Increased accuracy and speed of absorption cytometric DNA measurements by automatic corrections for nuclear darkness

We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.     

When Phase Contrast Fails: ChainTracer and NucTracer,Two ImageJ Methods for Semi-Automated Single Cell Analysis Using Membrane or DNA Staining

Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image analysis programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a cross-wall that is not detectable by phase contrast. We have developed ‘ChainTracer’, a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diameter. As a complementary analysis method we developed ''NucTracer'', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is especially useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and NucTracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.     

Discrepancies in ploidy determination due to specimen sampling errors

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.     

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.     

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.     

Dual wavelength scanning cytophotometry (Bicoscan).

A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot. To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other. The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other. The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO2) procedure for DNA in chicken erythrocytes and rat liver cells.     

Dual wavelength scanning cytophotometry (Bicoscan)

Summary A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot.To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other.The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other.The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO₂) procedure for DNA in chicken erythrocytes and rat liver cells.Supported by Grant Nr. 28-394 of the Praeventiefonds, The Hauge     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size,Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

Summary A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 x oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r=0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r=0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r=0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30–100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm× 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

Immunohistochemical Video-microdensitometry of Myocardial Collagen Type I and Type III

Collagen is an essential part of the cardiac interstitium. Collagen subtypes, their location, total amount and the architecture of the fibrillar network are of functional importance. Architecture in terms of density of the fibrillar network is assumed to be reflected by the intensity of immunohistochemical staining of collagen. The aim of this study was to evaluate a video-based microdensitometric method for quantifying density expressed as absorbance of collagen subtypes I and III stained with an indirect immunoperoxidase method in myectomy specimens of patients with hypertrophic obstructive cardiomyopathy. Various factors influencing the immunohistochemical staining product and the technical properties of the image analysis system were investigated. Linearity between collagen concentration and the absorbance of the immunohistochemical staining product was demonstrated for collagen I using a dot-blot technique. Immunohistochemical collagen staining and density measure ment were easily reproducible. The cardiac disability of the patients was assessed according to the New York Heart Association (NYHA) criteria. There was a significant increase in collagen type I density with higher NYHA class, whereas no significant association was found for total collagen area fraction. Thus, video-based microdensitometry gives further insight into the structural remodelling of myocardial collagens and reveals their significance in the process of heart failure in hypertrophic cardiomyopathy.     

Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images

Background

Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.

Methodology

We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells.

Significance

The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining     

Semiautomated computer-assisted image analysis to quantify 3,3'-diaminobenzidine tetrahydrochloride-immunostained small tissues

This work aimed to develop a technique to measure stained areas in images from sample tissue sections, namely when the structure of interest does not fill the entire image field of the microscope. We propose a semiautomated computer-assisted image analysis (SACAIA) method in which brightfield color images of 3,3'-diaminobenzidene tetrahydrochloride (DAB)-stained antigens are converted to their blue component and boundaries are delineated to extract the object of interest. The number of pixels of a defined color (elicited by DAB) is counted and used to measure the stained area relative to the total area of the tissue under study. The percentages of area stained with adenosine A(1) receptor were 40.76+/-2.08 and 42.44+/-2.26% for manual analysis and SACAIA, respectively (P=0.582). A strong linear correlation of A(1) receptor quantification was found (r=0.98, P<0.001, and 95% CI=0.97 to 0.99 for manual method; r=0.99, P<0.001, and 95% CI=0.98 to 0.99 for SACAIA method). The extent to which misclassification affected staining quantification was evaluated by Bland-Altman analysis, indicating that this method can be applied accurately to quantify the immunohistochemical staining area (occupied by a specific antigen) in small sample tissues that do not fill the entire image field of the microscope.     

Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.     

Histophotometric estimation of volume density of collagen as an indication of fibrosis in rat liver

The development of fibrosis in the liver of 16 rats treated for 1, 2, 3 or 4 weeks with CCl4, has been followed with chemical hydroxyproline determination and histophotometric analysis of histological sections stained with Sirius Red F3BA in saturated aqueous picric acid. The readings were taken with a scanning and integrating microphotometer and corrected for picric acid absorbance as a measure for mean protein mass per unit area of the section. It appears that the integrated absorbance readings of Sirius Red absorbing material in the section show a highly significant correlation with the hydroxyproline determinations. It is concluded that picrosirius photometry can be used to give a measure of the volume density of collagen in sections. An advantage of the photometric assay is that measurements are taken on the basis of the microscopic image, so that it is also possible to estimate collagen density in a selected area, e.g. a tumour formation amidst normal tissue, or to exclude necrotic areas.     

Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein

A method has been developed to determine the relative or actual number of attached cells in microtiter plate wells without making direct cell counts. The procedure is based upon staining total cellular protein with Coomassie brilliant blue G-250, followed by measurement of absorbance at 630 nm in a spectrophotometer designed to read each well of a 96-well microtiter plate. No destaining of cells is required. A linear correlation exists (r = 0.970) between cell number and absorbance over a useful range. Intraplate well-to-well variation is acceptable (CV = 0.101). This method was used to measure the proliferative response of human vascular smooth muscle cells to human serum. It should be useful in other assays involving proliferation of attached cultured cells.     

Quantitative method for in vitro matrigel invasiveness measurement through image analysis software

The determination of cell invasion by matrigel assay is usually evaluated by counting cells able to pass through a porous membrane and attach themselves to the other side, or by an indirect quantification of eluted specific cell staining dye by means of optical density measurement. This paper describes a quantitative analytical imaging approach for determining the invasiveness of tumor cells using a simple method, based on images processing with the public domain software, ImageJ. Images obtained by direct capture are split into the red channel, and the generated image is used to measure the area that cells cover in the picture. To overcome the several disadvantages that classical cell invasion determinations present, we propose this method because it generates more accurate and sensitive determinations, and it could be a reasonable option for improving the quality of the results. The cost-effective alternative method proposed is based on this simple and robust software that is worldwide affordable.