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Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique.

Kim DJ, Kim GS, Hyung JH, Lee WY, Hong CH, Lee SK - Nanoscale Res Lett (2013)

Bottom Line: Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported.Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Chung-Ang University, Seoul 156-756, Republic of Korea. sangkwonlee@cau.ac.kr.

ABSTRACT
Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

No MeSH data available.


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SEM images of the CD4 T cell and QNPA. (a, b, c) SEM images (top and tilt views) of the CT4 T cell on the QNPA substrates before and after FIB ion milling, respectively. (d, e) Cross-sectional SEM images of QNPA without and with surface-bound T cell, respectively. (f) Overlapped images of QNPA from only QNPA and from QNPA covered by the cell. All cells were highlighted in blue, while the Pt was in purple, for clear differentiation.
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Figure 4: SEM images of the CD4 T cell and QNPA. (a, b, c) SEM images (top and tilt views) of the CT4 T cell on the QNPA substrates before and after FIB ion milling, respectively. (d, e) Cross-sectional SEM images of QNPA without and with surface-bound T cell, respectively. (f) Overlapped images of QNPA from only QNPA and from QNPA covered by the cell. All cells were highlighted in blue, while the Pt was in purple, for clear differentiation.

Mentions: To investigate cross-sectional CTF of T cells on STR-functionalized QNPA substrate, we utilized both a high-performance etching and imaging scheme from FIB and FEM-based commercial simulation tools. In this regard, we first carried out the cross-sectional etching of the surface-bound T cells on QNPA substrates to assure CTFs exerted on the T cells. Figure 4a,b,c shows SEM images (top, tilt, and cross-sectional views) of the cell on the QNPA substrates before and after Ga+ ion milling process of dehydrated CD4 T cell using FIB technique, respectively. These figures show that the captured T cells on STR-functionalized QNPA were securely bound on the surface of QNPA. In addition, to further evaluate the deflection of the QNPA shown in Figure 4e, we took cross-sectional images both from only QNPA substrate (‘A’ region in Figure 4a) and from the CD4 T cell bound on the QNPA (‘B’ region in Figure 4c) as shown in Figure 4d,e, respectively (enlarged images of the cross-sectional views). This result exhibits that each nanopillar was clearly bended to the center region as shown in the overlapped images (Figure 4f). Accordingly, we can straightforwardly extract the deflection distance of each nanopillar, which is the key parameter to derive the CTFs with FEM simulation, from the SEM observation. According to the maximum bending distance (x) and the corresponding bending force (f) [18,29]f = (3EI / L3)x, where E is the elastic modulus of quartz nanopillar, I is the area moment of inertia, L is the height of the nanopillar, and x is the bending distance, the CTF (f) required to bend a nanopillar can be derived from the lateral displacement (x) of a nanopillar parallel to the quartz substrate. For this purpose, we then carried out FEM simulations using commercial COMSOL Multiphysics® (COMSOL AB, Stockholm, Sweden) software using the experimental measurements from the SEM observation and mechanical properties of the quartz nanopillar. Using a nonlinear model in COMSOL Multiphysics® software, we derived the relationship, which is served for the calibration to quantify the CTF of the cells, between the lateral deflection distance and CTFs of the CD4 T cell acting on the QNPA substrates as shown in Figure 5a. As a result, Figure 5b shows the cross-sectional CTF distribution of the CD4 T cell on STR-QNPA substrates, exhibiting that the CTFs at the edge of the cells are much stronger than those at center part of the cells. The values of CTFs for the captured CD4 T cells on STR-functionalized QNPA substrates are determined to be in the range of 0.1 to 2.1 μN, while the deflection distances were determined to be 0.2 to 3.69 μm, just after 20 min of incubation. Li et al. reported that the CTFs between the L929 cells and silicon nanowire arrays were in the range of 2.7~4.3 μN when cultured for 2 to 36 h, which is 1.3~1.6 times higher in CTFs as compared to our observation in maximum CTFs of CD4 T cells on QNPA substrates [18]. Our previous results [23] suggested that the traction force on the nanostructured substrates increased with increasing incubation times, which is in good agreement with previous results in cell migration with an increase in culture times [18]. As a result, the values of CTFs of the captured CD4 T cell on STR-functionalized QNPA substrate with short periods of incubation (<20 min) are much lower than those from other cells for long periods of incubation (>30 h).


Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique.

Kim DJ, Kim GS, Hyung JH, Lee WY, Hong CH, Lee SK - Nanoscale Res Lett (2013)

SEM images of the CD4 T cell and QNPA. (a, b, c) SEM images (top and tilt views) of the CT4 T cell on the QNPA substrates before and after FIB ion milling, respectively. (d, e) Cross-sectional SEM images of QNPA without and with surface-bound T cell, respectively. (f) Overlapped images of QNPA from only QNPA and from QNPA covered by the cell. All cells were highlighted in blue, while the Pt was in purple, for clear differentiation.
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Related In: Results  -  Collection

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Figure 4: SEM images of the CD4 T cell and QNPA. (a, b, c) SEM images (top and tilt views) of the CT4 T cell on the QNPA substrates before and after FIB ion milling, respectively. (d, e) Cross-sectional SEM images of QNPA without and with surface-bound T cell, respectively. (f) Overlapped images of QNPA from only QNPA and from QNPA covered by the cell. All cells were highlighted in blue, while the Pt was in purple, for clear differentiation.
Mentions: To investigate cross-sectional CTF of T cells on STR-functionalized QNPA substrate, we utilized both a high-performance etching and imaging scheme from FIB and FEM-based commercial simulation tools. In this regard, we first carried out the cross-sectional etching of the surface-bound T cells on QNPA substrates to assure CTFs exerted on the T cells. Figure 4a,b,c shows SEM images (top, tilt, and cross-sectional views) of the cell on the QNPA substrates before and after Ga+ ion milling process of dehydrated CD4 T cell using FIB technique, respectively. These figures show that the captured T cells on STR-functionalized QNPA were securely bound on the surface of QNPA. In addition, to further evaluate the deflection of the QNPA shown in Figure 4e, we took cross-sectional images both from only QNPA substrate (‘A’ region in Figure 4a) and from the CD4 T cell bound on the QNPA (‘B’ region in Figure 4c) as shown in Figure 4d,e, respectively (enlarged images of the cross-sectional views). This result exhibits that each nanopillar was clearly bended to the center region as shown in the overlapped images (Figure 4f). Accordingly, we can straightforwardly extract the deflection distance of each nanopillar, which is the key parameter to derive the CTFs with FEM simulation, from the SEM observation. According to the maximum bending distance (x) and the corresponding bending force (f) [18,29]f = (3EI / L3)x, where E is the elastic modulus of quartz nanopillar, I is the area moment of inertia, L is the height of the nanopillar, and x is the bending distance, the CTF (f) required to bend a nanopillar can be derived from the lateral displacement (x) of a nanopillar parallel to the quartz substrate. For this purpose, we then carried out FEM simulations using commercial COMSOL Multiphysics® (COMSOL AB, Stockholm, Sweden) software using the experimental measurements from the SEM observation and mechanical properties of the quartz nanopillar. Using a nonlinear model in COMSOL Multiphysics® software, we derived the relationship, which is served for the calibration to quantify the CTF of the cells, between the lateral deflection distance and CTFs of the CD4 T cell acting on the QNPA substrates as shown in Figure 5a. As a result, Figure 5b shows the cross-sectional CTF distribution of the CD4 T cell on STR-QNPA substrates, exhibiting that the CTFs at the edge of the cells are much stronger than those at center part of the cells. The values of CTFs for the captured CD4 T cells on STR-functionalized QNPA substrates are determined to be in the range of 0.1 to 2.1 μN, while the deflection distances were determined to be 0.2 to 3.69 μm, just after 20 min of incubation. Li et al. reported that the CTFs between the L929 cells and silicon nanowire arrays were in the range of 2.7~4.3 μN when cultured for 2 to 36 h, which is 1.3~1.6 times higher in CTFs as compared to our observation in maximum CTFs of CD4 T cells on QNPA substrates [18]. Our previous results [23] suggested that the traction force on the nanostructured substrates increased with increasing incubation times, which is in good agreement with previous results in cell migration with an increase in culture times [18]. As a result, the values of CTFs of the captured CD4 T cell on STR-functionalized QNPA substrate with short periods of incubation (<20 min) are much lower than those from other cells for long periods of incubation (>30 h).

Bottom Line: Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported.Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Chung-Ang University, Seoul 156-756, Republic of Korea. sangkwonlee@cau.ac.kr.

ABSTRACT
Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

No MeSH data available.


Related in: MedlinePlus