| AVS 65th International Symposium & Exhibition | |
| Applied Surface Science Division | Tuesday Sessions |
| Session AS+BI-TuM |
| Session: | Applied Surface Science: From Electrochemistry to Cell Imaging, a Celebration of the Career of Nicholas Winograd |
| Presenter: | Gregory L. Fisher, Physical Electronics |
| Authors: | G.L. Fisher, Physical Electronics C.E. Chini, University of Illinois at Urbana-Champaign B. Johnson, Colorado State University M.M. Tamkun, Colorado State University M.L. Kraft, University of Illinois at Urbana-Champaign |
| Correspondent: | Click to Email |
A goal of cellular imaging is to ascertain the composition of organelles, e.g. lipid profiling or pharmaceutical efficacy. To date most MS imaging of organelles is accomplished by stable isotope labeling because the imaging ion beam produces primarily (di)atomic ions. Such analyses are void of desired molecular specificity. We employed a TOF-TOF imaging capability [2] to achieve molecular specificity and conjectured that an ER-Tracker stain would yield characteristic molecular ions with which to image the endoplasmic reticulum (ER) and ER tubules.
We used human embryonic kidney (HEK) cells that had a high number of ER tubules near the plasma membrane (PM). Experimental cells were transfected to express GFP-Kv2.1 fluorescent ion channels. The cells were stained with ER-Tracker which selectively labels the ER. Control specimens were neither transfected nor stained.
We observed by simultaneous MS imaging and tandem MS imaging, in both the positive and negative ion polarities, the atomic and molecular moieties characteristic of an ER-Tracker stain localized to the ER and ER tubule structures. The ion species used for tandem MS imaging of the ER and ER tubules, namely F-, C6H5-, C5H5+ and C17H15N2O+, were shown irrefutably via the product ion spectra to arise solely from the ER-Tracker stain. Two-dimensional (2D) imaging revealed intersection of some ER tubules at the PM. Three-dimensional (3D) visualization via depth profile analysis, carried out to a depth of ≈ 40 nm from the PM, revealed additional ER tubules just under the PM. Some ER-Tracker was observed in the PM indicating ER tubule contact with the PM to form ER-PM junctions. We were able to confirm the presence and position of the PM owing to the presence of characteristic lipids, lipid fragments and fatty acids which were imaged in parallel. The observed tubule features were imaged at an effective lateral resolution of 137 nm and had measured diameters in the range of approximately 500 nm to 2 μm corresponding well with previous studies [3] and present total internal reflection fluorescence (TIRF) observations. More than a dozen control cells were analyzed, and neither atomic nor molecular moieties characteristic of the ER-Tracker were observed to be present. Our next aim is to visualize the ER within entire cells and to assess the lipid composition at different locations within the ER. By extension, with organelle-specific stains, we can apply this TOF-SIMS tandem MS imaging method to aspects of pharmaceutical delivery and metabolism.