Optimizing grid preparation methods for TEM imaging of amyloid-forming proteins

Area of Science:

• Biophysics
• Biochemistry
• Microscopy

Background:

• Transmission electron microscopy (TEM) and Thioflavin T (ThT) assays are standard for amyloid fibril studies.
• Discrepancies between ThT fluorescence and TEM can arise from limitations in TEM visualization, particularly sample preparation.
• Grid preparation significantly impacts fibril deposition and retention, affecting imaging outcomes.

Purpose of the Study:

• To compare five grid preparation methods for efficient detection and visualization of human islet amyloid polypeptide (hIAPP) fibrils.
• To assess methods based on detection speed, morphological representation, fibril abundance, and grid contamination.
• To evaluate the best methods for early aggregate detection and application to amyloid-beta 42 (Aβ42) fibrils.

Main Methods:

• Comparative analysis of five distinct grid preparation protocols for TEM imaging.
• Assessment of droplet-deposition (Method 2) and centrifugation-based loading (Method 3) protocols.
• Application of optimized methods to hIAPP and amyloid-beta 42 (Aβ42) protein samples.

Main Results:

• Methods 2 and 3 demonstrated the most efficient fibril detection and morphological representation.
• Method 2 provided the best compromise between rapid detection, simplicity, and low grid contamination.
• Optimized methods were effective for visualizing early hIAPP aggregates and Aβ42 fibrils.

Conclusions:

• Grid preparation protocols critically influence TEM observations of amyloid fibrils.
• Method 2 (droplet deposition) is recommended for its efficiency, simplicity, and minimal contamination.
• This study offers practical guidance for selecting optimal TEM grid preparation for amyloid fibril imaging.