Improved spatial resolution of infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry imaging using a reflective objective

Abstract

Rationale The level of visual detail of a mass spectrometry image is dependent on the spatial resolution with which it is acquired, which is largely determined by the focal diameter in infrared laser ablation‐based techniques. While the use of mid‐IR light for mass spectrometry imaging (MSI) has advantages, it results in a relatively large focal diameter and spatial resolution. The continual advancement of infrared matrix‐assisted electrospray ionization (IR‐MALDESI) for MSI warranted novel methods to decrease laser ablation areas and thus improve spatial resolution. Methods In this work, a Schwarzschild‐like reflective objective was incorporated into the novel NextGen IR‐MALDESI source and characterized on both burn paper and mammalian tissue using an ice matrix. Ablation areas, mass spectra, and annotations obtained using the objective were compared against the current optical train on the NextGen system without modification. Results The effective resolution was determined to be 55 μm by decreasing the step size until oversampling was observed. Use of the objective improved the spatial resolution by a factor of three as compared against the focus lens. Conclusions A Schwarzschild‐like reflective objective was successfully incorporated into the NextGen source and characterized on mammalian tissue using an ice matrix. The corresponding improvement in spatial resolution facilitates the future expansion of IR‐MALDESI applications to include those that require fine structural detail.

Maximized Spatial Information and Minimized Acquisition Time of Top-Hat IR-MALDESI-MSI of Zebrafish Using Nested Regions of Interest (nROIs)

“…A solid state 2970 nm laser (JGM Associates, Inc., Burlington, MA) fires downward toward the sample. In the down tube, the laser is first expanded by a −75 mm plano-concave beam expander (LC5401-E, Thorlabs; Newton, NJ), collimated by a +250 mm plano-convex lens placed 175 mm below (LA5255, Thorlabs), reshaped into a top-hat beam by a custom ZnSe DOE (HOLO-OR, FO-1933 #2), and focused onto the sample by a Schwarzschild-like reflective objective (LMM15X-P01, Thorlabs) . The laser energy at the focal plane was measured with a Nova II power meter and energy sensor (Ophir Optronics, LLC; North Logan, UT).…”

“…In the down tube, the laser is first expanded by a −75 mm planoconcave beam expander (LC5401-E, Thorlabs; Newton, NJ), collimated by a +250 mm plano-convex lens placed 175 mm below (LA5255, Thorlabs), reshaped into a top-hat beam by a custom ZnSe DOE (HOLO-OR, FO-1933 #2), 7 and focused onto the sample by a Schwarzschild-like reflective objective (LMM15X-P01, Thorlabs). 10 The laser energy at the focal plane was measured with a Nova II power meter and energy sensor (Ophir Optronics, LLC; North Logan, UT). Approximately 0.65 mJ per burst was applied to the sample (7−9 pulses) due to the low energy transmission of the objective (≈11%) and the higher fluence at smaller beam diameters.…”

“…Therefore, there is motivation to decrease the top-hat spot size to achieve higher-spatial-resolution top-hat MSI. With the success of achieving 50 μm spot sizes using a reflective objective that has shorter working distance, theoretically, the spatial resolution of top-hat IR-MALDESI-MSI can be improved by a combination of both optics. , However, increasing the spatial resolution will increase the acquisition time of imaging a comparable area. Eq describes the relative acquisition time ( t r ) of imaging the same area using a larger ( D o ) and smaller ( D i ) step size .…”

Automatic z-Axis Correction for IR-MALDESI Mass Spectrometry Imaging of Uneven Surfaces

“…The source setup was described in detail elsewhere. 30 The two optics used in this work, a Schwarzschild-like reflective objective 31 and a diffractive optical element, 32 were optimized previously. All the experiments were conducted on the NextGen source coupled to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”

High-Resolution Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Tissues

“…19 Infrared matrix assisted electrospray ionization (IR-MALDESI) has been used for tissue imaging with 55 µm resolution. 20 Meanwhile, liquid extraction surface analysis (LESA), 21 and liquid microjunction surface sampling probe (LMJ-SSP), 22 have been used for imaging with a spatial resolution of 1 mm and 0.5 mm, respectively. Recent developments of ambient ionization imaging platforms based on nanospray desorption electrospray ionization (nano-DESI), [23][24][25][26] single probe, 27 fiber probe laser ablation dielectric barrier discharge, 28 and tapping-mode scanning probe electrospray ionization 29 have enabled imaging of biological samples with a spatial resolution of better than 10 µm.…”