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METHODS Mass spectra over a mass range from 100 m/z to 1200 m/z were acquired using a prototype MALDI source on a SYNAPT G2-s mass

spectrometer (Waters, Manchester), (Figure 1). Ion images with a pixel size of 50µm x 50µm were generated from a 3mm x 4mm

rectangular area of paper containing a printed character.

Standard MALDI Imaging.

The paper sample was mounted on a standard MALDI plate and an optical image obtained using a flat bed scanner. This image

was used to identify a region of interest (ROI) using the High Definition Imaging (HDI) software (Waters, Manchester) (Figure

1) and generate a pattern for the instrument stage to follow. The MALDI imaging experiment was performed and the acquired data

processed to generate an ion image. The ions associated with the ink image as opposed to the paper could then be identified.

INTRODUCTION Imaging mass spectrometry of biological tissues using MALDI1 has become an established technique to determine the localization of chemical species within tissue sections. Demands for better spatial and mass resolving power drive the trend for larger data sets and increased time for data acquisition and processing.

Continued enhancements in hardware, such as higher repetition rate lasers and faster computer processors aid in shortening experiment times. Improvements in acquisition methods and data handling can also reduce the time spent obtaining the mass spectra and condense the data, to allow more efficient processing of the data.

During a targeted ion imaging experiment, the region of interest may be defined by the presence of an ion with a known m/z. Evaluation of novel algorithms which address size of the dataset and experiment time are presented.

TARGETED ION IMAGING MS: EVALUATION OF NEW ALGORITHMS TO REDUCE DATA SIZE AND NUMBER OF ACQUISITIONS.

Paul Murray, Richard Chapman, Jeff Brown, Keith Richardson, Chris Jones, Emmy Hoyes, Rennie Birch.

Waters Corporation, Floats Road, Manchester M23 9LZ, UK.

References

1. R. M. Caprioli, T. B. Farmer and J. Gile, Anal. Chem.. 1997; 69(23):4751-60.

2. http://arduino.cc/en/

RESULTS Standard MALDI Imaging.

The original acquisition resulted in a total of 4920 spectra, one

from each of the 50µm x 50µm pixels in the defined ROI.

Figure 3 shows the 25 combined spectra from both the ink and

the paper. Comparison of these reveal peaks that are common to both, and can be attributed to the paper (e.g. m/z 334.9); and

peaks that are only present in the inked regions (e.g. m/z 443.1).

The ion images showing the distributions of these two m/z ions are shown in Figure 4.

which, 1249 spectra were identified as containing the target

ion above the threshold level. This is a reduction in the total number of scans acquired to 35% of the standard imaging

technique, and a fall to 25% of the number of stored spectra compared with the original experiment.

Figure 7 shows the locations on the sample that the algorithm interrogated. The grid pattern followed by the initial survey

used to locate the target ion can be clearly seen, as can the edge bordering the recognized regions of interest.

Because the code must interrogate each spectrum in real time in order to determine its subsequent movement of the stage, it

is possible to generate an ion image in real time, as is demonstrated in Figure 8, where the ion image was captured

at different stages of the acquisition.

DISCUSSION In an MS imaging experiment, usually either one of two primary question is being asked: “What is in the region of

interest?”; or “Where is the ion of interest located?”.

To answer the first question, there is no option other than to identify the region and analyze each pixel within this area,

which can result in large datasets and take significant instrument time to acquire.

However if the experiment is to locate specific ions of interest then there are alternative approaches. One method is to

acquire the imaging mass spectral data in the normal manner and then filter the spectra according to the desired criteria to

reduce the size of the data set.

Another technique would be to enable the instrument to search

for spectra containing the relevant ions and use this to direct the stage motion during acquisition, decreasing the number of

pixels interrogated, and the size of the final stored files.

The savings in file size and number of pixels will be dependent

upon the distribution of the target species.

CONCLUSION Targeted mass ion imaging mass spectrometry.

Filtering of mass spectral imaging datasets post acquisition can effectively reduce the size of data

stored.

Real-time targeted mass search algorithm decreases

the number of pixels acquired.

Both approaches retain the ion image quality.

Real-Time Targeted Ion Imaging.

The search algorithm completed the data acquisition over the 3mm x 4mm rectangular area after collecting 1723 spectra, of

Figure 1. Schematic of the MALDI Synapt instrument and the HDI

imaging software.

Figure 8. Images of the 443.1 m/z ion map at different times during the data acquisition using the real-time targeted ion imaging algorithm .

Figure 4. Ion images of the (a) m/z 334.9 ion from the paper and

the (b) m/z 443.1 ion from the ink.

File Size Reduction.

After removing mass spectral data from pixels where the

intensity of the target ion was below the threshold, the data

was reduced from an original size of 3.04 GB to 1.03 GB

(34% of the original size) whilst the ion image remained

virtually identical (Figure 5) to the original.

The effect on the total ion count (TIC) from each pixel can be

seen in Figure 6 where the TIC from the first 1000 pixels are

shown, both (a) before and (b)

after applying the filtering algorithm. Figure 7. Locations of all the mass spectra acquired where the tar-

get ion intensity was found to be below (red) and above (blue) threshold .

0

0.5

1

1.5

2

2.5

3

3.5

4

0 0.5 1 1.5 2 2.5 3

Below

Above

Figure 5. Ion image after filter-

ing the data with the file size re-duction algorithm.

OVERVIEW

Comparison of standard imaging methods

with two algorithms for targeted ion

imaging approaches.

Removal of mass spectral data not

relevant to the desired information.

Data-directed sample and laser control to

minimize the required number of

acquisitions.

Figure 2. Flow diagram of the targeted acquisition control.

Figure 3. Combined spectra from 25 scans from (a) plain paper

and (b) the printed image.

File Size Reduction.

Using the data obtained from the original imaging experiment, a target ion m/z from the ink was identified (m/z 443.1). The

spectra from each pixel in the data set were checked using the file size reduction algorithm, post acquisition, for the presence of

this ion above a threshold intensity of 6000 (arbitrary units). Mass spectral data were removed for pixels where the target ion

was present below this level, and the data were then written to a new file.

Real-Time Targeted Ion Imaging.

WRENS (Waters Research Enabled Software) is an in-house software package allowing instrument parameters to be changed

between each scan. An Arduino™ (an open-source electronics prototyping platform)2 was used to synchronize the sample stage

and laser controls with the MS scans of the mass spectrometer.

The target ion and threshold level, used previously to reduce the file size, was coded into the algorithm and a fresh sample loaded.

Whilst the x-y stage and the laser firing were controlled externally by WRENS, the mass spectral data were acquired with

MassLynx (Waters, Manchester). The code controlling the stage movement and laser fire followed the flow diagram shown in

Figure 2. The initial survey acquisition mode involved the stage moving in a grid pattern with pixels 500µm apart, whilst the high

resolution mode acquired adjacent pixels with a pitch of 50µm.

By recording the locations and intensities, the algorithm could

construct an ion image in real-time. Once all pixels adjacent to locations where which the target ion was observed had been

interrogated, the stage returned to a grid pattern until either another pixel satisfied the threshold condition; at which point the

high resolution acquisition resumed; or the whole area had been

surveyed.

Figure 6. Total ion counts for the first 1500 pixels from the mass

spectral imaging acquisition (a) before, and (b) after running the file size reduction algorithm.