Correlative Light and Electron Microscopy (CLEM) is an imaging technique that combines the functionality of light microscopy with the high resolution of electron microscopy. However, a major limitation of CLEM in biological applications is the need to dry or cryogenically freeze samples, which not only complicates experimental procedures, but is also likely to introduce artifacts.
The Poseidon System holder enables wet samples to be imaged with electron microscopy. Therefore, cellular receptors that are tagged with nanoparticles, such as quantum dots (QDs), can be imaged with nanometer resolution under physiologically relevant conditions.
In this experiment, COS-7 fibroblast cells were directly cultured on Poseidon System E-chips™ sample supports. To label the epidermal growth factor (EGF) receptors, the cells were incubated for a period of 5 minutes in a 5 nM CdSe QDs solution coupled to EGF.
The cells were then washed, adhered with glutaraldehyde, and stored in phosphate buffered saline after labeling. The E-chips™ containing the cells were inverted and placed in a glass bottom culture dish to obtain fluorescence images.
A wide field fluorescence microscope fitted with an oil immersion objective was used to record all images. The E-chip™ was then placed in the Poseidon System holder with a liquid thickness of 5 µm and embedded into an FEI CM200 electron microscope. The sample was imaged in scanning TEM, and a 2 µL/min liquid flow rate was maintained during the entire imaging session.
The electron microscopy images were correlated with their corresponding location in the light microscopy image. The E-chip™ window has a rectangular design that was used as a reference system for matching position coordinates between images.
The direct interference contrast image of a cell present on the E-chip™ window is shown in Figure A, and the equivalent quantum dot fluorescence is shown in Figure B. As shown in Figure C, the area indicated with an arrow was using scanning TEM.
The individual QDs can be seen as yellow-green spots spread all over the cell (false colorized to improve contrast). Cellular contours can be seen as regions of blue shading. By using the 25-75% edge width of line scans taken over 10 QDs, the spatial resolution was determined to be 3 nm.
The study results show the efficiency of the Poseidon System for CLEM imaging of protein distribution on entire cells. The achieved resolution is adequate to distinguish different size, shape, and electron density among nanoparticles to facilitate complex imaging studies.
This information has been sourced, reviewed and adapted from materials provided by Protochips.
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