Flujo de trabajo guiado por cryo-CLEM para la evaluación y optimización cuantitativa de la calidad de especímenes de...

Cryo-correlative light and electron microscopy (cryo-CLEM) is an essential technique for precisely targeting regions of interest in vitrified biological specimens for in situ cellular cryo-electron tomography (cryo-ET). Despite its widespread use, the potential of cryo-CLEM remains underexploited because its imaging performance under cryogenic conditions has not been characterized. Here, we evaluated the imaging capabilities of two commercially available cryo-CLEM systems-a stand-alone Leica EM cryo-CLEM microscope and the integrated Thermo Fisher Scientific iFLM coupled to a cryo-FIB platform-by quantifying their spatial resolution and signal-to-noise ratio (SNR) using vitrified cellular specimens expressing GFP-labeled proteins. The EM cryo-CLEM setup provided higher SNR and faster acquisition via tile imaging, whereas the iFLM offered a wider field of view, revealing complementary strengths between the two instruments. Furthermore, we established a quantitative correlation between fluorescence intensity and lamella thickness, demonstrating that cryo-CLEM imaging can serve as a practical indicator of lamella quality during early specimen preparation, prior to cryo-ET data collection. Collectively, our results show that cryo-CLEM not only facilitates precise targeting but also provides quantitative feedback for optimizing lamella quality, thereby streamlining the overall cryo-ET workflow.