High-speed MALDI-TOF imaging mass spectrometry: rapid ion image acquisition and considerations for next generation instrumentation

Abstract

A prototype matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer has been used for high-speed ion image acquisition. The instrument incorporates a Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz and continuous laser raster sampling for high-throughput data collection. Lipid ion images of a sagittal rat brain tissue section were collected in 10 min with an effective acquisition rate of roughly 30 pixels/s. These results represent more than a 10-fold increase in throughput compared with current commercially available instrumentation. Experiments aimed at improving conditions for continuous laser raster sampling for imaging are reported, highlighting proper laser repetition rates and stage velocities to avoid signal degradation from significant oversampling. As new high spatial resolution and large sample area applications present themselves, the development of high-speed microprobe MALDI imaging mass spectrometry is essential to meet the needs of those seeking new technologies for rapid molecular imaging.

Figure 1

A 100 μm spatial resolution lipid ion image of a sagittal rat brain tissue section using typewriter continuous laser raster sampling acquired in 10 min. (a) Representative spectrum is shown. The ion image overlay (b) of signal from m/z 734.4 PC(32:0) (c), m/z 788.5 PC(36:1) (d), and m/z 806.5 PC(38:6) (e) highlights the differentiation of the spatial distributions for the selected ions. These results correlate to the H and E stained serial tissue section highlighting structural difference between grey matter, white matter and granular cells in the cerebellum (f). Important instrumental parameters: 3 kHz laser repetition rate, 5 mm/s sample stage velocity, and 60 laser shots/spectrum hardware average

Figure 2

Low quality ion image (m/z 734.4) of a sagittal rat brain tissue section using serpentine raster sampling showing “stripes.” Image quality is diminished by striping caused by differences in ion signal intensity dependent on the direction of sample stage motion. Important instrumental parameters: 3 kHz laser repetition rate, 6 mm/s sample stage velocity, 50 laser shots/spectrum hardware average, 100 μm spatial resolution

Figure 3

An illustration of continuous laser raster MALDI sampling highlighting the number of laser shots/unit area for a representative laser frequency (3 kHz), laser spot diameter (50 μm) and stage velocity (5 mm/s). After an initial distance roughly equal to the diameter of the laser beam, the sampling conditions become constant (blue circle) with a maximum of 30 shots/unit area. Laser shot overlap decreases for areas further away from the center of the raster path (grey bar)

Figure 4

Theoretical calculations for the number of laser shots/unit area as a function of (a) sample stage velocity, (b) laser pulse repetition rate and (c) laser beam diameter. 50 μm laser beam diameter was assumed for a and b. A sample stage velocity of 5 mm/s was used for plot c