Three-dimensional binding of epidermal growth factor peptides in colonic tissues produced from rotating bioreactor

Epidermal growth factor peptide binding was analyzed on primary cultures of colonic cells and along crypts by fluorescent laser-scanning confocal microscopy, using a three-dimensional image analysis software (Quant3D, Linux/Unix). Structural, proliferative units from primary cultures grown in rotating bioreactor for 41 d were arranged according to a tubular symmetry or on a parallelepiped sheet. Mean width, height, and depth of 23 tissue-like masses (+/- standard error) were 125 microm (+/-16), 152 microm (+/-23), and 29 microm (+/-3), respectively. Mean density of nuclei in tissue-like masses, expressed as the number of nuclei per cubic millimeter (+/- standard error of the mean), was 1.8 x 10(5) (+/-0.7 x 10(5)) nuclei per cubic millimeter, which corresponded to a density that was five to six times lower than that estimated for the colonic crypt isolated by chelation. Spots of high epidermal growth factor (EGF) peptide binding that corresponded to microlesions in crypt monolayers or to active colonization of microcarriers by epithelial and stromal cells in tissue-like masses were observed. The relative intensities of EGF peptide binding that were obtained below cell position 8 on crypts were very homogeneous and were representative of the profile obtained with crypts isolated from adult rats adapted to a normal diet and used to develop primary cultures of colonocytes in our laboratory. A microscopic multidimensional analytic system to record the expression profiles of biomarkers along intestinal tissues should enhance the use of primary cultures of colonocytes for in vitro testing of new food products.     

Cochlea in old world mice and rats (Muridae)

Morphometric analysis of the cochlea was performed in wild and laboratory murids: Mus musculus, Apodemus sylvaticus, Rattus rattus, R. norvegicus, NMRI mouse, and Wistar rat. Results are based on light microscopic examination of surface specimens and serial sections and on three-dimensional computer reconstruction. The cochleae have 1.75-2.2 coils. The length of the basilar membrane varies from 6.0 to 12.1 mm. Mean density of outer hair cells ranges between 363 and 411, inner hair cells 98 and 121, neurons 1,230 and 1,760 per 1 mm. Following parameters change from base to apex: basilar membrane width 66.0 (+/- 8.2) to 175.0 (+/- 24.7) microns, basilar membrane thickness 17.0 (+/- 2.6) to 1.9 (+/- 0.1) microns, width of triad of outer hair cells 13.2 (+/- 0.7) to 28.8 (+/- 4.4) microns. The given numbers are mean "murid" values (with respective standard deviations). Maximum of dimensions of scalae is located at 10-15%, that of density of outer hair cells at 65%, density of inner hair cells at 2.8 mm, maximum of innervation density at 40-60% from the base. The following parameters are correlated with pinna size: length and maximum width of basilar membrane, dimensions of scalae, total number of receptors, and probably resolution capabilities. The following parameters are correlated with body size: maximum width of triad of outer hair cells, density and total number of neurons, ratio of neurons to receptors, apicobasal difference in basilar membrane stiffness and width of triad of outer hair cells; inversely proportional is receptor density and ratio of outer to inner hair cells and probably low-frequency cut-off. Thickness, and minimum width of basilar membrane and triad of outer hair cells and probably high-frequency cutoff are species-specific and independent of pinna or body size. The parameters mentioned indicate that the examined murids are acoustically unspecialized mammals and their cochleae approximate the generalized plan for a mammalian cochlea. Differences between domesticated and wild murids are stated.     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

Analysis of immunohistochemical label of Fos protein in the suprachiasmatic nucleus: comparison of different methods of quantification

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.     

Prognostic value of cytokeratin 19 fragment (CYFRA 21-1) and cytokeratin-positive cells in bone marrow samples of breast cancer patients

The aim of this study was to investigate the relationship between the detection of micrometastatic cells by immunocytochemistry (ICC) with an anticytokeratin antibody and cytokeratin fragment (CYFRA 21-1) expression detected by an immunofluorescent assay in bone marrow of breast cancer patients. Micrometastatic CK+ cells were screened with a pancytokeratin antibody A45 B/B3 from bone marrow aspiration samples of 102 breast cancer patients (65 primary tumors, 10 local recurrences and 27 distant metastases). CYFRA 21-1 levels were assessed in bone marrow supernatant of these patients before collection of the mononucleated interface cells on a Ficoll-Hypaque density gradient and in 20 control patients. CYFRA 21-1 and CK+ cell detection by ICC were both correlated with clinical stage. CYFRA 21-1 was significantly elevated in patients with micrometastatic disease detected by ICC: 4.77 ng/mL (+/- 10.87 SD) versus 1.00 ng/mL (+/-1.36 SD) in patients with negative ICC (p=0.01). In univariate analysis, a CYFRA 21-1 value > or =1 ng/mL and the presence of CK+ cells were associated with a poorer survival for patients with stage I to III breast cancer (n=65). On multivariate analysis, only pathological nodal status and presence of CK+ cells in bone marrow were independent prognostic factors for overall survival. In conclusion, in this series CYFRA 21-1 was correlated with detection of CK+ cells by ICC in bone marrow, but cannot replace ICC. The presence of CK+ cells in bone marrow remains a strong independent prognostic factor in primary breast cancer.     

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.     

The micronucleus assay in exfoliated buccal cells: application to occupational exposure to polycyclic aromatic hydrocarbons.

Many polycyclic aromatic hydrocarbons (PAHs) have been identified as cancer-inducing chemicals for animals and/or humans. Also, there is sufficient evidence that exposures in the occupational settings are carcinogenic or probably carcinogenic to human. Engine exhaust and used engine oils are major PAH sources in engine repair workshops and traffic. Analysis of micronucleus (MN) in exfoliated buccal cells is a sensitive method for monitoring genetic damage in human populations. In our study, we used three different occupational groups (Group 1; engine repair workers, Group 2; taxi drivers, Group 3; traffic police) and two controls (Control I for Group 1 and Control II for Group 2 and Group 3) for the exposed groups. We analysed MN frequencies in exfoliated buccal cells and compared the exposed groups (Group 1; n=34, Group 2; n=17, Group 3; n=15) and subjects not occupationally exposed to PAH (Control I; n=28, Control II; n=20). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 1 and Control I were 0.07+/-0.05 and 0. 05+/-0.04, respectively (p>0.05; Table 2). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 2, 3 and Control II were 0.12+/-0.05, 0.10+/-0.05 and 0.03+/-0.03, respectively (p<0. 0001, p<0.05; Table 2) Smokers and nonsmokers do not differ with respect to the incidence of MN in all groups.     

Image morphometry of acute leukemias. Comparison between lymphoid and myeloid subtypes

OBJECTIVE: To determine if image morphometry has any role in distinguishing blasts of acute lymphoblastic leukemia (ALL-L2) from those of acute myeloid leukemia (AML-M1) and (AML-M2). STUDY DESIGN: Ten cases each of ALL-L2, AML-M1 and AML-M2 diagnosed according to the French-American-British criteria were studied. In all cases May-Grünwald-Giemsa-stained bone marrow aspiration smears were obtained. At least 100 blast cells from each case were subjected to analysis randomly with an image cytometer using Leica Quantimet 600 software (Cambridge, U.K.). The area, convex area, length, width, perimeter, convex perimeter, roundness, total optical density, average optical density and pixel grey value variance of the nuclei were measured by random selection of cells using a 40:1 objective (1 pixel = 0.446 micron). RESULTS: Mann Whitney's nonparametric test showed that there was considerable overlap of morphometric variables between the 3 subtypes. Though statistical significance was found in "roundness" between blasts of AML-M1 and ALL-L2, power analyses (sample size of 100 blasts of each subtype) did not show sufficient power for this variable. However, between blasts of ALL-L2 and AML-M2, "average optical density" and "pixel grey value variance" were statistically significant with full power using power analyses. CONCLUSION: Image morphometry may be helpful in differentiating blasts from lymphoid and myeloid leukemic subtypes.     

Bone marrow-derived side population cells are capable of functional cardiomyogenic differentiation

It has been reported that bone marrow (BM)-side population (SP) cells, with hematopoietic stem cell activity, can transdifferentiate into cardiomyocytes and contribute to myocardial repair. However, this has been questioned by recent studies showing that hematopoietic stem cells (HSCs) adopt a hematopoietic cell lineage in the ischemic myocardium. The present study was designed to investigate whether BM-SP cells can in fact transdifferentiate into functional cardiomyocytes. Phenotypically, BM-SP cells were 19.59% (+/-)9.00 CD14+, 8.22% (+/-)2.72 CD34+, 92.93% (+/-)2.68 CD44+, 91.86% (+/-)4.07 CD45+, 28.48% (+/-)2.24 c-kit+, 71.09% (+/-)3.67 Sca-1+. Expression of endothelial cell markers (CD31, Flk-1, Tie-2 and VEGF-A) was higher in BM-SP cells than whole BM cells. After five days of co-culture with neonatal cardiomyocytes, 7.2% (+/-)1.2 of the BM-SP cells expressed sarcomeric alpha-actinin as measured by flow cytometry. Moreover, BM-SP cells co-cultured on neonatal cardiomyocytes fixed to inhibit cell fusion also expressed sarcomeric alpha-actinin. The co-cultured BM-SP cells showed neonatal cardiomyocyte-like action potentials of relatively long duration and shallow resting membrane potential. They also generated calcium transients with amplitude and duration similar to those of neonatal cardiomyocytes. These results show that BM-SP cells are capable of functional cardiomyogenic differentiation when co-cultured with neonatal cardiomyocytes.     

A rearranging ligand enables allosteric control of catalytic activity in copper-containing nitrite reductase

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.     

Induction of micronuclei by 153Sm-EDTMP in peripheral blood lymphocytes of patients with bone metastases.

The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.     

Resolution and synthesis of optically active alcohols with immobilized water-soluble proteins from green pea, soybean and buckwheat as new bio-catalysts

Kinetic resolution of racemic alcohols, (+/-)-1-(4-substituted phenyl)ethanol and (+/-)-1-(2-naphthyl)ethanol, was done with immobilized green pea, soybean, or buckwheat proteins. The resolution was done stereoselectively by oxidizing only one enantiomer of a racemic alcohol to leave an optically active alcohol with a high purity. In addition, each protein could be reused consecutively at least three times without any decrease of yield or optical purity.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.     

Penetration of Pseudomonas aeruginosa by sodium chloride and its relation to the mechanism of optical effects

When cells of Pseudomonas aeruginosa were suspended in solutions containing increasing concentrations of NaCl, the optical density (OD) of the suspensions measured within 30 sec was found to increase in proportion to the increase in salt concentration. Measurement of intracellular fluid volumes indicated that the volume of the cells decreased roughly in proportion to the increase in salt concentration. After the initial increase in optical density, there was a slow decrease at all concentrations of NaCl tested except the highest, 500 mm. Metabolic inhibitors such as sodium azide and 2,4-dinitrophenol prevented the decrease. Direct analysis showed that the Na(+) and Cl(-) concentrations in the cells were 86 and 77%, respectively, of the concentrations of the ions in the suspending medium after 1 hr. Measurement of the (22)Na space in packed cells showed that Na(+) penetrated the total fluid space in the packed cells. The penetration of (22)Na was not prevented by the presence of metabolic inhibitors or by 500 mm NaCl in the suspending medium. The results indicate that the OD increases produced in suspensions of P. aeruginosa by NaCl are not due to the osmotic action of the salt. The subsequent optical density decreases observed are under metabolic control.     

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

Early effects of chemical carcinogens as compared to induced cell proliferation. II. Automated image analysis

Static automated image analysis was applied to study early variations of chromatin structure in Feulgen-stained liver nuclei from rats injected i.p. with a single dose of dimethylnitrosamine (DMNA), a well known hepatocarcinogen. An increase of nuclear area and a correspondent decrease of average optical density (integrated optical density/area) was observed, as compared with controls, in nuclei from rats treated with 5.4 mg/kg of DMNA. These findings, which were comparable with those induced by partial hepatectomy, indicate the existence in DMNA-treated cells of a chromatin DNA relaxation similar to the G0-G1 transition previously described for human diploid fibroblasts stimulated to proliferate. Because similar results were independently obtained by flow microfluorimetry, it seems reasonable to hypothesize that chromatin decondensation could be a prerequisite for cancer induction.     

Mitotic delay in lymphocytes from BRCA1 heterozygotes unable to reduce the radiation-induced chromosomal damage

Double strand breaks (DSB) are critical lesions involved in the formation of chromosomal aberrations. In response to DNA damage, the cell has mechanisms of repair and cell-cycle control to maintain the genome integrity in which BRCA1 gene is implicated. In the present study an evaluation of the radio-induced damage in G(2) phase of the cell cycle in lymphocytes from BRCA1 heterozygotes is presented. For this purpose Calyculin-A-based premature chromosome condensation (PCC) combined with mitotic arrest has been applied to examine with conventional cytogenetics the damage in G(2) and M phase cells, and to evaluate the G(2)-to-M phase transition. Irradiated peripheral blood lymphocytes from seven heterozygote females (BRCA1(+/-)) and seven control females (BRCA1(+/+)) have been analyzed. The mean proportion of G(2) cells in BRCA1(+/-) was significantly higher than in BRCA1(+/+), indicating a higher G(2) delay after IR exposure in cells from BRCA1(+/-) females. On the other hand, whereas the mean frequency of chromatid breaks (chtb) in G(2) cells was not statistically different between both groups, the mean frequency of chtb in M cells of the BRCA1(+/-) group was significantly higher than in the BRCA1(+/+) one. Moreover, the mean proportion of M cells with aberrations was significantly higher in BRCA1(+/-) than in BRCA1(+/+) suggesting that in spite of the higher G(2) delay of BRCA1(+/-) more damaged cells are able to pass the G(2)-to-M transition.     

Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.