Macrophage uptake of nanotubes

There is ongoing growth on 3D printing technology, which will deeply change the world in the future. Successful and potential applications can be found in medical industry, aerospace and aviation industries and automotive industry [1]. 3D printing is a part of process called additive manufacturing where adding the materials layer by layer. The main steps of printing 3D objects can be divided to the model creation and printing. Initially, the blueprint of object is created in software. Then the file of object model is sent to the printer where printing the object by laying down successive layers of materials until the final formed object. Different printing technologies differ on the way building the layers. Two main methods are commonly used in 3D printing process. The first way is to add layers using molten material, like fuse deposition modeling (FDM). The second way is to lay down the layers of photo-sensitive material where UV light or other similar light source suddenly cure the deposits, like polyjet printer. Here is the process on how FDM works. Materials, like plastic filament unwound from the spool and are driven into the extrusion head. The nozzle melts the material with molten state and extrude them onto the platform. The material quickly hardens after coming from the nozzle. Both the platform and the nozzle can be translated in lateral position. After the entire object is printed, the object is separated with the plastic platform. Polyjet printer jets the material with tin droplets form, followed by immediately curing UV light and builds the layer by layer structure. The common materials used in FDM are polylactic acid (PLA), acrylonitrile-buta diene styrene (ABS). The common material used in polyjet printer is liquid polymer (LP). PLA is a biopolymer made from renewable materials such as cornstarch or sugarcane. ABS is an oil-based plastic, which is tough and can be used as the plastic object in the everyday life. Liquid polymer is the photosensitive polymer that can be cured by the light. ABS has the amorphous structure, but PLA and liquid polymer possess some extent of crystallinity.

Scanning electron microscope (SEM) is one kinds of electron microscope that the image is formed by scanning across the sample by the tightly focused beam of electrons. The interaction of beam of electrons with sample produces various types of signals. The signals used in this project include secondary electron (SE), backscattered electron (BSE) and X-ray signals. SEs locate closer to the surface than that of BSEs. SEs and BSEs can be used to form different imaging modes, SE and BSD modes, which are capable of presenting various surface information. X-ray signals are used to identify the compositional information of the sample. X-ray energy dispersive spectrometry (EDS) is a device that detecting x-rays, changing the x-ray energy into electrical charges, processing the electrical charges by signals, identifying x-ray energy by signals and finding the element of the sample corresponds to the specific energy.

Atomic force microscopy (AFM) is one type of the scanning probe microscopes (SPMs). The image formed by this kind of microscope is to raster scan the certain area of the sample by the probe. The probe can measure many kinds of properties, such as height, friction and magnetism. The typical configuration of AFM consists of the cantilever, the piezoelectric component above the cantilever that oscillating the cantilever at certain frequency, the sharp tip at the open end of the cantilever acting as the probe, the quadrant photo detector that measures the deflection of the cantilever and the sample plane. The quadrant photo diode detector can measure the displacement of the cantilever with respect to the equilibrium position and convert those deflection into the electrical signal, which is proportional to the intensity. The operation of the AFM includes contact mode and non-contact (tapping) mode. In this project, the tapping mode is used, which prevents the contamination of the tip by contacting with the specimen.

Stereo pairs is the technique that generating 3D images using SEM, which can recover the third dimension in SEM. The principle of Stereo-pair technique origins from the ways human eyes view the world. Human eyes can distinguish the depth information of objects, resulting from the slightly distance between human left and right eyes. So Stereo pair 3D anaglyphs have two images with the slightly horizontal shift between them. The method of manipulating the Stereo pair using SEM in this project is to slightly tilt the second image after the first image.

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Experimental Procedure

1. Sputtering Coating

The first step is the physical preparation of the samples. Stick small amount of the carbon double faced adhesive tape onto the top surface of the sample stub. Put the three cubes, ABS, PLA and LP cubes onto the tape. 3 sample stubs were placed in the bell jar. After putting the cap onto the instrument and opening the tank that is connected to the sputter instrument, switch on the system and click the button of pressure with certain indicating color. Wait until the pressure decreases around 100 mTorr. Rotate the knob to increase and decrease the pressure but control the value that is less than 300 mTorr. Repeat the above process in 3 times. Adjust the time for 60 seconds and the current level with 15~20 mA, wait for the gold layer that deposits on the samples with 6 nm thickness. BSD image of LP cube has the thickness of coating layer with 12 nm.

2. Scanning Electron Microscope (SEM)

SEM used in this project is the Zeiss-Leo DSM982 instrument at the institute of optics at University of Rochester. After loading the samples in the SEM, the standard procedures are followed to view the images on the computer screen and to take several images. Samples are seen at SE and BSD modes. The BSD mode does not need to adjust focus, which is distinct to SE mode. So when lowering the WD and adding the BSD stage, turn off the camera, set large aperture, gain with the high value and reduce the scan rate. Next square the certain area in the image, adjust the numerical aperture with the large value and do X-ray compositional analysis.

3. Atomic Force Microscope (AFM)

AFM used in this project is the NT-MDT AFM at the University of Rochester. The procedure of operating AFM is generalized as following. Turn on the computer and the controller box, open AFM application in the computer, waiting for the initialization indicating ‘OK’. Open the door, then place the sample on the sample plane, flip over the probe head and close the door. Open the camera view on the screen and the laser aiming window. Rough cantilever finding followed by fine auto search. The optimal value is around 18. Find the resonance peak with approximately 300 kHz and choose mode, here is the semi-contact mode. Land the probe on the surface, square area of interest on the sample. Set the scan rate and form image. The 3D image and roughness analyze can be set in the data field.

4. Stereo Pairs - 3D Anaglyphs

Take the image as the usual procedure in SEM. Tab the “scan rotate” button and tilt the layer as horizontal. Mark the location of particles using the crosshair and shift the crosshair to the center. Tilt the sample at small degree that the mark moves to left a little bit, move the mark back to the center again. Do this shift several times until the tilt degree reaches around 3 degrees. Load the two images in photoshop (PS) software, transparent the left image as red and the right image as green. Overlay the two images and generate one 3D image.

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Results and Discussion

1. Secondary Electron (SE) Imaging

Fig. 1(a) is the SEM image of ABS cube in side view. In this image, multiple layers and the infilled layer can be seen clearly, which manifest the way to manufacture this cube. The extrusion head followed the square path deposits the molten strips of ABS material layer by layer, the stacked strips make the hollow cube. Then the infilled layer follows the zigzagged path to fill in the interior of the hollow cube. There are some air gaps that shown in black voids (label as flattened boundary) in the image. these voids have similar width, which is the obvious boundary between the strip of molten material and the infilled layer. Zigzagged path of the infilled layer can be identified as the black voids at the same certain interval (label as zigzagged path). In the left side of the image, the strips at the edges are not flattened. The reason probably is caused by the interaction of strips and infilled material, because some parts of infilled material that are not hardened completely migrate to the space where the parts of strips occupy, resulting the same space has extra material and showing as the round bump (label as small bump). The other possible reason is the oscillation of the extrusion head, rendering small fluctuations in the square contour. “Fillet” phenomenon can be seen at the round corner of the cube. This is the common way in mechanical engineering to reduce high stress concentrated on one point and distribute the stress over the larger area. Fig. 1(b) is the SEM image of ABS cube in another side view. The remaining traces that pulling up the cube from the plastic bed are seen obviously, which are the depressed parts in the strip and the distortion of the infilled material.

Fig. 1(a) Fig. 1(b)

Figure 1. (a) SEM image of ABS cube in side view. (b) SEM image of ABS cube in another side view

Fig. 2(a) is the SEM image of PLA cube in side view. The multiple strips of PLA and infilled material are made separately as the same manufacturing process as ABS cube. In this side, the quality of the strips are good, which have flattened surface of each strip and show only two small bumps (label as small bump). There are no air gaps in the infilled material. The path of making infilled material can be assumed to follow the parallel line with 45 raster degree (label as parallel path). The layer of the infilled material in the image is the bottom layer where attaches to the plastic bed. Because the trace that the layer is teared off can be seen at the right bottom of the image (label as tearing off trace). Fig. 2(b) is the SEM image of PLA cube at another side view. In this portion of PLA cube, the quality is horrible. The trace of joint of the extruded strip can be seen in the red mark (label as 1). It is assumed that the square contour for the strip starts from and ends to this area (label as 1). The heights of different strips vary a lot, demonstrating the uniformity of extruded material through the extruded head is bad (label as 2). Maybe this is caused by the coarse machine used for fabricating this PLA cube. Furthermore, the extra material with odd shape is probably caused by tearing off the cube from the plastic bed where this part of material sticks too firmly with the board and the distortion is too much after detaching from the bed.

Fig. 2(a) Fig. 2(b)

Figure 2. (a) SEM image of PLA cube in side view. (b) SEM image of PLA cube in another side view

Fig. 3 is the SEM image of LP cube at the side view. The width of one layer line is so tiny with only 0.0011in. Without viewing the image using SEM, the layer cannot be seen in the naked eye. The layer thickness in LP is only one tenth of that in ABS. so the resolution is much higher and the printing speed is slower in LP printer. This cube is formed by the process that the nozzle jets the layer of tiny liquid droplets and UV light instantly cures the material.

Figure 3. SEM image of LP cube in side view.

Fig. 4.1(a) is the SE image of ABS viewing the magnified layer and the boundary between layers at the closed view. Overall, the layer thickness is uniform, except for some tiny part in one layer that occupying a little bit more space in another layer, as shown in label 1. This is caused by the slightly more quantity of the extruded material. The boundary of two layers in the image with enhanced magnification is straight. Fig. 4.2(a) is the SE image of PLA at the view of layers and the boundary between layers at the closed view. The stuff shown in label 1 and the tiny lines between boundaries shown as mark 2 are possibly caused by the attachment of layers when the increased flow of PLA is extruded from nozzle. Because PLA goes through more phase transition than ABS when heated and becomes much more liquid.
This stronger binding between layers leads to the stronger strength in PLA than that in ABS. Fig. 4.3(a) is the SE image of LP at 600x magnification. The holes result probably from the way of processing this material. Because the nozzle sprays the little droplets at once, the droplets cannot exactly cover every area in the platform.
The visible particles are derived from dust.

Fig. 4.1(a) Fig. 4.2(a) Fig. 4.3(a)

Figure 4. 1 (a) SE image of ABS layer. 2 (a) SE image of PLA layer. 3 (a) SE image of LP layer.

2. Backscattered Electron (BSE) Imaging

BSD images of ABS and PLA layers in Fig. 4.1(b) and Fig. 4.2(b) show only the slightly contrast, because polymers have no elements with large atomic number. Fig. 4.3(b) is the BSD image of LP. This is the image obtained with more gold coating of 12 nm. So the bright particles are gold with larger atomic number. The layers are covered by coating and are invisible. The problems with the image at Fig. 4.2(b) are including charging effect, low brightness. Because the gold coating thickness is low with 6 nm and the aperture adjusts to the largest value in order to obtain BSD image.

Fig. 4.1(b) Fig. 4.2(b) Fig. 4.3(b)

Figure 4. 1 (b) BSD image of ABS layer. 2 (b) BSD image of PLA layer. 3 (b) BSD image of LP layer.

Generally, the coating with 6 nm thickness is not enough for ABS and PLA cubes, because slightly bright area can always be seen on the random SE or BSD image. However, the coating thickness with 6 nm is sufficient for observing the layer texture in LP cube.

3. EDS

Fig. 4.1(c), Fig. 4.2(c) and Fig. 4.3(c) are XRD images of ABS, PLA and LP cubes at 6 nm gold coating. The content of oxygen element decreases in the order of PLA, LP and ABS. These materials are polymers, so the elements consisting of these materials are mostly carbon and oxygen. Au element results from gold coating. The reason why only carbon element shows up in Fig. 4.1(c) is possibly induced by the extremely high content of carbon element and other peaks of elements, such as oxygen and gold are so tiny. So “peak ID” didn’t found. In Fig. 4.2(c), other elements maybe induced by the impurity of nozzle when fabricating the material.

Fig. 4.1(c)

Fig. 4.2(c)

Fig. 4.3(c)

Figure 4. 1 (c) XRD image of ABS layer. 2 (c) XRD image of PLA layer. 3 (c) XRD image of LP layer.

4. Atomic Force Microscope (AFM)

Fig. 5(a) is the AFM image of ABS cube in two dimensional (2D) view. The strips of layers combine in the ways that the slightly bright layer with the slightly dark one. These layers are different with the strips in SEM image. The scan area is 30 µm * 30 µm, but the thickness of strips in SEM image of ABS cube is greater than 200 µm. so the scan area in AFM image is only a small portion of strips in SEM image. These layout of layers in AFM image is possibly caused by the ways making original strings of ABS material before fabricating 3D printing polymer, like the ways of twining the strings used in guitar. Fig. 5(b)-(c) are the AFM images of ABS cube in different 3D views. Z axis directly tell the height of the different place on surface. Fig. 5(d) is the roughness graph. It is shown that most parts of surface have the height ranging from -20 to 40 nm, except for the height with -145 nm in the place investigated. The roughness average is 26. 410 nm in ABS cube.

Fig. 5(a) Fig. 5(b) Fig. 5(c)

Fig. 5(d)

Figure 5. (a) AFM image of ABS layer in 2D view (b) AFM image of ABS layer in 3D view (c) AFM image of ABS layer in another 3D view
(d) graph of surface roughness of ABS layer

Fig. 6(a) is the AFM image of PLA cube in 2D view. The different color in the image indicates the height variance. The height differs with no regular pattern. The following 3D images of PLA cube give the intuitive sense of the rough and uneven surface that AFM scans in Fig. 6(b) and 6(c). The roughness graph in Fig. 6(d) gives the large range of surface height, varying from -600 to 450 nm, which is around 25 times as the value of surface height in ABS cube. The roughness average is 0.246 µm in PLA.

Fig. 6(a) Fig. 6(b) Fig. 6(c)

Fig. 6(d)

Figure 6. (a) AFM image of PLA layer in 2D view. (b) AFM image of PLA layer in 3D view (c) AFM image of PLA layer in another 3D view
(d) graph of surface roughness of PLA layer

Fig. 7(a) and 7(b) is the AFM image of LP cube in 2D and 3D view. The color as a whole is consist with dark red. The holes in dark color can be explained as the lack of droplets of LP when the nozzle sprays at once. The heights of surface range from 30 to -30 nm. The roughness average is 9.699 nm in LP.

Fig. 7(a) Fig. 7(b)

Fig. 7(c)

Figure 7. (a) AFM image of LP layer in 2D view. (b) AFM image of LP layer in 3D view (c) graph of surface roughness of LP layer

Hence, the degree of smooth of surface can be placed in the sequence of LP, ABS and PLA. The SEM and AFM images both confirm this conclusion. The average roughness can be used to explain charging effect resulting from the non-uniform coating. If the coating process is imagined as the formation of the small islands, the rise and fall of the surface results in the different stacking thickness and shape of the small islands, so the coating is not uniform where some places have the build-up static electrons.

5. Stereo Pairs - 3D Anaglyphs

Figure 7. Stereo Pair of PLA layer

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