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Seungik son.
1 Department of Future Convergence Engineering, Kongju National University, Cheonan 1223-24, Korea; moc.liamg@32rlddmtsht
2 Department of Mechanical and Automotive Engineering, Kongju National University, Cheonan 1223-24, Korea
This experimental study investigated the effect of laser parameters on the machining of SS41 and SUS304. The metallic materials play an important role in engineering applications. They are widely used in high-tech industries such as aerospace, automotive, and architecture. Due to the development of technology and high-tech industrialization, the various processing technologies are being developed with the requirement of high precision. However, the conventional cutting process is difficult to meet high precision processing. Therefore, to achieve high precision processing of the SS41 and SUS304, laser manufacturing has been applied. The current study investigated the process quality of laser cutting for SS41 and SUS304, with the usage of a continuous wave CO 2 laser cutting system. The experimental variables are set to the laser cutting speed, laser power, and different engineering materials. The results are significantly affected by the laser parameters. As the result, the process quality of the laser cutting has been observed by measuring the top and bottom kerf widths, as well as the size of the melting zone and Heat Affected Zone (HAZ) according to volume energy. In addition, the evaluation of the laser processing parameters is significantly important to achieve optimal cutting quality. Therefore, we observed the correlation between the laser parameters and cutting quality. These were evaluated by analysis of variance (ANOVA) and multiple regression analysis. The experimental results of kerf top, kerf bottom, melting width, and HAZ on the laser parameters are properly predicted by multiple regression. In addition, the effect of laser parameters on the materials is determinant by the percentage of contribution of ANOVA.
There are various metallic materials used for production in the industrial fields. Among the metallic materials commonly used in industry, SS41 and SUS304 are the most widely used. SS41 is a structural steel containing Si and Mn. It is widely used in various fields such as aerospace, automobiles, ships, and construction due to its great mechanical properties and low cost. SUS304 is stainless steel that has high corrosion resistance due to containing Cr component. It is generally used for various applications without surface treatment because the metallic materials have low thermal deformation. It is challenging to machine SS41 and SUS304 with high precision using conventional techniques such as mechanical cutting, drilling milling. The features of the mechanical method have critical processing problems such as tools wearing [ 1 ]. However, the limitation of mechanical processing can be solved by laser processing. Thus, the laser machining using CO 2 laser is used as an alternative to the conventional method. Furthermore, the manufacturer prefers to use high-power laser processing rather than mechanical processing because the laser processing has more advantages than mechanical processing. Laser machining can be performed on various materials without tool wear and additional cost. The method is non-contact processing, which provides flexibility in processing [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. Among the laser system used by industries, the CO 2 laser has more economical than other laser systems. In addition, the laser system has high stability during the cutting or drilling processes of the significantly thick materials. Even non-metallic materials can be easily processed using a CO 2 laser. The special concern for manufacturers using laser cutting is to maximize productivity with the high quality of components produced through the high-power laser cutting process. However, to improve product quality and productivity, the effects of laser parameters on the material should be considered as major issues. In addition, to control the influence of the laser beam, the laser parameters must be selected appropriately. Indeed, adjustable laser parameters include laser power, cutting speed, assist gas pressure, and stand-off distance.
To maintain high precision and good quality process, the laser parameters applied to the process should be properly selected, but the effect of the parameters is difficult to predict. Besides, many manufacturers spend a lot of time and effort to determine the laser parameter which suitable for the process. In the previous studies, experiments were carried out according to specific laser parameters, and there was a comparative analysis of the effect of each parameter on the processing quality. Lamikiz et al. [ 16 ] suggested the optimum working areas and cutting conditions for the laser cutting of steel. The main experimental parameter was the thickness of the material and the results showed a remarkable different behavior between the thinnest and the thickest sheets. Kaebernick et. al. [ 17 ] described a monitoring technique in the laser cutting. The analytical techniques proved that the surface roughness was improved by controlling laser pulses. Rajaram et. al. [ 18 ] studied the effect of parameters on the characteristics of steel specimens. The material was cut through a CO 2 laser cutting system and cutting results were analyzed with kerf width, surface roughness, and heat-affected zone. The material which was cut using the CO 2 laser showed different results depending on the change of parameters. Yilbas [ 19 ] suggested that various parameters were affected during the laser cutting process and then, the laser power and the cutting speed for the kerf width were examined. It was confirmed that the kerf width increased with the combination of the laser power and the energy coupling factor. Anghel et. al. [ 20 ] demonstrated the experiment of laser cutting on 304 stainless steel miniature gear. In the experiment, the CO 2 laser system was employed to cut the miniature. The effects of laser parameters on average surface roughness (R a ) had been investigated on the surface of craters and cracks.
The previous studies have done significant investigations on the influence of laser parameters in the laser cutting process to materials. However, there is a lack of experimental studies on comparing laser cutting of SS41 and SUS304 under different laser parameters. In this study, we studied the effect of high-power laser parameters on the different metallic materials. Multiple regression and analysis of variance (ANOVA) are used to predict the kerf width, melting width, and Heat Affected Zones (HAZ) generated after laser cutting. In addition, these are used to investigate the effects of parameter and interaction between parameters. In this paper, we firstly describe the material properties, experimental equipment, and laser parameters. Then, the experimental results are discussed. Finally, conclusions are summarized.
In the present study, a continuous wavelength CO 2 laser system, which has a maximum laser power of 4.4 kW (Bylaser 4400, Bystronic, Niederönz, Switzerland), was used for the cutting process. During the experiment, the stand-off distance of the laser is set to constant, and the spot diameter is fixed at 2 mm. In addition, the laser cutting process depends on assistance gases. The assistance gases, N 2 and O 2 , are common assistance gasses used for laser cutting on stainless steel or carbon steel [ 21 , 22 ]. When cutting with O 2 gas, in the case of SS41, it is easily heated up to vaporizing temperature, thus, the material is also easily cut by a laser beam. In addition, when the SUS304 is processed using N 2 gas, the oxidation can be protected during laser cutting. At the cutting process of the SS41 and SUS304, the assistance gases are used by the constant pressure of O 2 and N 2 , respectively, to maintain high processing quality. Table 1 shows the laser parameters applied to SS41 and SUS304. Different laser powers and cutting speeds were conducted to cut the materials in the experiment. The laser parameters are set in the range where the material was completely cut. Table 2 shows the chemical composition of the materials used in the experiment. In order to analyze the experimental results, the kerf widths generated after the cutting process are measured on both top and bottom surfaces [ 23 ]. In addition, melting width and Heat Affected Zone (HAZ) formed in the bottom surface of the materials are measured using an optical microscope (Dino-lite AM4013MZT4, AnMo Electronics Corporation, New Taipei, Taiwan). The schematics of the kerf widths, melting width, and HAZ are shown in Figure 1 . The kerf widths are the part where the laser is irradiated, and the material is completely cut-off. The kerf widths are measured in the kerf top and kerf bottom. The melting width is defined by the width of the materials with melting marks as in Figure 1 HAZ is the region where the microstructure of the materials has changed.
Measurement method of SS41 and SUS304 after laser cutting ( a ) top surface ( b ) bottom surface ( c ) top surface ( d ) bottom surface.
Laser parameters.
SS41 | SUS304 | |
---|---|---|
Laser Power [W] | 1000–3700 | 2100–3900 |
Cutting Speed [mm/s] | 2000–4100 | 2000–3500 |
Assistance Gas | O | N |
Gas Pressure [bar] | 3 | 3 |
Thickness [mm] | 2 | 2 |
Materials chemical composition.
C | Si | Mn | P | S | Ni | Cr | |
---|---|---|---|---|---|---|---|
SS41 Properties [%] | 0.14~0.22 | 0.3 | 0.36~0.65 | 0.045 | 0.05 | ||
SUS304 Properties [%] | 0.08 | 1.00 | 2.00 | 0.45 | 0.30 | 8.00~10.50 | 18.00~20.00 |
3.1. analysis of kerf width in ss41 according to volume energy.
The experimental results of laser cutting on metallic materials (SS41 and SUS304) are investigated. The kerf width of the top and bottom surface, melting width, and HAZ are analyzed according to volume energy. Volume energy is also an important parameter in the laser cutting process which is used to understand the interaction between laser and materials [ 24 ]. The volume energy ( E volume ) is a parameter that represents the irradiated laser per unit volume, and it is calculated by the laser power divided by the laser scanning speed and the laser beam size.
where P laser is the laser power [W], V s is the cutting speed [mm/min], and A is the spot area of the laser beam [mm]. Experimental results are analyzed through E volume to identify the effect on the laser powers and cutting speed.
The effect of E volume on the kerf widths of the top and bottom surface is shown in Figure 2 . The measurements of the kerf widths are conducted on both top and bottom sections of the cutting material. Each data point represents the different laser power and is obtained by averaging all measured data. The kerf widths of the top and bottom surface increase with increasing E volume . Generally, the measured kerf widths on the top surface are slightly larger than those on the bottom surface. This happens due to various reasons, such as loss of intensity of the beam, defocusing of the laser beam, or loss of gas pressure. In addition, the kerf widths of the top and bottom surface increase with increasing laser power. At the laser power of 3700 W, the kerf widths of the top and bottom surface are observed with the largest widths of 905 μ m and 675 μ m , respectively. In the interaction between laser and materials, it is evident that kerf widths are affected by E volume . As the E volume increases, the material is rapidly heated. In addition, the materials are evaporated and removed easily on the top surface. Therefore, a larger kerf width of the top surface is formed than the kerf width of the bottom surface.
Variation of ( a ) kerf top and ( b ) kerf bottom in SS41 according to E volume .
The effect of E volume on the melting width of the bottom surface is shown in Figure 3 . Each measured data is obtained by averaging melting width. Melting is the area where the material melts due to the laser irradiation, and melting occurs around the kerf width. At most of the laser powers applied in the experiment, melting width increases with increasing E volume . At the laser power of 3700 W, the melting width is observed with the largest width of 917 μm. The E volume is directly proportional to laser power. As the laser power increases, the thermal energy entering the materials increases so the melting width is observed with the largest value. In short, the laser beam including the laser power and cutting speed directly affect the material.
Variation of melting width in SS41 according to E volume .
The effect of E volume on the kerf widths of the top and bottom surface are shown in Figure 4 . The kerf width on SUS304 is measured in the same method as SS 41. The kerf widths of the top and bottom surface also increase with increasing E volume . The kerf widths on the top surface are slightly larger than those on the bottom surface. At the laser power of 3100 W, the kerf widths of top and bottom are observed with the largest width of 796 μm and 375 μm, respectively. As mentioned, the difference between top and bottom can be caused by various factors, such as loss of intensity of the beam, defocusing of the laser beam, or loss of gas pressure for the thickness of the materials. In the case of the trend on kerf widths, kerf widths of the top and bottom surface are observed to increase with increasing E volume . The specimen is heavily influenced by the laser beam and rapidly heats up to the vaporization temperature of the material. As the laser power increases, the laser beam entering the material increases so the kerf widths of the top and bottom surface also increase.
Variation of ( a ) kerf top and ( b ) kerf bottom in SUS304 according to E Volume .
The effect of the E Volume on HAZ is shown in Figure 5 . HAZ is the area in which the microstructure of a material is changed by heat input. If the microstructure changes, a microcrack occurs in the processed material, it causes a partial breakdown of the product and deteriorates the quality. Therefore, it is important to reduce the HAZ during the laser cutting so that micro-cracks can be avoided. As observed from the experimental results, the effect of the E Volume on the HAZ also increases with increasing E Volume . The maximum width of HAZ is 800 μm at 3500W and the minimum width of the HAZ is 550 μm at 2100 W. This can be related to the heat input entering the material. E Volume is proportional to laser power. As the laser power increases, the heat entering materials increase and the spread of heat damage also increase. Therefore, the HAZ increases with increasing laser power.
Variation of the Heat Affected Zone (HAZ) in SUS304 according to E Volume .
The effect of the E Volume on HAZ is shown in Figure 5 . HAZ is the area in which the microstructure of a material is changed by heat input. If the microstructure changes, a microcrack occurs in the processed material, it causes a partial breakdown of the product and deteriorates the quality. Therefore, it is important to reduce the HAZ during the laser cutting so that micro-cracks can be avoided. As observed from the experimental results, the effect of the E Volume on the HAZ also increases with increasing E Volume . The maximum width of HAZ was 800 μm at 3500 W and the minimum width of the HAZ was 550 μm at 2100 W. This can be related to the heat input entering the material. E Volume is proportional to laser power. As the laser power increases, the heat entering materials increase and the spread of heat damage also increase. Therefore, the HAZ increases with increasing laser power.
In this section, the regression analysis of laser power and cutting speed in the laser cutting process is performed. Multiple regression analysis is a mathematical model for indicating the suitability of the relationship between the independent and dependent variable [ 25 ]. In the case of the regression model, if the high order equation is used regardless of experiment data, the determination coefficient always increases. This problem is called “overfitting”. If the regression model becomes overfitting, the prediction of experimental results through the regression model becomes meaningless. Thus, the regression equation used in this study is the quadratic regression model and the equation for the regression model is followed by:
where β is the regression coefficient and can be calculated using the least-squares method, X i and X j are the independent variables of this regression equation and these are laser power and cutting speed, respectively, y is the dependent variable and represents measured data. The second-order regression model has been developed for kerf top width, kerf bottom width, melting width, and HAZ using data from the experiments. To calculate the regression coefficient β, the coefficients of the quadratic regression model are calculated. In addition, the determination coefficient ( R sq -value) and the adjusted determination coefficient ( R adj ) are calculated to check whether the data predicted by the regression model is appropriate. When the determination coefficient is close to 1, the accuracy of regression model is estimated to be suitable. The regression coefficients are determined by the t-test. The ‘SE Coef’ represents the standard error of the coefficient, and it is useful for making up a confidence interval and performing a hypothesis test. The t-test is a statistical method of the standardized value which is calculated from experimental data. The T-statistic is used to measure the magnitude of variation for the experimental data. It is calculated from experimental data to compare the null hypothesis. Each term of coefficients is tested by the null hypothesis according to the p -value. The null hypothesis is statistical proof to determine that the regression model is statistically significant. It can be determined by statistical evidence when the experimental data is meaningful. In general, a low p -value (<0.05) indicates that the predicted model can be meaningful in the experimental data. The regression coefficient suitability and coefficient of determination are shown in Table 3 and Table 4 .
The regression coefficient of SS41.
-Value | ||||
388.6832 | 80.07202 | 4.85417 | 8.75 × 10 | |
0.2657 | 0.033199 | 8.00299 | 4.35 × 10 | |
−0.00855 | 0.044628 | −0.1916 | 0.848694 | |
−3 × 10 | 6.2 × 10 | −4.87501 | 8.11 × 10 | |
−8.5 × 10 | 7.18 × 10 | −0.1179 | 0.906538 | |
−7.6 × 10 | 7.02 × 10 | −1.08053 | 0.28416 | |
= 0.90, (adj) = 0.89 | ||||
-Value | ||||
−5.9335 | 89.49774223 | −0.066297523 | 0.947357769 | |
0.2503 | 0.037857046 | 6.611759502 | 1.0722 × 10 | |
0.0723 | 0.05112402 | 1.413235703 | 0.16266912 | |
−3.289 × 10 | 7.25866 × 10 | −4.531773519 | 2.7836 × 10 | |
−1.372 × 10 | 8.75522 × 10 | −1.566708952 | 0.122356105 | |
6.355 × 10 | 8.60318 × 10 | 0.738711082 | 0.462915371 | |
= 0.89, (adj) = 0.88 | ||||
-Value | ||||
1030.875 | 112.6616 | 9.150191 | 4.76 × 10 | |
0.2174 | 0.046711 | 4.653414 | 1.81 × 10 | |
−0.4547 | 0.062792 | −7.24157 | 8.9 × 10 | |
−7.995 × 10 | 8.73 × 10 | −0.91606 | 0.363242 | |
8.1839 × 10 | 1.01 × 10 | 8.104238 | 2.91 × 10 | |
−2.8654 × 10 | 9.88 × 10 | −2.8996 | 0.005187 | |
= 0.86; (adj) = 0.85 |
Regression coefficient of SUS304.
-Value | ||||
853.0468 | 251.3251 | 3.394196 | 0.001371 | |
0.167979 | 0.129446 | 1.297679 | 0.200474 | |
−0.21867 | 0.11114 | −1.96752 | 0.054797 | |
−1.1 × 10 | 2.14 × 10 | −0.05021 | 0.960161 | |
4.6 × 10 | 1.84 × 10 | 2.498231 | 0.015886 | |
−5 × 10 | 1.76 × 10 | −2.82229 | 0.006871 | |
= 0.80, (adj) = 0.78 | ||||
-Value | ||||
−108.267 | 69.11328 | −1.56651 | 0.123665 | |
0.360518 | 0.035597 | 10.12777 | 1.32 × 10 | |
−0.05506 | 0.030563 | −1.8015 | 0.077778 | |
−5.3 × 10 | 5.9 × 10 | −8.97845 | 6.35 × 10 | |
8.64 × 10 | 5.06 × 10 | 1.707061 | 0.094141 | |
−8.1 × 10 | 4.83 × 10 | −1.67321 | 0.100659 | |
= 0.92, (adj) = 0.91 | ||||
-Value | ||||
2289.716 | 218.9419 | 10.4581 | 4.46 × 10 | |
−0.68795 | 0.112767 | −6.10062 | 1.64 × 10 | |
−0.40922 | 0.09682 | −4.22663 | 0.000103 | |
0.000132 | 1.87 × 10 | 7.041524 | 5.72 × 10 | |
6.28 × 10 | 1.6 × 10 | 3.913384 | 0.000281 | |
−1.4 × 10 | 1.53 × 10 | −0.91761 | 0.363319 | |
= 0.85, (adj) = 0.83 |
The results based on the regression model for kerf widths of top, bottom surface, and melting width on SS41 on the laser power and cutting speed are plotted in Figure 6 and mathematical equations are expressed in Equations (3)–(5), respectively. The regression model of kerf top is shown in Figure 6 a R sq and R sq (adj) of the kerf top are 0.90 and 0.89, respectively. When the determination coefficient is close to 1, the accuracy of the regression model is high. Therefore, the experimental data are suitable for the regression model. It also shows the most appropriate coefficient of determination among the regression models. In Figure 6 , it is found that the kerf top increases as increasing laser power. On the other hand, the variation of the kerf top is insignificant when the cutting speed increases. However, the kerf top increases when the laser power and cutting speed increase simultaneously. The regression model of kerf bottom is shown in Figure 6 b. R sq and R sq (adj) of kerf bottom are 0.89 and 0.88, respectively. This regression model is appropriate for the experimental data. It is also found that kerf bottom increase as the increasing laser power. However, the variation of kerf bottom is not variation when the cutting speed increases. It is also that the kerf top increases when the laser power and cutting speed increase simultaneously. This is similar to the experimental result of kerf top . The regression model for melting width is shown in Figure 6 c. The correlation model is suitable for experimental data. R sq and R sq (adj) were 0.86 and 0.85, respectively. This leads to the fact that the data used in the regression model were well-fitted. In the relationship of the laser parameters, the melting width increases as the increasing laser power. However, the melting width first decreases when cutting speed increases up to 3000 mm/min. After the cutting speed of 3000 m/mm, the melting width increases when the cutting speed increases. In addition, when the laser power and cutting speed increase simultaneously, the melting width increases.
Multiple regression of SS41 ( a ) kerf top , ( b ) kerf bottom , ( c ) Melting.
The regression model for kerf widths and HAZ on SUS304 is shown in Figure 7 . The regression model of kerf top is shown in Figure 7 a and mathematical equations are expressed in Equations (6)–(8), respectively. R sq and R sq (adj) are 0.80 and 0.78, respectively. The regression model is relatively suitable for experimental data. In the relation between laser power and cutting speed, it is found that the kerf top increases as the decreasing cutting speed but the variation of the kerf top is insignificant when the laser power increase. When the laser power and cutting speed increase simultaneously the variation of kerf top is relatively low. The regression model of kerf bottom is shown in Figure 7 b. R sq and R sq (adj) of kerf bottom are 0.92 and 0.91, respectively. The experimental data are suitable for the regression model. It is also the most appropriate decision coefficient among the regression models for SUS304. In the effects of laser power and cutting speed on kerf bottom , it is also found that kerf bottom increases as the increasing laser power. However, there is a little variation of the kerf bottom when the cutting speed increases. When the laser cutting speed increases up to 35,000 mm/min and the laser power increases up to 3000 W, the kerf bottom increases but, after 3000 W laser power, then it decreases slightly. The regression model for HAZ is shown in Figure 7 c. R sq and R sq (adj) are 0.85 and 0.83, respectively. This regression is in good agreement with the experimental data. In the relationship of the laser parameters, as the cutting speed increases, HAZ decreases rapidly. In addition, HAZ first decreases when laser power increases up to 2500 W but after laser power of 2500 W the HAZ increases with increasing the laser power.
Multiple regress of SUS304 ( a ) kerf top , ( b ) kerf bottom , ( c ) Heat Affected Zone.
In this section, the effect of the laser parameter is investigated through the analysis of variance (ANOVA). The ANOVA statistically analyzes the effect of each independent variable on the dependent variable during laser cutting. The advantage of ANOVA can be identified by the important factors for each independent variable, as well as the interaction effect of each parameter on laser cutting quality [ 26 ]. The variability of the experimental data can be determined by the percentage of contribution (PCR) of each independent variable. In addition, the results of the ANOVA are represented by the 95% confidence level ( p ≤ 0.05) and it is considered that the independent variable has a statistically significant effect on the experimental data. Table 5 and Table 6 for ANOVA results show Degrees of Freedom (DF), Sum of Squares (SS), Mean squares (MS), F ratio, and percentage of contribution (PCR). The SS is the sum of the squared deviations between the mean and the variance of each experimental data. The MS represents the estimate of the population variance. This is the corresponding sum of squares divided by degrees of freedom. The F ratio is the distribution ratio obtained through a comparison of variances. It is used to test whether the variance of each group is different and whether the population mean is different. The PCR is calculated based on the estimated variance components. The higher PCR indicates that the variability of the experimental data by independent variables increases. In the results of ANOVA, the P-value on the effect of each parameter and interaction effects between parameters are less than 0.05. This indicates that the parameters used have a significant effect on the experimental results.
SS41ANOVA table.
Source | SS | DF | MS | F Ratio | -Value | PCR [%] |
---|---|---|---|---|---|---|
Laser Power | 5.6 × 10 | 8 | 7.0 × 10 | 4.2 × 10 | <0.05 | 59.28 |
Cutting Speed | 1.2 × 10 | 7 | 1.7 × 10 | 1.0 × 10 | <0.05 | 12.48 |
Laser power × Cutting speed | 2.7 × 10 | 56 | 4.7 × 10 | 2.8 × 10 | <0.05 | 27.99 |
Error | 2.4 × 10 | 144 | 1.7 × 10 | 0.26 | ||
Total | 9.5 × 10 | 215 | ||||
Laser Power | 4.0 × 10 | 8 | 5.0 × 10 | 1.4 × 10 | <0.05 | 73.06 |
Cutting Speed | 3.1 × 10 | 7 | 4.4 × 10 | 1.2 × 10 | <0.05 | 5.63 |
Laser power × Cutting speed | 1.1 × 10 | 56 | 2.0 × 10 | 5.6 × 10 | <0.05 | 20.37 |
Error | 5.1 × 10 | 144 | 3.6 × 10 | 0.94 | ||
Total | 5.5 × 10 | 215 | ||||
Laser Power | 5.3 × 10 | 8 | 6.6 × 10 | 1.2 × 10 | <0.05 | 59.65 |
Cutting Speed | 1.1 × 10 | 7 | 1.5 × 10 | 2.7 × 10 | <0.05 | 12.08 |
Laser power × Cutting speed | 2.4 × 10 | 56 | 4.3 × 10 | 7.6 × 10 | <0.05 | 27.35 |
Error | 8.2 × 10 | 144 | 5.7 × 10 | 0.92 | ||
Total | 8.9 × 10 | 215 |
SUS304 ANOVA table.
Source | SS | DF | MS | F Ratio | -Value | PCR [%] |
---|---|---|---|---|---|---|
Laser Power | 9.0 × 10 | 9 | 1.0 × 10 | 1.2 × 10 | <0.05 | 9.93 |
Cutting Speed | 9.5 × 10 | 6 | 1.6 × 10 | 1.9 × 10 | <0.05 | 10.45 |
Laser power × Cutting speed | 7.1 × 10 | 54 | 1.3 × 10 | 1.6 × 10 | <0.05 | 78.33 |
Error | 1.2 × 10 | 140 | 8.4 × 10 | 1.29 | ||
Total | 9.1 × 10 | 209 | ||||
Laser Power | 1.9 × 10 | 9 | 2.1 × 10 | 3.9 × 10 | <0.05 | 38.03 |
Cutting Speed | 9.9 × 10 | 6 | 1.6 × 10 | 3.1 × 10 | <0.05 | 20.22 |
Laser power × Cutting speed | 2.0 × 10 | 54 | 3.6 × 10 | 6.9 × 10 | <0.05 | 40.25 |
Error | 7.4 × 103 | 140 | 5.3 × 10 | 1.52 | ||
Total | 4.9 × 10 | 209 | ||||
Laser Power | 6.0 × 10 | 9 | 6.7 × 10 | 4.3 × 10 | <0.05 | 22.39 |
Cutting Speed | 7.7 × 10 | 6 | 1.3 × 10 | 8.3 × 10 | <0.05 | 28.74 |
Laser power × Cutting speed | 1.1 × 10 | 54 | 2.0 × 10 | 1.3 × 10 | <0.05 | 40.78 |
Error | 2.2 × 10 | 140 | 1.6 × 10 | 8.09 | ||
Total | 2.7 × 10 | 209 |
The ANOVA results for SS41 are shown in Table 5 . ANOVA tables demonstrate the results of laser power, cutting speed, and laser power × cutting speed for the 95% confidence level ( p < 0.05). At the ANOVA table of kerf top , it shows that the most effective variable is laser power which was 59.28% of the PCR. The other variables affecting kerf top were cutting speed and laser power × cutting speed, which were 12.48% and 27.99% of PCR, respectively. At the ANOVA table of the kerf bottom , the laser power was the most effective variable, which was 73.06% of PCR. The other variables affecting kerf bottom were cutting speed and laser power × cutting speed, which were 5.63% and 20.37% of PCR, respectively. As a result of melting width, the PCR of the laser power, cutting speed, and laser power × cutting speed were found to be 59.65%, 12.08%, and 27.35%, respectively. The ANOVA results for SUS304 are shown in Table 6 . At the ANOVA table of kerf top , it shows that the most effective variable is laser power × cutting speed which was 78.33% of the PCR. The other variables affecting kerf top were laser power and cutting speed which were 9.93% and 10.45% of PCR, respectively. As the results of kerf bottom , it shows that the most effective variable was laser power × cutting speed, which was 40.25% of PCR. The other variables affecting kerf bottom were laser power and cutting speed which were 38.3% and 20.22% of PCR, respectively. At the ANOVA results of HAZ, the PCR of the laser power, cutting speed, and laser power × cutting speed were found to be 22.39%, 28.74%, and 40.78%, respectively. In the case of SS41 analyzed by ANOVA, the most effective variable of kerf top , kerf bottom , and melting was laser power. On the other hand, at the ANOVA results of SUS304, the most effective variable of the kerf top was laser power and the most effective variables of kerf bottom and HAZ was laser power × cutting speed. The most effective variables of experimental results were different. The reason why the effective variable is different is the mechanical or chemical properties of metallic materials are different. In the case of the chemical properties of materials, SUS304 includes the chemical composition of Ni and Cr. These components improve corrosion resistance and heat resistance. Especially, The Cr component interacts with the atmosphere of the O and then, the thin film is generated on the SUS304 surface [ 27 ]. This thin film can protect from the surface corrosion and heat damage and the effect of laser power might decrease due to the protecting thin film. Therefore, we assume that the effect of laser power affecting the material is low. The complex effect of laser power × cutting speed has more influence on the material than the effect of laser power. The influence of laser parameters on the components such Ni and Cr needs further study.
Nowadays, there are many types of laser systems, such as Nd:YAG laser or CO 2 laser. The CO 2 laser system has many advantages such as providing good processing quality and high processing efficiency [ 28 ]. To achieve improvement in product quality and productivity, the effects of laser parameters on the material should be considered as a major issue. In this study, the influences of the laser parameter, such as laser power and cutting speed on the SS41 and SUS304 are studied. The experimental results of laser cutting on metallic materials are analyzed through multiple regression and analysis of variance (ANOVA). The effects of each independent variable to output variables are analyzed. The conclusions of this experiment are as follows:
D.L. and S.S. conceived and designed the experiments; D.L. and S.S. performed the experiments; D.L., S.S. analyzed the data; D.L., S.S. wrote the paper. All authors have read and agreed to the published version of the manuscript.
The research described herein was sponsored by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; Ministry of Science, ICT and Future planning) (No. 2019R1A2C1089644). The opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the sponsors.
The authors declare no conflict of interest.
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In this section
Hussain, Mohammed A (1989) A Generalised Approach for the Prediction of Laser Cutting Parameters. PhD thesis, University of Glasgow.
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A design of a tasking and control environment for laser based manufacturing systems is proposed. This uses an empirical approach based on recording previous manufacturing experience in a database, in order that this can be used in planning and control of future processes. The work presented gives details of the partial implementation of this design for laser cutting systems. This makes use of Computer Aided Design and Manufacture, Computer Numerical Controlled laser cutting machines, database and computerised planning and control.
Item Type: | Thesis (PhD) |
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Qualification Level: | Doctoral |
Keywords: | Mechanical engineering |
Date of Award: | 1989 |
Depositing User: | |
Unique ID: | glathesis:1989-76840 |
Copyright: | Copyright of this thesis is held by the author. |
Date Deposited: | 14 Jan 2020 09:33 |
Last Modified: | 14 Jan 2020 09:33 |
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Open Access
Peer-reviewed
Research Article
Roles Data curation, Formal analysis, Validation, Writing – original draft
Affiliation College of Mechatronics and Automation, Huaqiao University, Xiamen, China
Roles Formal analysis
Affiliation College of Mechanical Information Science and Engineering, Huaqiao University, Xiamen, China
Roles Writing – review & editing
* E-mail: [email protected]
Laser microdissection technology is favored by biomedical researchers for its ability to rapidly and accurately isolate target cells and tissues. However, the precision cutting capabilities of existing laser microdissection systems are hindered by limitations in overall mechanical movement accuracy, resulting in suboptimal cutting quality. Additionally, the use of current laser microdissection systems for target acquisition may lead to tissue burns and reduced acquisition rates due to inherent flaws in the capture methods. To address these challenges and achieve precise and efficient separation and capture of cellular tissues, we integrated a digital micromirror device (DMD) into the existing system optics to modulate spatial light. This allows the system to not only implement the traditional point scanning cutting method but also utilize the projection cutting method.We have successfully cut various patterns on commonly used laser microdissection materials such as PET films and mouse tissues. Under projection cutting mode, we were able to achieve precise cutting of special shapes with a diameter of 7.5 micrometers in a single pass, which improved cutting precision and efficiency. Furthermore, we employed a negative pressure adsorption method to efficiently collect target substances. This approach not only resulted in a single-pass capture rate exceeding 90% for targets of different sizes but also enabled simultaneous capture of multiple targets, overcoming the limitations of traditional single-target capture and enhancing target capture efficiency, and avoiding potential tissue damage from lasers.In summary, the integration of the digital micromirror device into laser microdissection systems significantly enhances cutting precision and efficiency, overcoming limitations of traditional systems. This advancement demonstrates the accuracy and effectiveness of laser microdissection systems in isolating and capturing biological tissues, highlighting their potential in medical applications.
Citation: Zhou B, Huang C, Yi D (2024) Laser microdissection system based on structured light modulation dual cutting mode and negative pressure adsorption collection. PLoS ONE 19(8): e0308662. https://doi.org/10.1371/journal.pone.0308662
Editor: Wenxuan Liang, University of Science and Technology of China, CHINA
Received: March 25, 2024; Accepted: July 28, 2024; Published: August 26, 2024
Copyright: © 2024 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Cells, which are the fundamental units of biological processes, are essential for biomedical research [ 1 – 3 ]. However, it is important to acknowledge that cells do not exist in isolation. The presence of different cell types in a mixture often obscures the true nature of a lesion. As a result, it is now a major challenge for biological researchers to isolate single cells from specific anatomical regions in complex heterogeneous tissues. To address this problem, scientists have conducted extensive research that has led to the development of various cell separation methods [ 4 – 6 ]. Laser microdissection is a commonly used technique for non-contact method for microdissection and isolation of biological samples under a microscope. When laser microdissection is conducted, the targeted material absorbs UV laser energy, converting it into internal heat energy. As the material’s temperature rises, it eventually reaches a critical point, leading to vaporization on the material’s surface. The vaporized material is then expelled from the processing area, creating a precise cutting opening. Laser microdissection technology enables extremely fine cutting with smooth edges, free from burrs, and does not produce carbonization. This technique ensures sample purity and high precision, making it invaluable for research in molecular biology, cytogenetics, and related fields [ 7 – 10 ]. In a study by Laura W. Harris et al. [ 11 ], laser microdissection was utilized to isolate microvascular endothelial cells and neurons from postmortem brain tissues of both schizophrenic patients and healthy individuals. The objective of their investigation was to elucidate microvascular system dysfunction in schizophrenia patients. Similarly, Eulalie Buffin et al. [ 12 ] employed laser microdissection to procure Drosophila precursor cells, aiming to study the mechanisms involved in their specification. Another study conducted by Selda Aydin et al. [ 13 ] employed laser microdissection to procure lesions, facilitating the characterization of the TP53 mutation spectrum in malignant urothelial tissues of patients with aristolochic acid nephropathy in Belgium.
The laser microdissection system utilized in the aforementioned studies relied solely on mechanical motion for point-scanning cutting. Two prevalent approaches to point scanning cutting methods in laser microdissection systems include stage movement and galvanometer systems [ 14 – 16 ]. Nevertheless, the system’s limited mechanical movement accuracy may result in incomplete trajectories or low contour quality when cutting small targets. Consequently, it is imperative to develop a laser microdissection system that is both straightforward and capable of effectively isolating complex tissues. This advancement will significantly enhance the utility of laser microdissection technology.
A fully functional laser microdissection system must accurately isolate the desired tissue and capture anatomical targets with precision. Currently, various laser capture microdissection systems are available on the market, including those offered by prominent companies such as American Thermo Fisher, German Leica, German Zeiss, and German MMI [ 17 – 19 ]. These systems employ diverse collection methods. For instance, some systems utilize adhesion capture methods whereby infrared light illuminates a thermoplastic membrane placed on cells to facilitate cell adhesion and capture [ 20 ]. However, this method exposes target cells to temperatures of up to 90 degrees Celsius [ 21 ], potentially causing thermal damage and mechanical damage during the pulling process. Additionally, this collection method may pose contamination risks. Another approach is gravity-based collection, where the cut sample falls into a collection tube under the influence of gravity [ 22 ]. This method, however, is unsuitable for cell environments requiring culture media, and there is a risk of losing target cells due to an uncontrolled falling process. Moreover, there is a collection method employing a refocused ultraviolet cutting laser that emits laser pulses, with the pressure generated by these pulses ejecting the target into a container directly above. This method, however, may potentially damage cellular DNA and RNA. Therefore, there is an urgent need for a novel and more universal method to capture target tissues.
In order to tackle the aforementioned challenges, this paper proposes a laser microdissection system that utilizes digital micromirror device (DMD) technology. In this system, the incident laser beam is shaped by the DMD to accommodate complex cutting trajectories and target tissues of varying sizes. When dealing with small targets characterized by intricate curved trajectories, the laser beam can be precisely shaped to match the contour of the area to be cut. This enables one-time cutting without the need for mechanical movement, thereby enhancing both cutting accuracy and efficiency. The point-scanning cutting method remains suitable for cutting large volume targets or tissues with simple trajectories. Moreover, the device incorporates negative pressure to ensure stable capturing of targets of different sizes and can simultaneously capture multiple targets.
2.1 laser microdissection method based on dmd.
Fig 1 depicts the essential instruments and overall layout of the experimental platform for the laser micro-cutting system utilizing digital micromirror device (DMD) technology.
https://doi.org/10.1371/journal.pone.0308662.g001
The laser microdissection system detailed in this article comprises multiple components, including a UV laser, a beam expander, a digital micromirror device (DMD), a zoom lens group, and a microscope system. The laser implemented is a diode-pumped Q-switched solid-state laser produced by Spectra-Physics, specifically the EONE-349-120 model, featuring a center wavelength of 349 nm. The microscope system showcases an Olympus IX73 inverted fluorescence microscope, with technical specifications elaborated in Table 1 . The DMD device utilized is the DLP7001 from Texas Instruments, with comprehensive technical specifications outlined in Table 2 . Throughout the laser microdissection procedures, the UV laser beam is directed towards the DMD through a laser beam expander. Subsequently, the DMD shapes the incident beam and projects it based on the preloaded pattern. The laser beam undergoes zooming via a telescope system before being focused onto the tissue surface.
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To facilitate projection cutting, a diffractive spatial light modulator, specifically a digital micromirror device (DMD), was integrated into the system. The DMD offers several advantages over existing galvanometer systems in terms of spatial light modulation, including high-speed switching capabilities, high display resolution, precise phase control, and enhanced stability and reliability. Additionally, the DMD benefits from mass production and cost advantages. The principle of laser beam shaping in the DMD relies on controlling the state of its micromirror elements. By toggling the state of these elements, any desired projection shape can be achieved [ 23 ]. Fig 2 illustrates the fundamental process of DMD laser beam shaping. In panel (a), micromirrors are utilized to generate open "spots" with specific diameters. In panel (b), a closed-loop polygonal cutting trajectory can be attained by activating the target micromirror element while deactivating corresponding micromirror elements at other locations.
(a) Point cutting mode. (b) Nonmechanical motion cutting mode.
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The reflection produced by the DMD yields a two-dimensional pattern of spots known as diffraction orders. Depending on factors such as the pixel pitch, the tilt angle of the DMD micromirrors, the wavelength of the illumination, and the angle of incidence of the illuminating light, there can be a spectrum ranging from fully blazed to fully anti-glare conditions. A blaze condition occurs when a single diffraction order contains the majority of the energy within the entire diffraction pattern, representing the optimal scenario. Conversely, an anti-blaze condition arises when the four brightest orders in the diffraction pattern possess equal amounts of energy, a situation to be avoided in this system [ 24 – 26 ]. The diffraction diagram of the DMD is depicted in Fig 3 , while Fig 4 illustrates the energy distribution of DMD diffraction patterns in both blazed and non-blazed states. The color depth indicates the intensity of the energy within the diffraction orders.
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The blaze angle, denoted as i , represents the angle formed between the groove surface and the grating surface. The grating constant d , signifies the distance separating two adjacent grooves. The incident angle φ indicates the angle formed between the incident light and the normal to the grating plane. Finally, the diffraction angle θ , denotes the angle between the diffracted light ray and the normal line of the grating plane.
After the Digital Micromirror Device (DMD) shapes the laser beam, a suitable optical path is required to scale the shaped spot and achieve a sufficiently small cutting line width, which is crucial for effectively cutting small tissue samples. To maintain the stability of the laser microdissection system, this study employs a telescope system to scale the projected pattern, as illustrated in Fig 5 .
https://doi.org/10.1371/journal.pone.0308662.g005
The narrowed laser beam width is denoted by W , the number of opened micromirrors is denoted by n , and d represents the size of the micromirror. Additionally, f 1 represents the equivalent focal length of the system components, and f 2 represents the equivalent focal length of the objective lens. The distance f 3 between the two lenses is equal to the sum of f 1 and f 2 . In this system, f 1 = 1000 mm, f 2 = 10 mm, and the magnification obtained according to Eq 1 is 100 times.
The physical setup and principles of the negative pressure adsorption system used in this study are depicted in Figs 6 and 7 .The negative pressure adsorption system comprises a suction pipe, a vacuum generator, a PLC controller, an electromagnetic proportional valve, a filtration system, and a gas source. The inner diameter of the straw employed in the experiment was 4 mm, with its end covered by a PET film layer featuring multiple holes distributed across its surface, facilitating the absorption of target tissue without penetration. The vacuum generator employed was the ZK2G07R5ALA-06 model manufactured by SMC Company. Additionally, the solenoid proportional valve and filter system, also sourced from SMC, were utilized to regulate intake pressure and filter impurities, respectively. The PLC controller used was the FX3U-16MR/ES controller manufactured by Mitsubishi Company, tasked with controlling the vacuum generator’s operational status. The air source was provided by a vacuum compressor.
(a) Working diagram of the negative pressure adsorption system (b) Physical view of the negative pressure adsorption system (c) Enlarged view of the end of the actuator suction pipe.
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In this study, 6-week-old Balb/c mice were used. The experimental protocol involving animal subjects was approved by the Ethics Committee for the Management of Experimental Animals at Huaqiao University School of Medicine. To induce anesthesia, the mice were administered a mixture of ketamine (100 mg/ml) and diazepam hydrochloride (10 mg/ml) at a ratio of 10:1 via intraperitoneal injection while being housed in a well-ventilated cage. Anesthesia was confirmed by the absence of response and slow breathing. Subsequently, euthanasia was performed using cervical dislocation.
To extract brain tissue, the mouse’s head was rinsed with normal saline, and the skin was incised with scissors to expose the skull. The skull was carefully opened with a scalpel to reveal the brain tissue, which was then delicately removed using dissecting forceps. The extracted brain tissue was immediately immersed in 10% neutral buffered formalin for fixation, with a fixation duration of 24 hours at room temperature to ensure complete fixation. Following fixation, the tissue was washed three times with PBS buffer for 5 minutes each. Subsequently, the fixed brain tissue was gradually dehydrated in sequential ethanol solutions (70%, 80%, 90%, and 95% ethanol for 30 minutes each, followed by absolute ethanol for two 30-minute intervals), and then cleared in xylene twice for 1 hour each. The brain tissue was subsequently saturated by overnight immersion in molten paraffin wax, followed by embedding, sectioning into 5-micron thickness slices, and dewaxing. The sections were stained with hematoxylin for 5–10 minutes, rinsed with tap water, and then counterstained with eosin for 1–2 minutes before another rinse with tap water. Finally, the sections were sequentially rehydrated in different ethanol concentrations (70%, 80%, 90%, and 95% ethanol for 5 minutes each, followed by absolute ethanol twice for 5 minutes each) and xylene (two 5-minute soaks), before being sealed with neutral gum.
For immunohistochemical sections, mice were immobilized on an operating table, and the mammary gland area was cleansed with 70% ethanol before the mammary gland tissue was excised. The removed tissue was promptly fixed in 10% neutral buffered formalin for 24 hours at room temperature. Following fixation, the mammary gland tissue underwent gradual dehydration (30 minutes each in 70%, 80%, 90%, and 95% ethanol, followed by two 30-minute intervals in absolute ethanol), embedding, sectioning into 5-micron thickness slices, and dewaxing. Antigen retrieval was then performed in a 95°C water bath using 10 mM phosphate buffer (pH 6.0), followed by protein blocking with 5% bovine serum protein. Subsequently, primary antibodies against rabbit anti-cell markers were applied at appropriate concentrations and incubated overnight at room temperature. Following this, an appropriate amount of HRP-labeled secondary antibody was added and incubated for 1 hour at room temperature. Finally, DAB color reagent and a color-developing substrate were applied. The slices were then gradually dehydrated (1-minute incubations in 70%, 95%, and 100% ethanol), followed by sealing with hyaluronic acid ester.
3.1 laser microdissection experiment.
The experimental device’s actual image is depicted in Fig 8 .
https://doi.org/10.1371/journal.pone.0308662.g008
Utilizing the DMD blazed grating model, this article utilizes a laser wavelength of 349nm and a DMD micromirror size of 13.68μm. It is apparent that at a system incident angle of 24°, the majority of energy is concentrated on the zero-order diffraction, thereby achieving maximum energy distribution. This is illustrated in Fig 9(A) , demonstrating the concentration of energy in the zero-order diffraction at the mentioned incident angle. Conversely, Fig 9(B) depicts the non-uniform distribution of laser energy across various diffraction orders at alternative angles.
(a) Distribution of laser energy when the incident angle is 24°. (b) Distribution of laser energy at other angles.
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To validate the efficacy of the dual-mode cutting improvement system, verification tests were conducted on two materials: PET film and biological tissue. The experimental findings are presented in Figs 10 and 11 . Fig 10(A) demonstrates the feasibility of the telescope system in scaling patterns on 1.4 μm thick PET film. With a loaded spot diameter of 50 microns on the DMD, the system’s L1 lens focal length f1 is 1000mm, while the 40x objective lens has an equivalent focal length f2 of 10mm, resulting in a reduction factor of 100x. In the single-factor experiment, a minimum laser current of 3A, a repetition frequency of 1KHz, and a cutting speed of 30mm/s were selected for cutting biological tissue. At these settings, the theoretical line width is calculated to be 6.84 μm, closely matching the actual line width observed in Fig 10(A) (6.94 μm), thus meeting system requirements. Fig 10(B) illustrates the projected cross-section of a five-pointed star using a laser current of 3A and a pulse frequency of 1KHz, with 300 micromirrors spaced between two vertices horizontally. Moving to Fig 11 , the results of the cutting experiment on biological tissue are shown. Employing a laser current of 3A, a repetition frequency of 1KHz, and a point scanning cutting speed of 30mm/s, the system operates on 5μm thick biological tissue. In Fig 11(A) , the DMD utilizes 100 micromirrors in point cutting mode, while in Fig 11(B) , the letters are delineated by a width of 30 micromirrors.
(a) Point scanning cutting (b) Projection cutting.
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Figs 10 and 11 collectively validate the efficacy of the DMD-based laser microdissection method in both point scanning mode for cutting straight lines and projection cutting mode for one-time projection cutting of anisotropic patterns on PET films and biological tissues. Leveraging the DMD’s laser micro-cutting technique, precise contours can be accurately projected to the intended location, enabling swift and accurate cutting. This method surpasses traditional cutting approaches constrained by mechanical movement, allowing for enhanced cutting precision and the handling of smaller target sizes. Notably, existing laser micro-dissection systems typically feature a minimum cutting target larger than 10 microns [ 27 – 29 ].
In Fig 11(A) , noticeable dark burn marks were observed around the incision. This phenomenon primarily results from the Gaussian distribution of energy emitted by the ultraviolet laser used in this platform’s circular laser spot. The higher energy at the center of the spot directly vaporizes tissue to form the incision, whereas the lower energy distribution at the periphery of the circular spot fails to vaporize tissue completely, resulting only in surface burns and the formation of dark burn areas.
To mitigate the occurrence of dark burns from ultraviolet laser cutting in biological tissue, optimizing the energy distribution of the laser spot to achieve uniform energy across the entire circular spot can be effective in preventing localized burning. Additionally, optimizing the process parameters of laser microsurgery can help minimize the burn area as much as possible.
To further scrutinize the system’s cutting accuracy, the experiment utilized a 40x objective lens offering 100x magnification. A DMD with a side length of 13.68 μm was employed to generate a circular cutting pattern with a line width equivalent to 20 micromirror lengths, ensuring precise targeting. The cutting laser operated at a current of 3A with a pulse frequency of 1KHz. The experiment utilized 5-micron thick immunohistochemical sections of mouse breast tissue as samples. Fig 12 illustrates the cutting process alongside its corresponding outcomes.
https://doi.org/10.1371/journal.pone.0308662.g012
The experimental results showcase the capability of the DMD projection cutting mode to achieve smaller cutting sizes, with diameters measuring up to 7.5μm, surpassing those achievable with existing laser microdissection systems. This technological advancement facilitates the swift and precise isolation of minute biological tissues.
To efficiently transfer adsorbent substances into the designated container, the vacuum suction of the micro pipette should not be excessively strong. As long as it meets the minimum suction requirement, it suffices to fulfill the task. The pressure characteristic curve of the vacuum generator (refer to Fig 13 ) illustrates how the vacuum level fluctuates with changes in the supply pressure. In this investigation, we identified the optimal operating conditions for the vacuum generator, setting the supply pressure at 0.45 MPa. For this experiment, the adsorption film featured a pore size of 5 micrometers. Utilizing HE-stained 5-micrometer mouse brain tissue slices as samples, each side measuring 100 micrometers square, we conducted the experiment. Fig 14(A) displays the remaining tissue portion post-target tissue capture, while Fig 14(B) exhibits the captured target tissue released onto a glass slide.
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(a) the remaining tissue portion after the target tissue is captured. (b) the target tissue released on the slide after being captured.
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To assess the capture efficiency of the negative pressure adsorption system, we conducted experiments to determine capture rates across various target volumes. Traditional targets typically range from 10 μm to 100 μm. Therefore, for our experiments, we employed sample sizes of 30 μm, 60 μm, and 90 μm. The air compressor’s supply pressure was maintained at 0.45 MPa. Utilizing HE-stained 5 μm sections of mouse brain tissue as samples, we conducted multiple tests for each target size, repeating the process 50 times. The resulting capture rates were 98%, 94%, and 92%, respectively. As the sample area increases, the contact area between the sample and the slide’s film also expands. This increased contact leads to heightened adhesion, making sample capture more challenging and consequently decreasing the capture rate.
Due to the extensive surface area of the membrane at the target collection end, multiple targets can be captured simultaneously in a single operation without necessitating multiple movements. We verified the system’s capability to capture multiple targets simultaneously. Fig 15 illustrates the tissue conditions of HE-stained sections, each with a side length of 100 μm and a thickness of 5 μm from mouse brain tissue, distributed across various areas of the capture device and simultaneously captured by the system.
https://doi.org/10.1371/journal.pone.0308662.g015
This paper introduces a laser microdissection system integrating DMD for spatial light modulation, featuring dual cutting modes and a negative pressure adsorption collection method. By controlling the flip of the DMD micromirror element during cutting, the device offers two cutting modes. The one-time projection cutting mode enables swift cutting of shaped targets smaller than 10 μm. The negative pressure adsorption system exhibits capture rates of 98%, 94%, and 92% for three sizes of mouse brain tissue targets (30 μm, 60 μm, and 90 μm), respectively. Additionally, simultaneous capture of multiple targets was successfully demonstrated. Overall, these results suggest that our newly devised dissection device enhances dissection accuracy and effectiveness. The tissues and cells collected using this system hold significant potential for various downstream applications.
S1 fig. specific data of the experimental platform..
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Targets with a 30μm edge length captured under a 10x objective. Targets with a 60μm edge length captured under a 10x objective. Targets with a 90μm edge length captured under a 10x objective.
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