operates on echocardiographic dynamic loops for classifying the left ventricular regional wall motion (RWM) in a four-point scale. A non-selected group of 37 patients (2 and 4 chamber views) was studied. Each view was segmented according to the standardized segmentation using three manually positioned anatomical landmarks (the apex and the angles of the mitral annulus). The segmented data were analyzed by two independent experienced echocardiographists and the consensual RWM scores were used as a reference for comparisons. A fast and automatic parametric imaging method was used to compute and display as static color-coded parametric images both temporal and motion information contained in left ventricular dynamic echocardiograms. The amplitude and time parametric images were provided to a cardiologist for visual analysis of RWM and used for RWM quantification. A cross-validation method was applied to the segmental quantitative indices for classifying RWM in a four-point scale. A total of 518 segments were analyzed. Comparison between visual interpretation of parametric images and the reference reading resulted in an absolute agreement (Aa) of 66% and a relative agreement (Ra) of 96% and kappa (κ) coefficient of 0.61. Comparison of the automated RWM scoring against the same reference provided Aa = 64%, Ra = 96% and κ = 0.64 on the validation subset. Finally, linear regression analysis between the global quantitative index and global reference scores as well as ejection fraction resulted in correlations of 0.85 and 0.79. A new automated four-point scale scoring of RWM was developed and tested in a non-selected database. Its comparison against a consensual visual reading of dynamic echocardiograms showed its ability to classify RWM abnormalities.

Renal blood flow quantification in pigs using contrast-enhanced ultrasound: an ex vivo study.

PURPOSE: The aim of the study was to evaluate a new method for the quantification of renal blood flow using contrast-enhanced ultrasound (CEUS) in an ex vivo pig kidney model. MATERIAL AND METHODS: After approval by the animal ethics committee, 4 pig kidneys were explanted and perfused with Celsior liquid (Imtix Sangstat, Lyon, France) at different flow rates (30, 50, 70 and 90 ml/min) in an ex vivo phantom. A 50 % diluted solution of SonoVue (Bracco, Milano, Italy) was infused in the artery at 0.5 ml/min. CEUS was performed with an Aplio system (Toshiba, Nasu, Japan) using a broadband linear transducer and pulse subtraction imaging. A total of 152 destruction-reperfusion sequences were acquired and cine loops were digitally stored for further quantification. Three different ROIs were placed upon the anterior, posterior cortex and segmental artery. Signal intensity measurements were performed in linear units and perfusion parameters were automatically extracted using dedicated software. Curve fitting was performed using a monoexponential model in which a time delay parameter was introduced. This fit allowed the assessment of the local blood flow into the region of interest (called "contrast-enhanced blood flow" (CEBF)). The artery mean signal intensity was averaged from the ten frames prior to the destruction phase. The normalized CEBF (nCEBF) was calculated as the ratio between CEBF and the mean arterial signal intensity. The CEBF and nCEBF were compared to the true blood flow indicated by the pump flow rate. RESULTS: The CEBF was correlated to the true blood flow only for the posterior cortical ROI (R(2) = 0.45, p = 0.05). The normalization using arterial signals improved CEBF correlation to true blood flow: nCEBF became correlated to the true blood flow when considering all ROIs (R(2)= 0.94, p < 0.0001) and correlation was improved for both anterior and posterior cortical ROIs (R(2)= 0, 93, p = 0.0004; R(2)= 0, 90, p = 0.0005, respectively). However, a significant kidney-dependent effect was observed for the anterior cortical ROI (p = 0.017) but not for the posterior cortical ROI (p = 0.89). CONCLUSION: Normalization using arterial signals significantly improved the estimation of blood flow calculated with CEUS.

An automated quantification of the transmural myocardial infarct extent using cardiac DE-MR images.

Evaluating myocardial viability is an important prognostic factor in the follow-up of infarctions. Delayed Enhancement magnetic resonance (DE-MR) imaging allows precise delineation of the infarct transmural extent. Visual interpretation is the most commonly used method to assess the myocardial infarction (MI) transmural extent. This study proposes to automate the segmentation of the (DE) images prior to the estimation of the extent of infarcted tissue. Indeed the segmentation of the myocardium was performed using cine contraction images which present a high contrast between cavity and myocardium. After the segmentation, the segmental transmurality is estimated on a conventional five point scale. A head to head comparison was performed between visual and quantitative analysis of infarct transmurality on DE-MR imaging. Results on 921 sub-segments (9 patients) showed an absolute agreement of 80% and a relative agreement (with one point difference) of 97%.

[Tissue attenuation in small animals on contrast enhanced ultrasound]. / Prise en compte de l'atténuation ultrasonore en présence d'agents de contraste chez le petit animal.

Despite recent advances in contrast-enhanced ultrasound imaging, evaluation of tissue perfusion with contrast-enhanced ultrasound is still impaired by shadowing effects. These effects are particularly relevant in small animal studies due to high frequency imaging. Current methods of tissue attenuation correction are not suited for contrast-enhanced ultrasound examinations, because microbubble acoustic response to ultrasound waves is far more complex than that of tissues. A method allowing in vivo tissue attenuation correction in the presence of contrast agents is presented.

An automated myocardial segmentation in cardiac MRI.

In this paper we present an automatic approach to segment Cardiac Magnetic Resonance (CMR) images. A preprocessing step that consists in filtering the image using connected operators (area opening and closing filters) is applied in order to homogenize the cavity and solve the problems due to the papillary muscles. Thereby the GVF snake algorithm is applied with one point clicked in the cavity as initialization and an optimized tuning of parameters for the endocardial contour extraction. The epicardial border is then obtained using the endocardium as initialization. The performance of the proposed method was assessed by experimentation on thirty-nine CMR images. A high agreement between manual and automatic contours was obtained with correlation scores of 0.96 for the endocardium and 0.90 for the epicardium. Overlapping percentage, mean and maximum distances between the two contours show a good performance of the method.

A posteriori respiratory gating in contrast ultrasound for assessment of hepatic perfusion.

An original strategy is proposed to minimize the impact of respiratory motion on hepatic contrast-enhanced ultrasound studies. It is based on the a posteriori triggering of dynamic image sequences. It was tested on perfusion studies acquired with a high temporal resolution (8 images s-1) to enable parametric imaging. A respiratory component was first estimated by independent component analysis. The estimation of the local minima and maxima of this curve enabled us to select two subsets of frames, corresponding to the end-of-inspiration plane and to the end-of-expiration plane. Both subsets were simultaneously analysed using factor analysis of medical image sequences. This method identified the main contrast uptake kinetics and their associated localizations. The global strategy was validated firstly on a simulated study and then applied to 11 patients' studies. In both cases, the frame selection was judged relevant and a necessary preliminary step before applying methods of parametric imaging. In conclusion, the a posteriori gating method that is proposed is a first step towards local quantification of hepatic contrast-enhanced ultrasound studies.

Assessment of left ventricular contraction by parametric analysis of main motion (PAMM): theory and application for echocardiography.

The computerized study of the regional contraction of the left ventricle has undergone numerous developments, particularly in relation to echocardiography. A new method, parametric analysis of main motion (PAMM), is proposed in order to synthesize the information contained in a cine loop of images in parametric images. PAMM determines, for the intensity variation time curves (IVTC) observed in each pixel, two amplitude coefficients characterizing the continuous component and the alternating component; the variable component is generated from a mother curve by introducing a time shift coefficient and a scale coefficient. Two approaches, a PAMM data driven and a PAMM model driven (simpler and faster), are proposed. On the basis of the four coefficients, an amplitude image and an image of mean contraction time are synthesized and interpreted by a cardiologist. In all cases, both PAMM methods allow better IVTC adjustment than the other methods of parametric imaging used in echocardiography. A preliminary database comprising 70 segments is scored and compared with the visual analysis, taken from a consensus of two expert interpreters. The levels of absolute and relative concordance are 79% and 97%. PAMM model driven is a promising method for the rapid detection of abnormalities in left ventricle contraction.

Reduced capillary perfusion and permeability in human tumour xenografts treated with the VEGF signalling inhibitor ZD4190: an in vivo assessment using dynamic MR imaging and macromolecular contrast media.

We describe the use of perfusion-permeability magnetic resonance imaging (ppMRI) to study hemodynamic parameters in human prostate tumor xenografts, following treatment with the vascular endothelial growth factor-A (VEGF) receptor tyrosine kinase inhibitor, ZD4190. Using a macromolecular contrast agent (P792), a fast MR imaging protocol and a compartmental data analysis, we were able to demonstrate a significant simultaneous reduction in tumor vascular permeability, tumor vascular volume and tumor blood flow (43%, 30% and 42%, respectively) following ZD4190 treatment (100 mg/kg orally, 24 h and 2 h prior to imaging). This study indicates that MR imaging can be used to measure multiple hemodynamic parameters in tumors, and that tumor vascular permeability, volume and flow, can change in response to acute treatment with a VEGF signaling inhibitor.

Local reconstruction of stenosed sections of artery using multiple MRA acquisitions.

A method for reconstructing magnetic resonance angiography (MRA) volumes from successive acquisitions is described. The method is based on double oblique acquisitions of highly anisotropic MRA volumes, each of which corresponds to reduced k-space filling. These partial k-spaces are then combined to obtain a 3D k-space adapted to the frequency spread of the angiographic image of the stenosis. The SNR-resolution compromise of MRA is thus improved by focusing the acquisition on the most relevant k-space regions. The reconstruction is performed directly in k-space by averaging the partial k-spaces. The feasibility of the method was demonstrated in studies on a Lucite stenosis phantom, on MRAs of carotid arteries using three bolus injections, and on MRAs of renal arteries using a single contrast injection.

Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies.

BACKGROUND: Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence. METHODS: To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission. RESULTS: By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides. CONCLUSIONS: Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results.

[Image analysis in contrast echocardiography]. / Les traitements d'image en échocardiographie de contraste.

The visualization of contrast agents in echocardiography is obtained with complex acquisition sequences and their analysis is dependent on operator experience. In order to be more accessible, images analysis is essential and the main objective is to integrate qualitative and quantitative data in one single image.

[Value of factor analysis in a wall motion study: preliminary application in the detection of left ventricular segmental contraction ischemic disorders by echocardiography]. / Apport d'une analyse factorielle à l'étude de mouvements: application préliminaire à la détection par échocardiographie des troubles de la contraction segmentaire du ventricule gauche ischémique.

PURPOSE: Factor Analysis of Medical Image Sequences (FAMIS) was tested to study the regional wall motion of the left ventricle at echocardiography. MATERIALS AND METHODS: FAMIS analyzed the time signal curves of each pixel. One flat curve and one curve describing the contraction-relaxation of the left ventricle were first estimated. The contributions of the curve of each pixel to the two previous curves were computed, yielding two "factor images". The spatial distribution of positive and negative coefficients of the second factor image was analyzed. The evaluation was carried out on 222 segments (20 patients, 18 parasternal short-axis views, 17 apical four-chamber views, and 15 apical two-chamber views). A first echocardiographer reviewed the factor images and the reading was compared to the conventional reading of the cine-loops by two other echocardiographers. Each segment was scored as normal, hypokinetic, akinetic, or dyskinetic. RESULTS: On normal segments, the positive coefficients of the second factor image were on the inner side, the negative coefficients were on the outer side. Dyskinesis yields the opposite pattern. Hypokinesis and akinesis give intermediate images. An absolute concordance was obtained on 71.2% of all segments between the two types of reading. Larger discrepancies were found for akinetic and hypokinetic segments. CONCLUSION: FAMIS is a promising tool to study regional wall motion of the left ventricle.

Validation of myocardial perfusion reserve measurements using regularized factor images of H(2)(15)O dynamic PET scans.

UNLABELLED: The use of H(2)(15)O PET scans for the measurement of myocardial perfusion reserve (MPR) has been validated in both animal models and humans. Nevertheless, this protocol requires cumbersome acquisitions such as C(15)O inhalation or (18)F-FDG injection to obtain images suitable for determining myocardial regions of interest. Regularized factor analysis is an alternative method proposed to define myocardial contours directly from H(2)(15)O studies without any C(15)O or FDG scan. The study validates this method by comparing the MPR obtained by the regularized factor analysis with the coronary flow reserve (CFR) obtained by intracoronary Doppler as well as with the MPR obtained by an FDG acquisition. METHODS: Ten healthy volunteers and 10 patients with ischemic cardiopathy or idiopathic dilated cardiomyopathy were investigated. The CFR of patients was measured sonographically using a Doppler catheter tip placed into the proximal left anterior descending artery. The mean velocity was recorded at baseline and after dipyridamole administration. All subjects underwent PET imaging, including 2 H(2)(15)O myocardial perfusion studies at baseline and after dipyridamole infusion, followed by an FDG acquisition. Dynamic H(2)(15)O scans were processed by regularized factor analysis. Left ventricular cavity and anteroseptal myocardial regions of interest were drawn independently on regularized factor images and on FDG images. Myocardial blood flow (MBF) and MPR were estimated by fitting the H(2)(15)O time-activity curves with a compartmental model. RESULTS: In patients, no significant difference was observed among the 3 methods of measurement-Doppler CFR, 1.73 +/- 0.57; regularized factor analysis MPR, 1.71 +/- 0.68; FDG MPR, 1.83 +/- 0.49-using a Friedman 2-way ANOVA by ranks. MPR measured with the regularized factor images correlated significantly with CFR (y = 1.17x - 0.30; r = 0.97). In the global population, the regularized factor analysis MPR and FDG MPR correlated strongly (y = 0.99x; r = 0.93). Interoperator repeatability on regularized factor images was 0.126 mL/min/g for rest MBF, 0.38 mL/min/g for stress MBF, and 0.34 for MPR (19% of mean MPR). CONCLUSION: Regularized factor analysis provides well-defined myocardial images from H(2)(15)O dynamic scans, permitting an accurate and simple measurement of MPR. The method reduces exposure to radiation and examination time and lowers the cost of MPR protocols using a PET scanner.

Adaptive and self-evaluating registration method for myocardial perfusion assessment.

With the advent of ultra-fast MRI, it is now possible to assess non-invasively regional myocardial perfusion with multislice coverage and sub-second temporal resolution. First-pass contrast enhanced studies are acquired with ECG-triggering and breath holding. Nevertheless, some respiratory induced movements still remain. Myocardial perfusion can be assessed locally by parametric imaging methods such as Factor Analysis of Medical Image Sequences (FAMIS), provided that residual motion can be corrected. An a posteriori registration method implemented in the image domain is proposed. It is based on an adaptive registration model of the heart combining three elementary shapes (left ventricle, right ventricle and pericardium). The registration procedure is performed on a potential map derived from the distance map. To evaluate the quality of the registration procedure a superimposition score between the registration model and the contour automatically extracted in the sequence is proposed. Rigid transformation hypotheses and registration analysis provide an efficient and automatic method which allows the rejection of outlier images, such as: out of synchronisation images, out of plane acquisitions. When compared to a manual registration method, this approach reduces processing time and requires a minimal intervention from the operator. The proposed method performs registration with a subpixel accuracy. It has been successfully applied to simulated images and clinical data. It should facilitate the use of MR first-pass perfusion studies in clinical practice.

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342.

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells.

OBJECTIVE: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.