Unveiling the hidden secrets of cellular calcium within the delicate mitochondria of human retinal ganglion cells is no small feat. But here's where it gets controversial: traditional methods often fall short due to the extreme sensitivity of these living systems to radiation. So, how do we map the chemical composition of cryogenically frozen, hydrated cells without damaging them? This is the part most people miss—the intricate dance between technology and biology that makes it possible.
The challenge lies in the limited radiation resistance of hydrated living cells, which complicates the investigation of their chemical makeup. For instance, single-particle tomography typically requires a dose of around 40 e-/Ų, a fraction of the ~10⁷ to 10⁸ e-/Ų needed for electron energy loss spectroscopy (EELS) on inorganic samples. Why the disparity? It’s partly due to the ionization edges, which have smaller inelastic scattering cross-sections compared to elastic scattering. Older scintillator/CCD EELS detectors were inefficient, requiring higher doses to achieve a decent signal-to-noise ratio (SNR).
Enter the game-changer: integrating direct detection technology with the GIF Continuum® K3®. This innovation boosts collection efficiency while slashing the overall dose required for EELS testing. But how does this translate to real-world applications? This article showcases how the GIF Continuum and K3 can be combined to map calcium distribution within a biological cell, a task that was once deemed nearly impossible.
Materials, Methods, and Breakthroughs
Human retinal ganglion cells were cultivated on carbon grids and treated with an organic calcium phosphate solution. These hydrated samples were then plunge-frozen in liquid nitrogen and cryogenically transferred to a transmission electron microscope (TEM) using a 626 cryo-transfer holder. The (S)TEM operated at 200 kV, and Latitude® S was used to locate mitochondria on the TEM grid.
Once mitochondria were identified, the microscope’s mode switched to STEM. EELS spectrum images (SI) were captured at a dispersion of 0.9 eV/channel and a total energy range of 3,000 eV. To minimize multiple scattering from core-loss edges—a common issue in thicker samples (≥1.5 t/λ)—DualEELS™ was employed. The EELS SI data were acquired using the in-situ SI tool in DigitalMicrograph® (https://www.gatan.com/products/tem-analysis/digitalmicrograph-software), which stores each SI pass separately.
Each pass was scrutinized for H⁺ bubble production in the mitochondria, a critical indicator of radiation damage. With a pixel dwell duration of 1.0 ms and an 8 × 8 subpixel scan array, the total dose per pass was kept at 18.37 e-/Ų. H⁺ bubbles appeared after a cumulative dose of ~300 e-/Ų, prompting the exclusion of subsequent passes from the dataset. The first 15 passes were spatially aligned (drift-corrected) and combined using the in-situ technique.
Given the sample’s strict dosage limits, principal component analysis (PCA) was employed to denoise the spectra. A model was developed using multiple linear least squares fitting to address overlapping C K and Ca L₂,₃ edges, as well as plural scattering. Figure 1a displays an aligned and summed annular dark-field (ADF) image of the mitochondria, where bright, white contrasts indicate calcium-rich areas. Figure 1b confirms this, showing Ca accumulation in purple regions that align with the bright ADF areas.
Figure 1. a) Aligned annular dark-field (ADF) image of mitochondria. b) EELS elemental map highlighting Ca presence in purple regions. Image Credit: Gatan, Inc.
Summary and Implications
By combining the K3 camera’s sensitivity with the GIF Continuum’s high-performance optics, researchers demonstrated that the stark contrast in the ADF STEM image corresponds to Ca buildup within mitochondria. A traditional scintillator-based detector would have failed under such stringent dosage constraints.
The ability to save passes separately using in-situ spectrum imaging revealed how mitochondria respond to cumulative dosage and allowed the exclusion of H⁺ bubble-contaminated data. Latitude S streamlined the process by enabling a preliminary survey of the TEM grid via bright-field imaging, significantly reducing the time needed to locate mitochondria. For deeper insights, explore the publication in Structure.
Acknowledgments
Special thanks to Dr. Wah Chu and Dr. Gong-Her Wu for their collaboration, sample provision, and expertise in cryo-EM.
Reference
- Wu, G.-H. et al. (2025). Cryogenic electron tomography and elemental analysis of mitochondrial granules in human retinal ganglion cells, Structure, 33(10), pp. 1771-1780.e3. DOI:10.1016/j.str.2025.07.010. https://www.cell.com/structure/abstract/S0969-2126(25)00258-8.
About Gatan, Inc.
Gatan, Inc. (https://www.gatan.com/) is the global leader in manufacturing instrumentation and software that enhance electron microscope performance. Their products, compatible with all electron microscope brands, cover the entire analytical process—from specimen preparation to imaging and analysis. Serving industrial, governmental, and academic laboratories, Gatan supports research in metallurgy, semiconductors, biology, and more. Renowned for quality and innovation, Gatan is a trusted name in the scientific community.
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