Fig. 1: Transmission MALDI-2-MSI with integrated scanning microscopy for subcellular resolving power.

a A schematic drawing of the t-MALDI-2 setup illustrates the general layout. The sample is illuminated homogeneously by an LED ring. Light is collected by the microscope objective and focused onto the sensor of a digital camera. The MALDI laser-beam is co-aligned using a dichroic mirror and focused onto the sample by the same objective. Ions generated by the MALDI process with optional MALDI-2 are extracted to a mass analyzer. b A horizontal cut through the schematic displays the fluorescence microscope components. Fluorescence is excited by a fiber-coupled LED and filtered by an excitation filter, a dichroic mirror and an emission filter. ((a) and (b) are renderings created by the authors in Autodesk Fusion 360.) c To enable fluorescence microscopy and t-MALDI-2-MSI on the same sample, different staining steps are performed following a brief fixation. Samples are imaged with an external slide scanning fluorescence microscope, and are then prepared for MALDI-MSI by homogeneous coating with MALDI matrix (Supplementary Fig. 5 A, B, D). To enable high fidelity co-registration of the fluorescence microscopy image, a second fluorescence image is recorded inside the ion source prior to t-MALDI-2-MSI (Supplementary Fig. 1a, d-e).