Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

Chen, Yong; Cai, Jun; Zhao, Tao; Wang, Chenxi; Dong, Shuo; Luo, Sizuo; Chen, Zheng W.

doi:10.1016/j.ultramic.2004.11.019

Cited by 30 publications

(18 citation statements)

References 28 publications

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“…Figure 2A shows a 50×50 µm 2 AFM image, illustrating a small group of eight live cells. These cells are spherical in shape, with a dimension of 10-20 µm in diameter and 2.5±0.3 µm in height, representative of undifferentiated cells as reported by others [18]. When imaging an individual cell (Fig.…”

Section: Topographic Imaging Of Hes Cellssupporting

confidence: 74%

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

Qiu

Xiang

et al. 2008

Biochemical and Biophysical Research Communications

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Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the "turn-on" signal and understand the mechanism of early differentiation. Here we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at ~17800 epitopes and ~700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.

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Section: Topographic Imaging Of Hes Cellssupporting

confidence: 74%

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

Qiu

Xiang

et al. 2008

Biochemical and Biophysical Research Communications

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“…We made use of our expertise of ␥␦/␣␤ T-cell biology 17 and nanotechnology 18,19 to perform nanoscale immunofluorescence imaging of antigen-specific T-cell antigen receptor (TCR) response during the in vivo clonal activation-expansion. We first established a NSOM-and fluorescent quantum dot (QD)-based imaging system to generate a best-optical-resolution immunofluorescence imaging of a cell membrane protein.…”

Section: Introductionmentioning

confidence: 99%

“…Whereas home-made NSOM operating in liquid can yield images of biologic molecules, the current commercial NSOM instruments are all designed for in-air imaging, 12-15 posing a challenge for nanoscale imaging of cell membrane proteins. Although NSOM combined with some common fluorescent materials were used for imaging, 16 the absence of highly photostable fluorophores for use in NSOM is perhaps one of the major reasons why NSOM has not been reproducibly used for nanoscale imaging of functional cellular molecules.We made use of our expertise of ␥␦/␣␤ T-cell biology 17 and nanotechnology 18,19 to perform nanoscale immunofluorescence imaging of antigen-specific T-cell antigen receptor (TCR) response during the in vivo clonal activation-expansion. We first established a NSOM-and fluorescent quantum dot (QD)-based imaging system to generate a best-optical-resolution immunofluorescence imaging of a cell membrane protein.…”

mentioning

confidence: 99%

NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion

Chen

Shao

Ali

et al. 2008

Blood

Self Cite

106

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Nanoscale imaging of an in vivo antigenspecific T-cell immune response has not been reported. Here, the combined nearfield scanning optical microscopy-and fluorescent quantum dot-based nanotechnology was used to perform immunofluorescence imaging of antigen-specific T-cell receptor (TCR) response in an in vivo model of clonal T-cell expansion. The near-field scanning optical microscopy/quantum dot system provided a best-optical-resolution (Ͻ50 nm) nanoscale imaging of V␥2V␦2 TCR on the membrane of nonstimulated V␥2V␦2 T cells. Before Ag-induced clonal expansion, these nonstimulating V␥2V␦2 TCRs appeared to be distributed differently from their ␣␤ TCR counterparts on the cell surface. Surprisingly, V␥2V␦2 TCR nanoclusters not only were formed but also sustained on the membrane during an in vivo clonal expansion of V␥2V␦2 T cells after phosphoantigen treatment or phosphoantigen plus mycobacterial infection. IntroductionT-cell receptors (TCRs) play a crucial role in recognition of antigens and development of immune responses. Whereas immune events for TCR-mediated recognition, signaling, and activation are well described, 1-4 nanoscale imaging of immunobiology of antigenspecific TCR during the in vivo clonal T-cell expansion has not been studied. Because TCRs trigger downstream signaling and activation after antigen recognition, some unique TCR nanostructures may develop after TCR contact on Ag/antigen-presenting cell (APC) and thus contribute to selected functions, such as clonal expansion, effector function, contracting (clonal exhaustion), or differentiation. Whereas this presumption can be tested by imaging or visualization of antigen-specific TCR during the in vivo T-cell response, conventional imaging techniques using fluorescent or confocal microscopy do not have nanoscale optical resolution power to reveal individual TCRs and their dynamics during clonal expansion-maturation. [1][2][3][4] Nanotechology-based imaging may make it possible to reveal TCR nanostructures in the context of T-cell recognition of antigens and therefore provide new insight into T-cell response or ultimately immunity. 5 Nanotechnology is emerging as a multidisciplinary tool to advance life science and medicine. 6,7 However, nanoscale imaging or dissecting of functional biologic molecules in cells remain challenging. Near-field scanning optical microscopy (NSOM) has proved to be a useful nanotechnology tool for studying hard and flat materials, but its application in biomedical research is still limited. [8][9][10][11] Complicated natures of cell membranes or biologic molecules make it difficult for NSOM to generate high spatial resolution images. Whereas home-made NSOM operating in liquid can yield images of biologic molecules, the current commercial NSOM instruments are all designed for in-air imaging, 12-15 posing a challenge for nanoscale imaging of cell membrane proteins. Although NSOM combined with some common fluorescent materials were used for imaging, 16 the absence of highly photostable fluorophores for use in NSOM is perhap...

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“…It has also been used for simultaneous mechanical characterization of cells to create models for FEA of cell deformations with applied external forces (Charras and Horton, 2002;Ohashi, 2002). While the concurrent collection of detailed surface topography and mechanical properties are an advantage of AFM, the collection of images is slow, the true height of the cell is difficult to obtain, and cells must be fixed to image their internal structures (Chen et al, 2005). Furthermore, the need of AFM for a stylus protruding from above the sample precludes the use of closed flow chambers, simultaneous use of some other imaging modalities (e.g.…”

Section: Discussionmentioning

confidence: 99%

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy

Ferko

Patterson

Butler

2006

Microsc. Res. Tech.

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Optical-sectioning, digital fluorescence microscopy provides images representing temporally-and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling.

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Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

Cited by 30 publications

References 28 publications

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy

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