EELS elemental mapping of cryogenically frozen mitochondria in human retinal ganglion cells
Gatan instruments used
GIF Continuum® K3®, model 626 cryo-holder, DigitalMicrograph® software, and Latitude® S software.
Background
Investigating the chemical composition of cryogenically frozen samples that have remained hydrated is technically challenging due to the low radiation tolerance of hydrated biological cells. A typical dose for single particle tomography is ~40 e-/Å2. This dose is several orders of magnitude smaller than typically used for acquiring an electron energy loss spectroscopy (EELS) spectrum image (SI) from an inorganic sample, which can range from ~107 – 108 e-/Å2. This is partially due to the relatively small inelastic scattering cross-sections of the ionization edges compared to the elastic scattering cross-sections. In addition, previous generation scintillator/CCD EELS detectors were inefficient at converting fast electrons into readable signals, necessitating a higher dose to achieve a sufficient signal-to-noise ratio (SNR). Integrating the direct detection technology of the K3 with the GIF Continuum increases collection efficiency and reduces the overall dose of the EELS measurement. In this brief, we show that the combined capabilities of the GIF Continuum and the K3 allow us to measure the distribution of Ca within a biological cell.