Reducing the variability between constant-depth film fermenter experiments when modelling oral biofilm.

AIMS: The inherent instabilities associated with the development of multispecies biofilm communities within the constant-depth film fermenter (CDFF) and other microcosm systems can yield unacceptable variability between experiments, which could limit their potential applications in oral microbiology. The extent of this variability needs to be determined and a protocol developed which minimizes it. METHODS AND RESULTS: Two custom-made CDFFs were supplied concurrently with the same inoculation culture, begat from an aliquot of a saliva pool and artificial saliva growth medium via a dual-channel pump. Transformed log(10) data of the viable counts at fixed time points were analysed using the Bland-Altman approach to test for the levels of agreement between two CDFFs running concurrently and those CDFFs run in series. The coefficients(95%) of agreement were lower (i.e. less variable) in the concurrent model than when run in series for total counts of bacteria (1.238 vs 2.124), Lactobacillus spp. (0.517 vs 1.431) and Mutans streptococci (2.817 vs 3.864). Other measures of variability showed a similar trend. CONCLUSIONS: Operating CDFFs concurrently minimizes the degree of difference and variability between them. SIGNIFICANCE AND IMPACT OF THE STUDY: Operating CDFFs concurrently will improve the sensitivity for experiments that seek to determine the effects of a variable, such as a nutritional supplement or antimicrobial agent, and a control.

Photobleaching of red fluorescence in oral biofilms.

BACKGROUND AND OBJECTIVE: Many species of oral bacteria can be induced to fluoresce due to the presence of endogenous porphyrins, a phenomenon that can be utilized to visualize and quantify dental plaque in the laboratory or clinical setting. However, an inevitable consequence of fluorescence is photobleaching, and the effects of this on longitudinal, quantitative analysis of dental plaque have yet to be ascertained. MATERIAL AND METHODS: Filter membrane biofilms were grown from salivary inocula or single species (Prevotella nigrescens and Prevotella intermedia). The mature biofilms were then examined in a custom-made lighting rig comprising 405 nm light-emitting diodes capable of delivering 220 W/m(2) at the sample, an appropriate filter and a digital camera; a set-up analogous to quantitative light-induced fluorescence digital. Longitudinal sets of images were captured and processed to assess the degradation in red fluorescence over time. RESULTS: Photobleaching was observed in all instances. The highest rates of photobleaching were observed immediately after initiation of illumination, specifically during the first minute. Relative rates of photobleaching during the first minute of exposure were 19.17, 13.72 and 3.43 arbitrary units/min for P. nigrescens biofilms, microcosm biofilm and P. intermedia biofilms, respectively. CONCLUSION: Photobleaching could be problematic when making quantitative measurements of porphyrin fluorescence in situ. Reducing both light levels and exposure time, in combination with increased camera sensitivity, should be the default approach when undertaking analyses by quantitative light-induced fluorescence digital.

The evaluation of a novel method comparing quantitative light-induced fluorescence (QLF) with spectrophotometry to assess staining and bleaching of teeth.

This study reports the development and evaluation of a novel method using quantitative light-induced fluorescence (QLF), which enables its use for quantifying and assessing whole tooth surface staining and tooth whitening. The method was compared with a spectrophotometer to assess reliability. Two experimental phases, intrinsic stain formation and tooth whitening, were conducted in vitro on 16 extracted bovine teeth. Intrinsic stains were developed via access through lingual surfaces and root canals of these teeth using tea solution (2 g/100 ml, Marks and Spencer Extra Strong Tea, Marks and Spencer, London, UK) for 6 days. Stains were removed using 33% hydrogen peroxide (VWR Prolab, Leicestershire, UK) in cycles over 150 min. Stain development/whitening was monitored with QLF (Inspektor Research systems, Amsterdam, Netherlands) and spectrophotometry (Easy shade, Vita Zahnfabrik, Bad Säckingen, Germany). Parameters Delta F for QLF and Delta E for the spectrophotometer were obtained. The progression of stain intensity and removal observed by the methods were tested for correlation using Pearson's correlation coefficient. Intra-examiner reliability for each method was tested. QLF showed a high correlation with spectrophotometry for detecting and monitoring intrinsic tooth stain progression (Pearson coefficient r was -0.987 with correlation significant p < 0.0001). For stain removal, the Pearson coefficient (r) between both methods was -0.906 with no significance p = 0.094. The use of an external reference material in combination with the inner patch QLF analysis technique had the ability to detect and measure whole tooth surface staining and its removal longitudinally. The reliability of the method shows a potential clinical application.

In Vitro Red Fluorescence as an Indicator of Caries Lesion Activity.

This in vitro study examined the utility of comparing red fluorescence between active and inactive caries lesions and investigated whether changes in red fluorescence and fluorescence loss are influenced by lesion activity following remineralization. Sixty-two noncavitated smooth surface caries lesions on extracted human teeth were classified into active or inactive lesions using the Nyvad system prior to a 12-day pH-cycling procedure. Quantitative light-induced fluorescence-digital images were used to measure fluorescence parameters before and after pH cycling. At baseline, the intensity (ΔR) and area (AΔR) of red fluorescence were 1.5- and 2.2-fold higher in active lesions than in inactive lesions (p<0.05). The ratio of AΔR to lesion area was associated with classification of active lesions (odds ratio = 1.031; 95% confidence interval = 1.005-1.058). After pH cycling, the active lesions showed about 2- and 8-fold greater reductions in the median values of AΔR and fluorescence loss related to lesion volume (ΔQ) compared with inactive lesions (p<0.05). In conclusion, red fluorescence differs depending on lesion activity, and the red fluorescence area and lesion volume change following remineralization. The results suggest that measuring red fluorescence may be a useful way of objectively evaluating lesion activity of smooth surface lesions.

Error analysis of the microradiographic determination of mineral content in mineralised tissue slices.

The microradiographic method, used to measure the mineral content in slices of mineralised tissues as a function of position, is analysed. The total error in the measured mineral content is split into systematic errors per microradiogram and random noise errors. These errors are measured quantitatively. Predominant contributions to systematic errors appear to be x-ray beam inhomogeneity, the determination of the step wedge thickness and stray light in the densitometer microscope, while noise errors are under the influence of the choice of film, the value of the optical film transmission of the microradiographic image and the area of the densitometer window. Optimisation criteria are given. We used these criteria, together with the requirement that the method be fast and easy, to build an optimised microradiographic system.

Optimised microcomputer-guided quantitative microradiography on dental mineralised tissue slices.

It is well realised that the contact microradiographic method is the most direct method that gives position-dependent information on the mineral content of calcified tissue and its loss. We developed a microcomputer-guided microradiographic system which features fast operation by the experimenter with a low appearance of errors. Tooth tissue slices of 75 micron thickness are cut with a sawing machine. Images (Cu K alpha radiation) of the tooth slice and an aluminium step wedge (exposure 10-15 s) are made on film (Kodak SO-253). The images of step wedge and tooth slice are scanned in a densitometer (1 micron X 30 microns slit), which is fitted with an XY table (0.5 micron steps). A microcomputer (Apple IIe) is programmed to control the XY table and to record the optical film transmission. Scans of the images are plotted on the computer screen. The calibration step optical film transmission values are determined by the operator by adjusting a computer-generated bar onto the individual steps shown on the screen. The dose-density relation of the film is approximated by the program by a fourth-degree polynomial using the step-wedge data. The resulting curve is used to convert the tooth-slice data into a mineral volume percentage. To enable the calculation of total mineral loss (in kg m-2) (loss integrated over depth), the operator adds the assumed diagram for sound enamel onto the display. This is done by adjusting computer-generated bars to the scan of the tooth slice shown on the screen. The resolving power in the image made by the microradiographic system is 3 microns X 30 microns. On the basis of the analysis of random errors and a comparison with chemical analysis of tooth slices we claim that the error in mineral volume percentage amounts to 4% of its value. Starting with a microradiographic image of a tooth slice 5 min are required to obtain a microradiographic curve on paper and to obtain a value for mineral loss.

Longitudinal microradiography: a non-destructive automated quantitative method to follow mineral changes in mineralised tissue slices.

This paper presents a new powerful method, longitudinal microradiography (LMR), with which it is possible to follow the mineral content in a de- or remineralizing carious tooth sample as a function of position on the surface of the mineralizing tissue independent of the sample thickness. Planoparallel tooth slices (thickness 300-400 microns) are cut parallel to the labial tooth surface and then fixed on a PMMA holder. Repeated microradiographic images of the same tissue slice together with an aluminium step wedge are made on photographic film at different times of mineralisation. The images are scanned under a densitometer with a microcomputer and the mineral content per unit tooth area at the same discrete tooth-slice surface positions is calculated (maximum predefined area is 5 mm x 5 mm, 15 x 15 positions). The change of mineral in one tooth slice per unit tooth surface area can be calculated at different times of mineralisation locally, as well as averaged over the total tooth-slice surface area. The SD in average mineral change is 0.009 kg m-2, which corresponds approximately to the mineral loss involved with a 8 microns deep tooth enamel carious lesion. All results are stored on disc. In an example, the enamel surfaces (6 mm2) of five human tooth enamel slices were exposed to an acetic acid buffer solution (pH 4.8) containing Ca, P and F. The mineral content variations in the enamel sample were followed in time by longitudinal microradiography. Mineral content evaluation shows in all cases a gradual increase in mineral loss. 3D plots of the mineral loss per unit area against tooth surface position at different stages of demineralisation of the same sample show an excellent uniform local behaviour in time with all tooth slices.

Quantitative light-induced fluorescence (QLF): a tool for early occlusal dental caries detection and supporting decision making in vivo.

OBJECTIVES: This study reports the development and assessment of a novel method using quantitative light-induced fluorescence (QLF), to determine whether QLF parameters ΔF and ΔQ were appropriate for aiding diagnosis and clinical decision making of early occlusal mineral loss by comparing QLF analysis with actual restorative management. METHODS: Following ethical approval, 46 subjects attending a dental teaching hospital were enrolled. White light digital (WL) and QLF images/analyses of 46 unrestored posterior teeth with suspected occlusal caries were made after a clinical decision had already been taken to explore fissures operatively. WL and QLF imaging/analysis were repeated after initial cavity preparation. The type of restorative treatment was determined by the supervising clinician independent of any imaging performed. Actual restorative management carried out was recorded as fissure sealant/preventive resin restoration (F/P) or class I occlusal restoration (Rest.) thus reflecting the extent of intervention (=gold standard). All QLF images were analysed independently. RESULTS: The results showed statistically significant differences between the two treatment groups ΔF (p=0.002) (mean 22.60 - F/P and 28.80 - Rest.) and ΔQ (p=0.012) (mean 230.49 - F/P and 348.30 - Rest.). CONCLUSIONS: ΔF and ΔQ values may be useful in aiding clinical diagnosis and decision making in relation to the management of early mineral loss and restorative intervention of occlusal caries. CLINICAL SIGNIFICANCE: QLF has the potential to be a valuable tool for caries diagnosis in clinical practice.

The video camera compared with the densitometer as a scanning device for microradiography.

In the recent past image analysis systems, comprising a video (CCD) camera and dedicated software, have replaced densitometer-based systems to analyze mineral content profiles using transversal microradiography (TMR). The main reasons for the introduction of the CCD camera were the ease and speed at which it can be operated. The densitometer, as a scanning device for TMR, has in the recent years been validated and is in this study considered as 'gold standard'. Comparisons of the two scanning devices for measuring the optical density of microradiograms have never been reported in the literature. The focus of this study was on accuracy and reproducibility of the scanning devices with the emphasis put on possible limitations of the CCD camera relative to the densitometer. These include resolution, number of gray levels and homogeneity of illumination of the scan area. The microscope was arranged in such a way that the same area on the microradiogram could be assessed by both scanning devices. Three different types of lesions were analyzed: a subsurface lesion, a surface-softened lesion and a laminated lesion. Paired t tests showed no significant difference between the mineral content profiles produced by the two scanning devices. The integrated mineral loss values were calculated and compared with analysis of variance and showed no significant differences. It is therefore concluded that mineral content profiles and integrated mineral loss obtained by the CCD camera are as accurate and reproducible as those obtained by the densitometer.

Strategies for improving the assessment of dental fluorosis: focus on optical techniques.

In its milder forms, enamel fluorosis is characterized clinically by diffuse opacities. The appearance is due to optical properties of a subsurface or surface porous layer with lower mineral content. These areas usually have texture and color similar to those of initial caries lesions but generally another shape and location. Therefore, several optical methods, previously used to diagnose initial caries lesions, were applied to fluoride-induced opacities on extracted premolars and on incisors of four subjects in vivo. These methods included light-scattering measurements, white light illumination, violet light illumination, ultraviolet illumination, and laser fluorescence. Video images were captured with a charge-coupled-device (CCD) camera, digitized, and computer-processed. It is concluded that the light-scattering monitor can be used for the determination of the local porosity of fluorotic enamel and that the laser fluorescence method might be developed into a method applicable for the assessment of the severity of enamel fluorosis.

Laser fluorescence quantification of remineralisation in situ of incipient enamel lesions: influence of fluoride supplements.

The aims of this study were to test the laser fluorescence method for quantification of remineralisation in situ of enamel with incipient lesions and to evaluate the enhancement of remineralisation by fluoride (F) supplements when a F dentifrice was used on a regular basis by healthy young subjects. Enamel samples were cut from extracted premolar teeth with the natural surface kept intact, and subjected to a pH-cycling system to produce subsurface demineralisation in vitro. The enamel blocks were then inserted into composite holders bonded to the buccal surfaces of both upper first molars of 12 panelists. Each panelist participated in 3 separate experiments in a randomised order; each lasted for 5 weeks with washout intervals of 2 weeks between experiments; experiment 1: F dentifrice (0.145% F as NaF) only; experiment 2: F dentrifice + F lozenges (0.25 mg F x 6/day); experiment 3: F dentifrice + F chewing-gum (0.25 mg F x 6/day). Fluorescence radiance was measured before, during, and after in vitro demineralisation, and once a week during the in situ experimental periods. The enamel samples were then sectioned and analysed with transverse microradiography (TMR). Anova and Pearson correlation coefficient were used for the statistical analysis. At the end of the 5-week in situ periods, fluorescence radiance had been regained to a level of 80-100% of the value before in vitro demineralisation, indicating remineralisation of the enamel samples. There were no differences in the fluorescence radiance gain between the 3 different F exposures. However, there was a statistically significant time trend (p < 0.001), and a highly significant linear dependence (p < 0.001) between the final measurements obtained with the laser fluorescence method (LAF) and the data obtained from TMR, r = 0.76. It was concluded that: (1) with the sensitive LAF method it was possible to register the small changes in the enamel week by week during in situ remineralisation: (2) when F dentrifice was used regularly, F supplements such as F lozenges or F chewing-gum did not significantly enhance the in situ remineralisation of incipient enamel lesions in healthy, young adults with normal salivary flow.

Application of quantitative light-induced fluorescence for assessing early caries lesions.

Quantitative light induced fluorescence (QLF) is a nondestructive diagnostic method for the longitudinal assessment of early caries lesions in time. When a tooth becomes carious the fluorescence radiance at the location of the caries lesion decreases. The fluorescence image of enamel with incipient lesions can be digitized and then the fluorescence loss in the lesion can be quantified in comparison to the fluorescence radiance level of sound enamel. Changes in fluorescence radiance and lesion area can be followed in time to measure lesion development. The amount of fluorescence radiance loss is related to the mineral loss in the lesion. The technique can be used in vitro, in situ and in vivo to monitor mineral changes in lesions. Applications of QLF are found in the testing of products designed to inhibit demineralization and promote remineralization of caries. The method has been successfully applied to smooth surfaces as well as occlusal surfaces, but application on approximal lesions is not yet possible.

A longitudinal laser fluorescence study of white spot lesions in orthodontic patients.

Orthodontic treatment with fixed appliances increases the caries risk in young persons. The aim of this study was to apply a new caries diagnostic method, quantitative laser fluorescence, for longitudinal in vivo quantification of changes in incipient enamel lesions related to fixed orthodontic appliances. Seven young patients with active caries lesions disclosed at removal of the orthodontic brackets and bands were enrolled in the study. Caries preventive measures were intensified, including dietary advice, oral hygiene instructions, and the regular use of a fluoride dentifrice. The caries lesions were monitored with the quantitative laser fluorescence method after removal of the brackets and once a month thereafter. For each lesion, three quantities were measured: lesion area (mm2), mean fluorescence loss (%) over the lesion, and maximum loss of fluorescence (%) in the lesion. During a 1-year follow-up period, the areas of the lesions decreased and the enamel fluorescence lost was partly regained indicating that a remineralization process had occurred. It was concluded that quantitative laser fluorescence seems suitable for in vivo monitoring of mineral changes in incipient enamel lesions, and useful for the evaluation of preventive measures in caries prone persons, such as orthodontic patients.

Determination of mineral changes in human dental enamel by longitudinal microradiography and scanning optical monitoring and their correlation with chemical analysis.

Both longitudinal microradiography (LMR) and scanning optical monitoring (OM) are non-destructive methods for measuring mineral changes in dental tooth enamel slices with time at 169 locations on the slice. Average calcium losses from four human tooth enamel slices (300-400 micron thickness), etched in HClO4, were determined by LMR and chemical analysis (C). As predicted from theory, LMR and C correlate very well (r = 0.99), but the appearance of a systematic error of unknown source of 30% made with LMR, C, or both could not be avoided. Another, more complex, experiment concerned six human tooth enamel slices of the same thickness which were demineralized in an aqueous buffered acid solution containing Ca and PO4. From this experiment it was found that average calcium loss as measured by C and LMR correlated well with the optical scattering as measured with scanning OM (Spearman rank correlation rs approximately equal to 0.79). It was also found that three-dimensional plots of local calcium loss by LMR and scanning OM as a function of tooth slice surface position show a well-defined tooth-dependent increase due to local demineralization and rather similar behaviour with time. From the experiments it follows that LMR and scanning OM are reliable methods to determine the mineral change in a tooth tissue as a function of local tooth slice surface position and of time and that with LMR and scanning OM time- and position-dependent measurements with an oral device become feasible.

A model to investigate xerostomia-related dental caries.

The aim of this study was to develop an in vivo model in which onset, progression, and prevention of xerostomia-related dental caries can be studied. The progress of the caries process was investigated on ground and polished human enamel blocks placed in the lower denture of 7 edentulous subjects suffering from xerostomia. During a period of 6 weeks the samples were analyzed at weekly intervals by means of scanning optical monitoring, scanning longitudinal microradiography, scanning electron microscopy, and hardness measurements. Although the indentation length increased significantly within 2 weeks, no demineralization could be yet observed with scanning optical monitoring, longitudinal microradiography, and electron microscopy in most samples. Starting from the 3rd week a progressive demineralization could be observed by all methods. The mineral loss (hydroxyapatite) in the 3rd week was 0.0126 kg.m-2 (median) which increased to 0.0761 kg.m-2 after 6 weeks. Scanning electron microscopy showed a destruction which resembled that of natural xerostomia-related dental caries. From this study it may be concluded that the in vivo model developed is suitable for studying onset, progression, and prevention of xerostomia-related dental caries.

Comparison of laser fluorescence and longitudinal microradiography for quantitative assessment of in vitro enamel caries.

A new quantitative, non-destructive method using laser-induced fluorescence (LAF) was compared with longitudinal microradiography (LMR) for assessment of mineral changes in enamel slices using an in vitro caries model. Ten enamel slices, cut longitudinally from sound natural smooth surfaces of human teeth, were exposed to de- and remineralization in a pH-cycling model. The enamel slices were subjected to LAF and LMR measurements before and at 2, 4, 7, and 9 days of demineralization. For LAF, the average fluorescence radiance decreased during the demineralization period with 11% by day 2 and 49% by day 9. For LMR, the corresponding average loss of mineral content changed with 0.01 and 0.10 kg.m-2 over the same time period. The mineral losses in each individual enamel slice measured with the two techniques were strongly correlated, r = 0.97. The Spearman rank correlation coefficient for all LAF and LMR demineralization results was 0.86. The precision (coefficient of variation) for LAF was 3.1%, corresponding to 0.005 kg.m-2, and the repeatability error for LMR was 0.02 kg.m-2, indicating a lower discrimination threshold for LAF compared to LMR. It was concluded that the new, sensitive, non-destructive LAF method provides possibilities for further improvement in the quantification of initial caries lesions in natural smooth enamel surfaces for use in in vitro studies. Furthermore, it offers potential in in situ caries studies as well as a tool in the diagnosis of early enamel caries in vivo.

Mineral loss in incipient caries lesions quantified with laser fluorescence and longitudinal microradiography. A methodologic study.

The laser fluorescence method (LAF) was validated with longitudinal microradiography (LMR) for assessment of mineral loss in incipient caries lesions in human enamel. Fluorescence radiance scans and LMR recordings were made of 36 enamel slabs with incipient lesions. Original sound values for fluorescence radiance and enamel amount (kg.m-2) at the lesion site were reconstructed by a computer algorithm. Changes in fluorescence radiance and amount of enamel in each measuring point were calculated. The reconstruction method was tested on 20 sound enamel surfaces. The differences between measured and reconstructed values were -0.13 +/- 0.17% with LAF and 0.002 +/- 0.005 kg.m-2 with LMR. The repeatability of the caries quantification was tested by measuring one lesion 20 times. The fluorescence loss in this lesion was 18.2 +/- 1.0%. The enamel loss was 0.09 +/- 0.02 kg.m-2. The correlation between measurements with the two methods was r = 0.73. The non-destructive laser fluorescence method was concluded to be a sensitive and valid method for quantification of mineral loss in enamel caries lesions.

A new method for in vivo quantification of changes in initial enamel caries with laser fluorescence.

A new method for the in vivo assessment of changes in initial enamel caries lesions was developed and tested. A CCD camera equipped with a high-pass filter (lambda > 520 nm) collects the fluorescence image of carious teeth, illuminated intraorally with diffuse laser light (lambda = 488 nm). Incipient lesions show a loss in fluorescence to be expressed as a percentage of fluorescence radiance of sound tissue. A PC program (Inspektor, model QLF 1.0) is used for display, storage, and subsequent analysis of images. To enable the calculation of fluorescence loss, the fluorescence of sound tissue at the lesion site is reconstructed from the radiances of sound tissue bordering the lesion. This method was tested on 19 visually sound buccal surfaces in vivo. The differences between actual and reconstructed radiance was -1.6 +/- (SD) 1.1%, over areas varying between 8 and 14 mm2. The repeatability of the caries quantification was tested by measuring one arrested initial caries lesion 25 times in vivo. The lesion area was 0.56 +/- 0.20 mm2, and the loss of fluorescence was 17.6 +/- 0.7%, corresponding to a lesion depth of 17 +/- 2 microns. The new quantitative method was applied for the testing of an in vivo caries model using plaque-accumulating brackets on premolars scheduled for extraction. Videoimages were recorded in vivo before bracketing and 0, 2, 3, and 5 weeks after debracketing. Clear changes between the different time points were recorded for both lesion size and mineral content.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-induced fluorescence studies on dehydration of incipient enamel lesions.

Changes in the hydration state of enamel affect its optical qualities, such as light scattering and fluorescence. In this study, the rate of fluorescence loss was measured when incipient enamel lesions with different de-remineralization history were left to dehydrate. Four groups of lesions were studied. In groups A, B and C, the lesions were prepared in vitro in an acid-gel system. Group A was kept as control, and groups B and C were remineralized (4 weeks) without and with 1 ppm F in solution, respectively. Group D consisted of natural incipient lesions. Enamel fluorescence was measured for all lesions immediately after removal from water and subsequently at short intervals for 30 min. The change in fluorescence with dehydration varied between the groups. In lesions from groups A and B, it followed a double exponential decrease, while in lesions from groups C and D, it followed a mono-exponential decrease. In all groups, the fluorescence of sound surfaces declined mono-exponentially. The 'fractional fluorescence difference', defined as (L(sound) - L(carious) )/L(sound), became constant after periods of dehydration of about 5, 5, 20 and 5 min for groups A to D, respectively. The observation of the change of fluorescence with dehydration should be taken into consideration when planning studies that use fluorescence as an assessment method. However, it might also be used to gain insight into the properties for fluid transport inside the various lesions, relevant to de-remineralization or fluoride treatments.

Application of transverse microradiography for measurement of mineral loss by acid erosion.

This paper describes a novel application of transverse microradiography for the detection and quantification of mineral loss due to acid erosion in thin tooth sections. Sixty-four specimens were randomly divided into eight equal-sized groups and exposed to an orthophosphoric-acid-based erosive fluid (pH = 3) for 0, 0.25, 0.5, 1, 2, 5, 12, or 24 hrs. We made microdensitometric scans separately across both enamel and dentin to derive data for the total mineral loss and the minimum mineral content within the eroded area. We then analyzed specimens using a profilometer to determine (1) the area above a plot of penetration depth against distance and (2) the maximum depth of penetration. Correlation coefficients for comparisons between microradiographic and profilometric data for both enamel and dentin specimens varied between 0.87 and 0.96. Two-sample t tests demonstrated that the microradiographic technique could detect early erosion, i.e., discriminate between erosion times of less than 1 hr. It was concluded that this application of transverse microradiography was a useful and acceptable method for the measurement of early mineral loss in vitro, occurring as a result of acid erosion.