Marginal Adaptation and Quality of Interfaces in Lithium Disilicate Crowns — Influence of Manufacturing and Cementation Techniques
Purpose: To evaluate the cement line thickness and the interface quality in milled or injected lithium disilicate ceramic restorations and their influence on marginal adaptation using different cement types and different adhesive cementation techniques.
Methods and Materials: Sixty-four bovine teeth were prepared for full crown restoration (7.0±0.5 mm in height, 8.0 mm in cervical diameter, and 4.2 mm in incisal diameter) and were divided into two groups: CAD/CAM automation technology, IPS e.max CAD (CAD), and isostatic injection by heat technology, IPS e.max Press (PRESS). RelyX ARC (ARC) and RelyX U200 resin cements were used as luting agents in two activation methods: initial self-activation and light pre-activation for one second (tack-cure). Next, the specimens were stored in distilled water at 23°C ± 2°C for 72 hours. The cement line thickness was measured in micrometers, and the interface quality received scores according to the characteristics and sealing aspects. The evaluations were performed with an optical microscope, and scanning electron microscope images were presented to demonstrate the various features found in the cement line. For the cement line thickness, data were analyzed with three-way analysis of variance (ANOVA) and the Games-Howell test (α=0.05). For the variable interface quality, the data were analyzed with the Mann-Whitney U-test, the Kruskal-Wallis test, and multiple comparisons nonparametric Dunn test (α=0.05).
Results: The ANOVA presented statistical differences among the ceramic restoration manufacturing methods as well as a significant interaction between the manufacturing methods and types of cement (p<0.05). The U200 presented lower cement line thickness values when compared to the ARC with both cementation techniques (p<0.05). With regard to the interface quality, the Mann-Whitney U-test and the Kruskal-Wallis test demonstrated statistical differences between the ceramic restoration manufacturing methods and cementation techniques. The PRESS ceramics obtained lower scores than did the CAD ceramics when using ARC cement (p<0.05).
Conclusions: Milled restorations cemented with self-adhesive resin cement resulted in a thinner cement line that is statistically different from that of CAD or pressed ceramics cemented with resin cement with adhesive application. No difference between one-second tack-cure and self-activation was noted.SUMMARY
INTRODUCTION
Ceramic restorations are aimed at the esthetic, structural, and biomechanical recovery of dental elements. These materials constitute one of the main indirect alternative restorations, as they present favorable properties, such as chemical stability, biocompatibility, high resistance to compression, a thermal expansion coefficient near the dental structure, and optical properties that are similar to the dental tissues, making adequate esthetic and functional results possible.1-7
Resistance to fracture and marginal adaptation are among the key factors necessary for longevity and clinical success in indirect restorations.1-12 These materials can be classified into three groups: the siliceous, the aluminum-based, and the zirconia-based groups.2,13
In the siliceous group, lithium disilicate ceramics are widely used, as they contain favorable properties of high intrinsic resistance and adherence.6,7,13,14 These ceramics are processed using two methods: automation technology using the CAD/CAM system or isostatic injection by heat technology.
The composition of siliceous ceramics includes a silicon dioxide network that makes conditioning possible by means of hydrofluoric acid and the application of silane as a binding agent.13-15 The internal surface of the conditioned restoration is predisposed to the micromechanical and chemical interaction with the luting agent.13-15 In turn, silane is a bifunctional molecule with hydrolyzable monovalents that allow for primary chemical bonding between the inorganic materials of the ceramic (SiO2) and the organic portion of the resin material within its double bonding between carbons.13,14
Resin cements promote adhesive continuity between the tooth and the ceramic restoration, using a high content of silicon dioxide, promoting the sealing of the tooth/restoration interface.6,9,14-17 Such cementing agents can be classified according to the types of applied adhesion strategy,18 which include the conventional resin cements, used with etch-and-rinse adhesive systems; self-conditioning resin cements, associated with self-etch adhesive systems; and self-adhesive resin cements.18 This last type requires fewer steps during the cementation process and foregoes the need to treat the dentinal substrate separately, thereby reducing the work time and diminishing the sensitivity of the technique.7,16,19
Since the indirect restorations present an interface between the restoration and the dental structure, the vertical thickness of the cement line becomes a determining factor in establishing favorable characteristics of marginal adaptation.8-12 A wide line of a cementing agent exposed to the oral environment may result in periodontal problems and marginal staining, among other complications.20,21 To minimize these complications, the adhesive cementation techniques must follow an optimized and rational protocol that seeks to achieve predictable results.7,14,19,22 This process is influenced by such factors as the type of ceramic, the type of resin cement, the preparation and the appropriate cleaning of the dental substrate, the handling of the material, the activation of the adhesive/cement system, and the method used to remove the excess cement from the margins.14,19,22 Therefore, procedures that establish the moment and method of removing the excess cement are necessary in order to obtain the characteristics of complete sealing and quality of interface.22,23
Within this context, studies8-12,22-29 show that factors such as the ceramic restoration manufacturing method, the configuration of the crown margin, the space required for the cement, the type of cement, and the cementation technique used can all lead to different results in terms of marginal adaptation.
The present study aims to evaluate the cement line thickness and the interface quality in milled or injected lithium disilicate ceramic restorations and their influence on marginal adaptation using different cement types and different adhesive cementation techniques. The null hypotheses included the following: no differences would be detected between the manufacturing methods for ceramic restorations, between the cements themselves, or between the cementation techniques.
METHODS AND MATERIALS
The present study used 64 lower bovine incisors, which were stored in a 5% Chloramine-T solution. The teeth were cleaned using curettes and a prophylaxis brush with pumice and without fluoride (Pasta Prophy Zircate, Dentsply, Milford, CT, USA).
One base structure was created to fix the teeth, allowing for greater stability of the structures during optical microscopic analysis: mechanical retention was created in the roots prior to the inclusion of chemically activated acrylic resin (Jet, Clássico Dental Producs, São Paulo, SP, Brazil) in polyvinyl chloride (PVC) tubes (Tigre, Joinville, SC, Brazil). The teeth were placed with the long axis parallel to the height of the tube, with the cementoenamel junction positioned approximately 3 mm above the acrylic resin surface.
For the prosthetic preparation of the full crown, the specimens were attached to a lathe (Nardini–ND 250 BE, São Paulo, SP, Brazil) and were prepared under constant water cooling. The final dimensions of the preparations were 7.0 ± 0.5 mm in height, 8.0 ± 0.5 mm in cervical diameter, and 4.2 ± 0.5 mm in incisal diameter. For the preparations, this study used a coarse round end taper diamond bur number 5850.314.018 (Brasseler, Savannah, GA, USA) with 0.8-mm depth at the end line. The diamond bur was replaced after every five preparations. Next, the preparations were finished using fine round end taper diamond burs, numbers 4137 F and 4137 FF (KG Sorensen, Cotia, SP, Brazil) (Fig. 1A and 1B). All angles were rounded, and the preparation finish line was located 1.0 ± 0.2 mm above the cementoenamel junction.
Citation: Operative Dentistry 42, 2; 10.2341/15-288-L
The specimens were divided randomly into two groups according to the manufacturing technique of indirect restorations made of lithium disilicate ceramics, which was performed by means of the automation technique guided by the CAD/CAM system (IPS e.max CAD, Ivoclar Vivadent, Schaan, Liechtenstein) or isostatic injection by heat technology (IPS e.max Press, Ivoclar Vivadent).
Two types of resin cements were used to cement the ceramic crowns: dual-activation conventional cement (RelyX ARC, 3M ESPE, St Paul, MN, USA) and dual-activation self-adhesive cement (RelyX U200, 3M ESPE, Seefeld, Germany).
Two techniques were executed to remove the excess cement at the crown margins of the restoration after performing two activation methods: initial chemical activation or initial pre-activation for one second (tack-cure 1s) (Figure 2A and 2B). All of the materials used in this study are described in Table 1.
Citation: Operative Dentistry 42, 2; 10.2341/15-288-L
Preparation of the Lithium Disilicate Crowns—CAD/CAM System
The 32 ceramic crowns were manufactured by scanning each prepared tooth. The teeth received light air spray. Next, a uniform layer of contrast, without excess, was placed (IPS Contrast Spray, Ivoclar Vivadent), and the optical reading of the prepared teeth was performed with a laboratory scanner (InEos Blue, Sirona Dental Systems, Bensheim, Germany), transferred to a computer, and subsequently processed by an appropriate system software (Cerec inLab. SW4 version of application 4.0.2.45144, Sirona), in which a three-dimensional virtual model was created for each of the 32 teeth. Next, the external margin of the tooth and the preparation's finish line were defined, and the adaptation was measured along the entire extension of the crown.
Each IPS e.max CAD ceramic block was placed in the milling unit (In Lab MC XL–102591, Sirona), which had two diamond burs, one cylindrical, with a diameter of 1.2 mm, and the other conical. Next, from the digital model, the blocks were milled to create the crowns. The restorations were completely cleaned, and all of the residues from the milling additive from the CAD/CAM unit were fully removed. For the crystallization process, each crown was positioned on pins for crystallization (IPS e.max CAD Crystallization Pin, Ivoclar Vivadent), and its inner portion was filled with auxiliary firing paste (IPS Object Fix Putty/Flow, Ivoclar Vivadent) to the edge of the restoration margin.
Preparation of the Lithium Disilicate Crowns—PRESS SYSTEM
Thirty-two prepared teeth were impressed with polyvinylsiloxane (Express, 3M ESPE) through the double-impression technique in individual molds manufactured in PVC (Tigre). Type IV plaster (Fuji Rock, GC America, Aslip, IL, USA) was used to manufacture the dies. The teeth were stored in distilled water at 23°C ± 2°C until the cementation process. The dies received a layer of spacer (Spacelaquer Ducera Lay, Degussa Huls, Hanau, Germany) approximately 1 mm above the finish line. The dies were isolated with insulator (Die Lube, Dentaurum J.P. Winkelstroeter KG, Pforzheim, Germany), and wax patterns with 0.7-mm thickness were prepared over the master dies using a wax immersion unit (Hotty, Renfert, Hilzingen, Germany). Next, the wax patterns were included in a coating agglutinated by phosphate from the IPA system itself (PressVest Speed, Ivoclar Vivadent). After the coating was set, a sequence of two heating stages was applied, in which the temperature was increased from 58°C/min to 250°C and maintained for 30 minutes before being increased to 58°C/min to 850°C and maintained for one hour. After the pre-heating stage, the coating cylinders were immediately transferred to the EP500 press oven (Ivoclar AG, Ivoclar Vivadent). The temperature was 920°C and then compression was completed. The coating cylinders were removed from the oven and cooled for two hours. The cooled specimens were removed from the coating using an 80-μm glass bead blasting (Williams Glass Beads, Ivoclar North America, Amherst, NY, USA). The final dimension of the crowns was 7.0 ± 0.5 mm in height and 8.0 ± 0.5 mm in cervical diameter, with a thickness of 2.0 mm, measured with a digital caliper (Mitutoyo, Suzano, SP, Brazil).
Crown Cementation
All of the specimens were cleaned with a bristle brush and pumice without fluoride (Prophy Zircate, Dentsply, Rio de Janeiro, RJ, Brazil) and were washed with a water/air spray.
In the cementations using RelyX ARC, the tooth substrates were treated with an Adper Single Bond Plus adhesive system (3M ESPE). Phosphoric acid at 35% (Ultra-Etch, Ultradent do Brazil Ltda, Indaiatuba, SP, Brazil) was initially applied for 15 seconds on the enamel along the finish line, followed by 15 seconds on dentin, and was then washed for one minute with air/distilled water spray, removing the excess water with high-speed suction, and gently air-dried without desiccation. Next, two layers of the adhesive system were applied actively and the excess was removed with applicator tips (Cavibrush, FGM, Joinville, SC, Brazil), followed by evaporation of the solvent for five seconds. The photoactivation was carried out using a light-curing unit (650 mW/cm2, LED Bluephase, Ivoclar Vivadent) for 20 seconds.
For RelyX U200, after the teeth were cleaned, 35% phosphoric acid (Ultra-Etch, Ultradent do Brazil Ltda) was only applied to the enamel for 15 seconds, and they were next washed for one minute with air/distilled water spray, the excess water was removed with high-speed suction and then gently air-dried without desiccating. The prepared substrates presented the appearance of a slightly moist dentin surface.
The internal surfaces of the lithium disilicate ceramics were conditioned with 10% hydrofluoric acid11,30 (Condac Porcelana, FGM Brazil) for 20 seconds, followed by an air/distilled water spray for one minute. Next, 35% phosphoric acid was applied for 30 seconds,30 followed by air/distilled water spray for one minute. After this step, the crowns were placed in an ultrasound with distilled water for five minutes and dried in oil-free air. The silane agent (RelyX Ceramic Primer, 3M ESPE) was applied, and after five minutes the surface was dried with an air spray for 20 seconds.
Cementation Technique Using RelyX ARC and RelyX U200 with Initial Chemical Activation
The cements were handled and applied in a thin layer to the internal surface of the crowns. After three minutes of initial chemical activation, the excess resin cement was removed using applicator tips (Cavibrush, FGM) and later using a curette (Scaler H6/H7, BISCO Inc, Schaumburg, IL, USA). Next, photopolymerization was performed for 40 seconds, with one activation on each of four crown surfaces for a total of 160 seconds with the light-curing unit, using a “soft start” curing program that began with a low light intensity followed by its gradual increase. The light intensity was monitored with a radiometer (Bluephase Meter, Ivoclar Vivadent). The crowns were maintained in position under a constant 454 g pressure during the entire setting process, during the removal of the excess resin cement, and until photoactivation had been completed.
Cementation Technique Using RelyX ARC and RelyX U200 with Tack-cure 1s
With the exception of the initial chemical activation process of the resin cement, the cementation procedures were performed as described above. For this technique, the method of previous photoactivation was performed for one second for both resin cements at an intensity of 650 mW/cm2, in an exposure program at low power at buccal and lingual surfaces, at a distance of 3.0 mm from the activating tip to the full crown, using a light-curing unit. Next, photopolymerization was performed as described above.
Analysis of the Marginal Adaptation
To measure cement line thickness and to conduct the interface quality analysis, eight marks were made on the surfaces of the tooth roots, symmetrically placed 2.0 mm apical to the end of the tooth preparation; the tooth roots had been marked using a number 1011 spherical diamond bur (KG-Sorensen, São Paulo, SP, Brazil). The defined areas served as reference, and between these marks, four measurements were taken, totaling 32 measurements per specimen (Figure 3). The cement line thickness was evaluated through the vertical measurement of the cement line areas between the crown margins and the preparation finish line. To measure the marginal adaptation, an optical microscope was used (STM, Olympus Optical Co Ltda, Tokyo, Japan) at 30× magnification and with the images being displayed by the digital reading unit for coordinates X and Y (MMDC 201, Olympus Optical Co Ltda), with registered values in micrometers and with precision of 0.5 μm.
Citation: Operative Dentistry 42, 2; 10.2341/15-288-L
To analyze the interface quality, the following score values were used according to the cement line characteristics and sealing: 1 = deficient: irregularities were found with discontinuity; 2 = regular: minor irregularities existed but without the presence of discontinuity; and 3 = satisfactory: sealing presented a cohesive pattern, maintaining an integral margin along the entire extension.23
Scanning Electron Microscopy
The scanning electron microscope (SEM) micrographs were presented to demonstrate the various features found in the cement line, showing the measurement areas and interface quality. Sixteen crowns were used, eight crowns from each group, and they were subdivided by cement type and cementation method. One impression was taken from the prepared teeth using Express polyvinylsiloxane (3M ESPE) using the double-impression technique in individual molds manufactured in PVC (Tigre), and a replica was created using an epoxy resin material (EpoxiCure 2 Resin, Buehler An ITW Company, Lake Bluff, IL, USA). The epoxy resin models used to evaluate the restoration marginal adaptation were sputter-coated with gold (Balzers-SCD050, Oerlikon Balzers, Balzers, Liechtenstein) for 180 seconds at 40 mA. The images were obtained with a SEM (LEO 435 VP, LEO, Cambridge, UK) at 20 kV with magnification from 50× to 200×.
Statistical Analysis
To evaluate whether there was a difference in the mean values for the variable cement line thickness according to the manufacturing techniques of the ceramic restorations (CAD and PRESS), cements (ARC and U200), and cementing techniques (initial self-activation and the tack-cure), three-way analysis of variance (ANOVA) was applied. When ANOVA indicated a statistically significant difference in the mean values of the dependent variable cement line thickness, according to the combination of the analyzed factors (manufacturing techniques, cements, and cementing techniques), the multiple comparisons post hoc test between the different factor levels was made using the multiple comparisons Games-Howell test for heterogeneous variances, since the Levene test showed heterogeneous variances among the analyzed factors. The significance level for all tests was set at α = 0.05. For the variable interface quality that displayed an ordinal scale, the nonparametric Mann-Whitney U-test was used when the analysis involved each isolated factor. For treatment interactions, the Kruskal-Wallis test was applied. When the Kruskal-Wallis test indicated differences between the average scores of at least two treatments, comparison between them was made using the multiple comparisons nonparametric Dunn test. The significance level was set at α = 0.05. The statistical analysis was performed using the software IBM SPSS Statistics 22.0 (IBM Corp, Armonk, NY, USA).
RESULTS
Cement Line Thickness
The three-way ANOVA test presented statistical differences among ceramic restoration manufacturing methods and a significant interaction between the manufacturing methods and the types of cement (p<0.05).
The Games-Howell test demonstrated statistical differences for the CAD with regard to resin cement (p<0.05). The U200 resin cement presented lower values of cementation thickness when compared to the ARC resin cement in the CAD technique (p<0.05). When compared, the U200 cement for the two methods of CAD and PRESS manufacturing presented statistical differences (p<0.05). The lower values of marginal adaptation were obtained for the CAD technique. The average values are presented in Table 2.
Quality of the Interface Cement Line Thickness
The Kruskal-Wallis test and multiple comparisons nonparametric Dunn test presented statistical differences in the cementation techniques when comparing the manufacturing methods for ceramic restorations; the values for PRESS presented lower scores (p<0.05). The scores obtained for the cementation quality in the PRESS technique, using pre-activation for one second, presented statistical differences (p<0.05), showing higher scores both for the ARC cement as well as for the U200 cement, as compared to the conventional cementation technique (initial chemical activation). The means are presented in Table 3.
Correlation Between Cement Line Thickness and Quality of the Interface
The multiple comparison Games-Howell test of heterogenic variances showed that mean values of cement line thickness did not present significant differences between regular and deficient interfaces; however, a satisfactory interface quality presents a mean value of cement line thickness (μm) that is thinner compared to those of regular and deficient interfaces. The comparison is showed in Tables 2 and 3.
SEM
Illustrative SEM images, showing the locations of measurement and the interface quality, are shown in Figures 4 through 7.
Citation: Operative Dentistry 42, 2; 10.2341/15-288-L
Citation: Operative Dentistry 42, 2; 10.2341/15-288-L
DISCUSSION
The interface of indirect restorations can be considered acceptable when it presents cement line thickness values of approximately 120 μm and when the interface quality presents a uniform aspect without the presence of cracks or irregularities.5,9,23,31,32 According to these requirements, the elements evaluated in this study were within clinically acceptable limits.
The suggested null hypotheses were rejected for both the ceramic manufacturing techniques and the types of cement, given that a significant difference in the marginal adaptation could be observed.
The measurement of the vertical thickness of the cement line showed the best adjustment for the CAD crowns when compared to the PRESS crowns. These results corroborate with those of Ng and others,8 who compared the two methods of manufacturing, pointing out the difficulties associated with the PRESS method. The CAD ceramics required fewer laboratory steps, allowing for a greater control of the work stages.8,9 By contrast, the PRESS ceramics can undergo a greater influence from independent factors, such as the types of impression materials, the molding technique, the variation in temperature during the transport of the materials to the laboratory, the attainment of the plaster model, the laboratory states for the manufacturing of a piece, and the actions on the part of the operator.8,9,19
In the evaluation of the cements, the U200 presented lower thickness values of the cement line when compared to the ARC for the crowns manufactured using the CAD method. This fact may well be related to the stages of adhesion and photoactivation, in which the activation of the adhesive in the dental substrate, according to manufacturer instructions, was carried out prior to the placing of the indirect restorations, which may have caused an increase in the cement line thickness.7-9 In addition, one must consider the difficulty of limiting oneself to the thickness of the adhesive/cement layer, as this condition can accelerate the mechanism of fatigue and cause early failure.7,9 These results corroborate with findings from Borges and others,11 who compared the influence of different types of cements on the marginal discrepancy in pure ceramic systems. Clinically speaking, one must observe aspects such as the viscosity of the cement and its correct manipulation protocol.9,14,17,27
The marginal discrepancy of the two methods of cementation evaluated in this study was not affected by the initial chemical activation, nor by the tack-cure 1s, as shown in Table 3. It is suggested that the tack-cure 1s pre-activation method and the removal of its excess, with the aid of a brush and scaler, can lead to a faster and more efficient procedure, which is a clinically relevant consideration. Paying close attention to attainment of the activation time of only one second is highly recommended, since a time of greater than three to five seconds can produce areas of hard resinous cement on the surface, as compared to fluid cement in deeper regions, which can cause maladjustments and dislocations of the fluid and poorly activated cement areas. This type of pre-activation does not affect the intrinsic characteristics of the resinous cement, according to Flury and others,33 who held that the physical properties of the cements remain unaltered when activation is performed for five seconds, followed by the removal of the excess of cement and the final activation.22,33 In another study about the techniques for the removal of excess cement, Anami and others22 determined that the morphology of the restoration margin and the roughness of the interface affect the accumulation of bacterial biofilms, and these authors also obtained favorable results using brushes in the removal of excess resinous cement. This method, as compared to other such removal methods, better prevents against bacterial colonization in the adhesive interface.22 Other important aspects were reported by Conrad and others5 who explained that the exposure of a wide band of cementing agent to the oral fluids, as well as to mechanical wear, can lead to the dissolution or modification of the adhesive interface's surface aspect, in addition to allowing the formation of niches that promote the accumulation of bacterial plaque, marginal staining, periodontal problems, and restoration failures.5,20-22 Other relevant aspects include the final characteristics of prosthetic preparation, such as the conicity of the axial slopes, convergence of the walls, smooth surfaces, transitions, and rounded angles, as they promote the appropriate axis of the restoration's longitudinal insertion, thereby avoiding the formation of hydraulic pressure, favoring a better draining of the cement and an appropriate placing of the crown itself.14,17,20,24,25 Some studies2,7,9,17,18,33-35 have reported that the characteristics found in the preparations for adhesive cementation increase the resistance to fracture (tooth/restoration) by providing a more uniform distribution of strength and a lower concentration of stress. Moreover, the existence of cracks in the cementation line can generate a concentration of stress, which can reduce the restoration's final resistance.1,3,5,9,17 The tack-cure 1s and the use of a brush to remove the excess cement from the interfaces may also represent an interesting method, and these steps should be followed by a careful light irradiation protocol to obtain the optimal properties of the adhesion and from the resin cement.17,22,33-35 Given this, further research on this method is warranted, as the resinous cements can present behaviors that are different from their physical-chemical properties.
The interrelationship aspects of the criteria evaluated in this study suggest a greater durability and longevity of the adhesive interfaces, bearing in mind that these factors are limited to the stability and characteristics of each element involved.4,5,7,9,17,19,22,31,32
CONCLUSIONS
Within the limitations of this study, it can be concluded that
- 1.
Milled ceramic restorations cemented with self-adhesive resin cement resulted in a thinner cement line compared to the resin cement with adhesive application;
- 2.
No difference between tack-cure 1s and self-activation was noted; and
- 3.
The best interface quality is in fact related to the reduced thickness of the cement line.
(A and B) - The final dimensions of the preparations were (A) 7.0 ± 0.5 mm in height, (B), 8.0 ± 0.5 mm in cervical diameter, and (C) 4.2 ± 0.5 mm in incisal diameter.
(A and B) - Removal of the excess cement using the activation tack-cure. (A) Initial removal with application tips; (B) exploration and final removal with curette.
Illustrative image related to interface quality and measurement locations. (A) Tooth structure, (B) ceramic, (C) tooth/restoration interface, (D) marks for guidance, and (E) locations of measurement.
Sample and MEV image. (A) Tooth structure, (B) ceramic, (C) tooth/restoration interface, (D) marks for guidance, and (E) locations of measurement.
(A and B). Illustrative image related to interface quality, score 3 = satisfactory, in which case the sealing presents a standard of cohesion and integrated margin along the entire extension. (A) Magnification = 53×; (B) magnification = 200×.
Figure 6. (A and B). Illustrative image related to interface quality, score 2 = regular, in which case small irregularities exist but without the presence of discontinuity. (A) Magnification = 50×; (B) magnification = 200×.
Figure 7. (A and B). Illustrative image related to interface quality, score 1 = deficient, in which case irregularities were found with discontinuity. (A) Magnification = 52×; (B) magnification = 200×.
Contributor Notes