Three-Dimensional Mass Spectrometric Imaging of Biological Structures Using a Vacuum-Compatible Microfluidic Device

Three-dimensional (3D) molecular imaging of biological structures is important for a wide range of research. In recent decades, secondary-ion mass spectrometry (SIMS) has been recognized as a powerful technique for both two-dimensional and 3D molecular imaging. Sample fixations (e.g., chemical fixation and cryogenic fixation methods) are necessary to adapt biological samples to the vacuum condition in the SIMS chamber, which has been demonstrated to be nontrivial and less controllable, thus limiting the wider application of SIMS on 3D molecular analysis of biological samples. Our group recently developed in situ liquid SIMS that offers great opportunities for the molecular study of various liquids and liquid interfaces. In this work, we demonstrate that a further development of the vacuum-compatible microfluidic device used in in situ liquid SIMS provides a convenient freeze-fixation of biological samples and leads to more controllable and convenient 3D molecular imaging. The special design of this new vacuum-compatible liquid chamber allows an easy determination of sputter rates of ice, which is critical for calibrating the depth scale of frozen biological samples. Sputter yield of a 20 keV Ar₁₈₀₀⁺ ion on ice has been determined as 1500 (±8%) water molecules per Ar₁₈₀₀⁺ ion, consistent with our results from molecular dynamics simulations. Moreover, using the information of ice sputter yield, we successfully conduct 3D molecular imaging of frozen homogenized milk and observe network structures of interesting organic and inorganic species. Taken together, our results will significantly benefit various research fields relying on 3D molecular imaging of biological structures.