ABSTRACT
This study aimed to examine the performance characteristics of four high-frequency oscillatory-type ventilators, using an in vitro model of the intubated neonatal respiratory system. Each ventilator was examined across its operative range of settings and at varying model lung compliance (C) and resistance. The oscillatory pressure waveform was measured at the airway opening (Pao). Tidal volume (VT) and flow were determined from pressure changes within the model lung (DeltaPA). The spectral content of the Pao waveform differed between ventilators. The maximum ventilator VT ranged from 3.7 to 11.1 ml at 15 Hz and a mean airway pressure (Paw) of 12 cm H(2)O to oscillate a model lung (C = 0.4 ml/cm H(2)O) through a 3.0-mm internal diameter (i.d.) endotracheal tube (ETT). A small drop in C was associated with a decrease in VT and marked increase in DeltaPA from 0.1 to 0.8 ml/cm H(2)O. The influence of C on VT and DeltaPA and the pressure cost of ventilation (DeltaPA/f.VT(2)) was dependent on the oscillatory frequency, ETT inner diameter, and the specific ventilator used. Substantive differences exist between oscillatory ventilators that need to be considered in their clinical application. The rapid establishment of optimal lung volume and oscillatory frequency is important in minimizing barotrauma during high-frequency oscillatory ventilation.
Subject(s)
High-Frequency Ventilation , Respiratory Distress Syndrome, Newborn/therapy , Ventilators, Mechanical , Biomechanical Phenomena , High-Frequency Ventilation/instrumentation , Humans , Infant, Newborn , Lung/physiology , Lung Compliance , Models, Anatomic , Models, Theoretical , Tidal VolumeABSTRACT
Several attempts have been made at the removal of specific pathogens from the intestinal microflora using either bacteriophages or "predatory" bacteria such as Bdellovibrio spp. To date these attempts have had mixed success. A mechanism explaining these findings based on competitive hindrance by non-prey, or decoy species is put forward. It is shown that this hindrance tends to damp out predator-prey oscillations, and therefore reduces the probability of prey extinction. Possible experiments to verify this theory are discussed. The decoy effect may play a role in any system with high densities of bacteria or other particulate matter, such as activated sludge or biofilms.
Subject(s)
Bacteria , Bacteriophages/physiology , Bdellovibrio/physiology , Humans , Models, BiologicalABSTRACT
High inspired oxygen concentrations have recently been recommended to control Cheyne-Stokes respiration in adults, with the intention of averting periodic apnea and its attendant arterial desaturation. We report a case study on an infant presenting with recurrent apnea and cyanosis in which oxygen treatment led to a gross form of respiratory instability we call episodic breathing, in which a breathing phase of 60 to 90 s alternated with an apnea lasting up to 60 s. When oxygen was discontinued, a profound arterial desaturation developed before breathing recommenced and restored oxygen levels. We propose that episodic breathing is an unusual respiratory pattern that involves the central chemoreceptors and results from the ventilatory threshold (the central PCO(2) at which breathing starts) lying considerably above the apneic threshold (the central PCO(2 )at which breathing stops). This feature predisposes to lengthy periods of hyperpnea alternating with lengthy periods of apnea. We suggest that when the case infant returned to air during episodic breathing, termination of apnea was entirely dependent upon carotid body activity, which reached a sufficient level to restart breathing only when arterial desaturation was severe. We conclude that oxygen therapy involves potential risks when employed to treat respiratory disorders involving unstable breathing patterns in the infant.
Subject(s)
Cyanosis/physiopathology , Oxygen Inhalation Therapy , Sleep Apnea, Central/physiopathology , Adult , Animals , Carotid Body/physiopathology , Chemoreceptor Cells/physiopathology , Cheyne-Stokes Respiration/physiopathology , Disease Models, Animal , Diseases in Twins , Female , Follow-Up Studies , Humans , Infant , Infant, Newborn , Oxygen/blood , Polysomnography , Respiratory Center/physiopathology , SheepABSTRACT
Recovery of six anaerobic and five aerobic pathogens from viscose swabs and polyurethane swabs (Culturette EZ) was evaluated quantitatively, and transport in aerobic dry tubes, aerobic Amies transport medium (Transwab), and anaerobic universal transport medium (Port-a-Cul) was compared. The Culturette EZ in aerobic dry tubes gave the highest recovery levels. Data obtained with clinical specimens confirmed these results.
Subject(s)
Bacteria, Aerobic/isolation & purification , Bacteria, Anaerobic/isolation & purification , Culture Media , HumansABSTRACT
1. Arousal from sleep is an important protective response to hypoxia that becomes rapidly depressed in active sleep (AS) when hypoxia is repeated. This study questioned whether there might also be selective depression of cardio-respiratory responses to hypoxia during AS. 2. Nine newborn lambs (7-22 days of age) were studied over three successive nights. The first and third nights were baseline studies (inspired oxygen fraction, Fi,O2 = 0.21). During the second night, during every epoch of sleep, lambs were exposed to 60 s episodes of isocapnic hypoxia (Fi,O2 = 0.10). 3. During quiet sleep (QS), the probability of arousal in hypoxia exceeded the probability of spontaneous arousal (P < 0.001) throughout repeated exposures to hypoxia. Similarly, there were persisting increases in ventilation (135 +/- 25 %), blood pressure (3 +/- 1 %) and heart rate (3 +/- 1 %). 4. By contrast, rapid depression of all responses occurred during repetitive hypoxia in AS. Thus, the probability of arousal in hypoxia exceeded the probability of spontaneous arousal during the first 10 hypoxia exposures (P < 0.001) but not thereafter. Similarly, during the first 10 exposures to hypoxia, the changes in ventilation (88 +/- 15 %) and blood pressure (5 +/- 1 %) were greater than subsequent responses (P < 0.05). 5. We conclude that, when repeated, hypoxia rapidly becomes ineffective in stimulating protective arousal, ventilatory and blood pressure responses in AS, but not in QS. Selective depression of responses during AS may render the newborn particularly vulnerable to hypoxia in this state.
Subject(s)
Animals, Newborn/physiology , Hemodynamics/physiology , Hypoxia/physiopathology , Respiratory Mechanics/physiology , Sleep, REM/physiology , Sleep/physiology , Animals , Arousal/physiology , Blood Gas Analysis , Blood Pressure/physiology , SheepABSTRACT
This study investigated factors contributing to differences between mean alveolar pressure (PA) and mean pressure at the airway opening (Pao) during high-frequency oscillatory ventilation (HFOV). The effect of the inspiratory-to-expiratory time (I/E) ratio and amplitude of oscillation on the magnitude of - Pao (Pdiff) was examined by using the alveolar capsule technique in normal rabbit lungs (n = 4) and an in vitro lung model. The effect of ventilator frequency and endotracheal tube (ETT) diameter on Pdiff was further examined in the in vitro lung model at an I/E ratio of 1:2. In both lung models, fell below Pao during HFOV when inspiratory time was shorter than expiratory time. Under these conditions, differences between inspiratory and expiratory flows, combined with the nonlinear relationship between resistive pressure drop and flow in the ETT, are the principal determinants of Pdiff. In our experiments, the magnitude of Pdiff at each combination of I/E, frequency, lung compliance, and ETT resistance could be predicted from the difference between the mean squared inspiratory and expiratory velocities in the ETT. These observations provide an explanation for the measured differences in mean pressure between the airway opening and the alveoli during HFOV and will assist in the development of optimal strategies for the clinical application of this technique.
Subject(s)
High-Frequency Ventilation , Pulmonary Alveoli/physiology , Respiratory Mechanics/physiology , Airway Resistance/physiology , Animals , Humans , In Vitro Techniques , Infant, Newborn , Intubation, Intratracheal , Lung Compliance/physiology , Models, Biological , Pressure , RabbitsABSTRACT
1. Arousal from sleep is an important protective mechanism that is depressed by repeated episodes of hypoxia. We aimed to determine how rapidly arousal depression occurs during repeated hypoxia and to determine if the depression is sleep state specific. 2. Three successive 12 h overnight sleep recordings were performed in six newborn lambs instrumented to record sleep state, blood pressure, heart rate and blood gases. The first (control) and third (recovery) nights were baseline studies (inspired oxygen fraction, FI,O2 = 0.21) to determine the spontaneous arousal probability. During the second (test) study night, lambs were exposed to a 60 s episode of isocapnic hypoxia (FI,O2 = 0.10; inspired carbon dioxide fraction, FI,CO2 = 0.03) during every epoch of sleep. 3. During quiet sleep (QS), the probability of arousing to hypoxia (56%) remained significantly higher than the probability of arousing spontaneously (18%) throughout the repeated hypoxic exposures (chi(2) = 81.5, P < 0.001). By contrast, during active sleep (AS) arousal rapidly became depressed with repetition of the hypoxic stimulus; the probability of arousal in hypoxia (52%) was significantly higher than the probability of spontaneous arousal (12%) during the first ten hypoxic exposures (chi(2) = 18.2, P < 0.001), but there was no difference thereafter. 4. We conclude that, when repeated, moderate hypoxia very rapidly becomes ineffective as an arousing stimulus in AS, but not in QS. These results suggest that the arousal mechanism is particularly vulnerable to failure during AS.
Subject(s)
Animals, Newborn/physiology , Arousal/physiology , Hypoxia/psychology , Sleep/physiology , Animals , Blood Gas Analysis , Hemodynamics/physiology , Sheep , Sleep, REM/physiologyABSTRACT
1. Oxygen administration is thought to suppress periodic breathing (PB) by reducing carotid body activity, and yet earlier experiments in neonates have shown that PB incidence may be increased following the application of hyperoxia. To clarify this paradox, we studied the changes in the pattern of PB that occur following administration of oxygen in a lamb model of PB. 2. PB was induced in eleven of seventeen anaesthetized lambs following passive hyperventilation with air. When oxygen was administered during PB, the pattern was first enhanced, as evidenced by a sudden decrease in the ratio of the ventilatory duration to the apnoeic pause duration, and then suppressed, as evidenced by a progressive return to stable breathing which was associated with an increase in minute ventilation. 3. Five of the six lambs that did not show PB following passive hyperventilation with air could be made to do so if oxygen was substituted for air as the inspired gas following passive hyperventilation. 4. Five of the eleven lambs that showed PB following hyperventilation with air responded to the application of oxygen during PB by switching to a gross form of episodic breathing consisting of long apnoeic pauses followed by equally long periods of breathing during which minute ventilation fell progressively with time. 5. We conclude that when applied against a background of arterial hypoxaemia, oxygen has a destabilizing influence on ventilation in that (a) it accentuates the unstable breathing that occurs during PB, (b) it induces PB in lambs that exhibited stable breathing in air, and (c) it may precipitate episodic breathing.
Subject(s)
Apnea/physiopathology , Hyperventilation/physiopathology , Oxygen/pharmacology , Respiratory Mechanics/drug effects , Sheep/physiology , Animals , Animals, Newborn , Calibration , Carbon Dioxide/blood , Oximetry , Oxygen/bloodABSTRACT
1. Five lambs (19-27 days old) were studied to determine the effects of feeding on cardiorespiratory function. 2. Each lamb was instrumented to record cardiac output, aortic and pulmonary artery pressure and arterial and mixed venous oxyhaemoglobin saturations (Sa,O2 and Sv,O2). 3. During feeding, arterial haemoglobin desaturated and resaturated sequentially during the periods of sucking and non-sucking. The nadir of these Sa,O2 desaturations (83 +/- 2%, mean +/- S.E.M.) was significantly lower than the baseline value (92 +/- 2%, P < or = 0.05, ANOVA). Sa,O2 returned to the baseline level between periods of sucking. Sv,O2 also decreased (55 +/- 3% baseline, 46 +/- 3% sucking, P < or = 0.05) but, in contrast to Sa,O2, it remained significantly lower than baseline levels in the pauses between periods of sucking. 4. Arterial pressure increased during feeding (94 +/- 4 mmHg baseline, 113 +/- 6 mmHg feeding, P < or = 0.05), while heart rate and cardiac index did not change. 5. Total body oxygen consumption rose during the pauses between sucking periods (10.9 +/- 1.1 ml O2 min-1 kg-1 baseline, 13.9 +/- 1.2 ml O2 min-1 kg-1 non-sucking, P < or = 0.05) and was provided for by a significant increase in total body oxygen extraction as systemic oxygen transport was unchanged. 6. Our results reveal that during feeding in young lambs oxygen consumption increases and body stores of oxygen (e.g. Sv,O2) become depleted; this combination may promote rapid arterial desaturation and cyanosis during feeding.
Subject(s)
Aorta/physiology , Hemoglobins/metabolism , Oxygen Consumption , Oxygen/blood , Oxyhemoglobins/metabolism , Pulmonary Artery/physiology , Analysis of Variance , Animals , Animals, Suckling , Blood Pressure , Cardiac Output , Eating , Female , SheepABSTRACT
To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients.
Subject(s)
Cisplatin/pharmacology , DNA Adducts/analysis , Animals , Cells, Cultured , Fluorescent Antibody Technique, Indirect , Humans , Immunohistochemistry/methods , Lymphocytes , Mouth Mucosa/cytology , Platinum/analysis , Rabbits , Video RecordingABSTRACT
In order to investigate the mechanisms underlying periodic breathing (PB), we studied the initiation of breathing after passive hyperventilation in 14 anaesthetised 10-20 day old lambs. Eight of the lambs exhibited PB following post-hyperventilation apnea (PHA), with an epoch duration of 82.4 +/- 14.2 sec (mean +/- SEM), a cycle duration of 9.7 +/- 0.7 sec and a ratio of ventilatory duration to apnea duration (V-A ratio) of 1.24 +/- 0.32. The remaining lambs showed stable breathing patterns following PHA. The ventilatory response to isocapnic hypoxia was significantly greater in the group that had PB (-7.2 +/- 1.0 ml min-1% Sao2-1 kg-1) than in the animals that did not (-2.5 +/- 1.0 ml min-1%Sao2-1 kg-1). Using experimentally determined ventilatory response curves to O2 and CO2 we calculated that the swings in Sao2 and Paco2 during PB generated chemical drive that accounted for only 16.2% of the ventilatory oscillations observed during PB. Much of the remaining drive appeared to originate in the 'switch-on' characteristics of the respiratory controller, in lambs that exhibited periodic breathing, when breathing began after PHA ventilation jumped abruptly from zero to 55.1% of the eupneic ventilation. The magnitude of this jump in ventilation accounted for 51.9% of the amplitude of ventilatory oscillations that occur during PB. We speculate that this previously unrecognised feature of the respiratory controller, together with an elevated sensitivity to hypoxaemia, play crucial roles in generating PB in the infant.
Subject(s)
Drive , Respiratory Mechanics/physiology , Sheep/physiology , Animals , Apnea/blood , Apnea/physiopathology , Blood Gas Analysis , Calibration , Carbon Dioxide/blood , Hyperventilation/physiopathology , Hypoxia/physiopathology , Oximetry , Oxygen/bloodABSTRACT
Digital image processing (DIP) of bacterial smears is a new method of analysing the composition of the gut microbial flora. This method provides the opportunity to compare and evaluate differences in the complex highly concentrated anaerobic fraction of gut microbial flora, based on micromorphological differences. There is ample evidence that this fraction can be characterized as related or unrelated to the host organism by its immunogenicity. In this study germfree ND2 mice were associated with either related (rodent) SPF microflora (SPF-MF) or unrelated human MF (HUM-MF). DIP analysis was performed on original SPF-MF and HUM-MF and on the faeces of ex-germfree mice 4 weeks after association. The micromorphological pattern of highly concentrated anaerobic bacteria in faeces of HUM-MF associated ex-germfree mice was significantly different from SPF-MF associated counterparts with regard to the scores for elongation (P < 0.01) and morphological variety (P < 0.05). Moreover, gross morphological variability was present between individual HUM-MF associated mice but not between individual SPF-MF associated animals. No differences were found between original SPF and HUM-MF. The data are discussed with regard to differences in the presence of (non-)immunogenic bacteria and the ability for related and unrelated flora to colonize the murine gut. This study provides evidence that murine host specificity of microbial flora may not only be reflected in the number of non-immunogenic bacteria but also in the micromorphological pattern of highly concentrated anaerobic bacteria in faeces measured by DIP analysis.
Subject(s)
Bacteria, Anaerobic , Feces/microbiology , Intestines/microbiology , Adult , Animals , Humans , Image Processing, Computer-Assisted , Male , Mice , Rats , Rats, Wistar , Signal Processing, Computer-Assisted , Species Specificity , Specific Pathogen-Free OrganismsABSTRACT
The progressive decrease in the periodic cycle duration (PCD) of periodic breathing with postnatal age in term infants has been previously reported by a number of authors and is thought to be associated with peripheral chemoreceptor maturation. We hypothesized that a similar decrease should be observed in preterm infants. Therefore, in this study we measured the changes in PCD with postnatal age in a small group of preterm (n = 4) infants followed longitudinally (36 afternoon nap studies) over the first 6 mo postnatally. PCD declined in these infants from 17.1 +/- 3.3 s (mean +/- 2 SD) at 9 d to 9.8 +/- 3.2 s (mean +/- 2 SD) at 105 d. The regression slope was -0.072 s/d. Beyond 105 d there was no change in PCD up to 6 mo postnatally. We found no significant difference between active and quiet sleep. These results are similar to results previously published in term infants but apparently contradict recent data on a group of preterm infants. Possible reasons for this discrepancy are discussed. By examining long epochs of periodic breathing in these infants we also identified characteristic changes in PCD and V/A ratio, defined as the duration of the ventilatory period divided by the duration of the apneic interval. V/A ratio fell from the start of an epoch from 1.21 +/- 0.08 (mean +/- SEM) to a minimum of 0.62 +/- 0.03 and then increased again to 0.8 +/- 0.05 at the end of the epoch.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Infant, Premature/physiology , Periodicity , Respiration/physiology , Age Factors , Chemoreceptor Cells/growth & development , Chemoreceptor Cells/physiology , Female , Humans , Infant , Infant, Newborn , Infant, Premature/growth & development , Male , Models, Biological , Respiratory Mechanics/physiology , Respiratory Physiological Phenomena , Respiratory System/growth & development , Respiratory System/innervation , Sleep/physiologyABSTRACT
While arterial oxygen desaturation during apnea is a common occurrence in adults and infants, the factors determining the rate of desaturation are poorly understood. We describe a theoretical model which suggests that arterial desaturation during an apneic episode occurs in two stages. In the initial stage (stage 1) the oxygen store in the lung is depleted, while in the second phase of the desaturation process (stage 2) tissue oxygen needs are met predominantly by depletion of the blood store. Our model predicts that preapneic venous oxygenation (SvO2) will strongly influence the rate of the desaturation in stage 1 but not in stage 2. We therefore examined the effect of changing preapneic SvO2 on the rate of arterial oxygen desaturation (SaO2) during stage 1 and stage 2 apnea in anaesthetised 10-20 day-old lambs. Preapneic arterial oxygen saturation was maintained constant. In agreement with the model's prediction there were two stages to the desaturation process and during stage 1 a significant increase in SaO2 was observed when preapneic SvO2 was lowered; SaO2 was -3.1 +/- 0.4%.sec-1 when SvO2 = 47.4 +/- 2.1% increasing to -5.8 +/- 0.7%.sec-1 when SvO2 = 28.3 +/- 1.2%. During stage 2, SaO2 was -1.62 +/- 0.07%.sec-1 and was independent of preapneic SvO2, also in accord with the model's prediction. In order to assess whether the accelerated desaturation rate we observed in stage 1 could have resulted from a decline in lung volume during apnea rather than lower levels of SvO2, we repeated the experiment with CPAP applied. Under these conditions SaO2 continued to be greater at lower preapneic SvO2 levels. In summary, lowered preapneic SvO2 has a potent influence on SaO2 during stage 1 of the desaturation process but not during stage 2.
Subject(s)
Models, Biological , Oxygen/blood , Sleep Apnea Syndromes/physiopathology , Animals , Animals, Newborn , Hypoxia/physiopathology , SheepABSTRACT
Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.
Subject(s)
Bifidobacterium/genetics , In Situ Hybridization, Fluorescence/methods , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Base Sequence , Bifidobacterium/classification , Bifidobacterium/isolation & purification , DNA Probes/genetics , DNA, Bacterial/genetics , Feces/microbiology , Humans , Molecular Sequence Data , RNA, Bacterial/isolation & purification , RNA, Ribosomal, 16S/isolation & purification , Species SpecificityABSTRACT
In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographic staining model in human skin, using structural EBMZ antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and suggests an effective lower limit of some 60-65 nm. Although this limit is not reached under present conditions, our results show that it is possible to distinguish topographic differences of antigen distributions with an upper resolution limit of 200 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topographic reference markers in future applications of the technique aiming at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Preliminary results on this topic are most promising indeed.
Subject(s)
Antigens, Surface/analysis , Basement Membrane/immunology , Fluorescent Antibody Technique , Photomicrography/methods , Skin/immunology , Frozen Sections , Humans , Video RecordingABSTRACT
In this second report on the fluorescence overlay antigen mapping (FOAM) technique, we highlight some of the errors that may influence faithful color rendition of slide preparations using triple antigen immunofluorescence staining. Reliable interpretation of multicolor fluorescence images requires that the observer can unambiguously assign each color in these images to the presence of a specific combination of the labeled antigens. This is possible only when the image fidelity meets certain standards. The present study concentrates on color fidelity which is easily undermined by spectral matching errors, image contrast errors, and exposure time errors. Evaluation of these errors, using the photomicrographic overlay variant of FOAM, showed the potential unreliability of the simultaneous use of multiple fluorophores for immunofluorescence microscopy. The procedures described here may serve as a solid starting point in formulating technical conditions that allow reliable color rendition in multicolor immunofluorescence microscopy. Furthermore, these procedures can be adapted to studies other than the analysis of basement membrane zone antigens, to which they have been first applied.
Subject(s)
Basement Membrane/cytology , Color , Fluorescent Antibody Technique , Frozen Sections , Humans , Microscopy, Fluorescence , Photomicrography/methods , Skin/cytologyABSTRACT
A microbiological image processing system has been developed, which is used to determine both morphometry and fluorimetry data on (mainly faecal) bacteria. A special program for display of these data has been developed for this system. Besides probability density estimates of any one or two parameters and scatter plots of any two or three parameters in the distribution, it can generate scatter plots using the actual bacterial shapes, rather than arbitrary symbols. A particularly powerful feature of the program is the possibility to overlay a contour plot of the probability density distribution of the bacteria in parameter space, with a scatter plot of the actual bacterial shapes involved. In this paper, the program's organization, options, and the algorithms used are discussed.
Subject(s)
Bacteria/ultrastructure , Computer Graphics , Image Processing, Computer-Assisted , Algorithms , Factor Analysis, Statistical , Fluorometry , Probability , SoftwareABSTRACT
Several techniques that use computer analysis of microscopic images have been developed to study the complicated microbial flora in the human intestine, including measuring the shape and fluorescence intensity of bacteria. These techniques allow rapid assessment of changes in the intestinal flora and could apply equally to other complex microbial ecosystems.
Subject(s)
Bacteria/cytology , Image Processing, Computer-Assisted , Intestines/microbiology , Feces/microbiology , FluorometryABSTRACT
An image processing system with applications in bacterial (immuno-)fluorescence measurement has been developed. To reach quantitative results, correction for non-uniformities in system sensitivity, both as a function of time (calibration for drifts) and as a function of image coordinates (shading correction), is essential. Both problems can be handled simultaneously by acquiring images of a uniformly fluorescent, solid standard as a reference image. To measure bacterial fluorescence, the average fluorescence intensity of isolated areas of interest (the bacteria) is computed, and corrected using the reference images. Two shading correction methods are theoretically and experimentally compared: direct averaging in the corrected image, and (weighted and unweighted) averaging using the raw image and a separate shading image to determine the weights and correct for shading during the averaging. The latter method proved computationally 3.5-6.5 times faster on average and reduced propagation of truncation errors during computation, resulting in 40% less noise, for 8-bits/pixel images.