DESI Mass Spectrometry Imaging (MSI)

Fig. 1

Parts of the Waters DESI sprayer

2.4 Software

1.

Prosolia Omni Spray 2D: software to control the DESI source for DESI sprayer optimization (Prosolia Inc., Indianapolis, IN, US).
2.

MassLynx v4.1: software to control the DESI source for DESI imaging experiments and to control the mass spectrometer (Waters, Corporation, Wilmslow, UK).
3.

High Definition Imaging (HDI) 1.4: software to set up DESI imaging experiment, processing, and visualization of the DESI imaging data (Waters, Corporation, Wilmslow, UK).

3 Methods

3.1 Sprayer Construction

1.

Screw the positioning nut at the back of metal rear housing.
2.

Feed the cut end of the emitter through the metal emitter guide tube and the ferrule.
3.

Place the assembly (emitter, guide tube, and ferrule) into the rear housing, allowing the emitter to be positioned against the positioning nut. Tighten the two metal parts together with spanners so that the ferrule has the emitter held firmly (see Note 2 ).
4.

Place the metal assembly into the main PEEK body from the back.
5.

Carefully place the gas cone to the front of the main PEEK body, avoiding damaging the emitter tip (see Note 3 ). Wind the spray cone using a 5 mm spanner until the emitter starts to protrude. The fully assembled sprayer is shown in Fig. 2. Remove the positioning tip from the back of the sprayer.

Fig. 2

Fully assembled Water DESI sprayer
6.

Mount the sprayer assembly using the mounting bracket onto the arm of the DESI source (see Note 4 ).
7.

Connect the High voltage cable, gas, and solvent fittings (see Note 5 ).

3.2 DESI Signal Optimization

A critical aspect to a successful DESI experiment is the production of a focused, co-axially aligned spray . This is governed by the relationship between the solvent flow, nebulizing gas flow, the nozzle protrusion, the nozzle to emitter tip distance, and applied high voltage (see Fig. 3). Once the sprayer parameters are optimum the final task is to optimize the geometric parameters, i.e., the sprayer height above the surface and the distance between the sprayer and the inlet capillary.

Fig. 3

Schematic of DESI with the Waters sprayer showing the gas, solvent, and high voltage applied onto the sprayer with the capture of the desorbed charged droplets into the metal capillary and into the mass spectrometer API source

Typical starting positions are described in Table 1.

Table 1

Starting DESI imaging settings

Height of emitter above surface	1–2 mm
Emitter to inlet distance	5 mm
Sprayer angle	75°
Solvent flow	1 2-μL/min
Gas pressure	5–7 bar
Emitter protrusion	0.2–2 mm
Solvent composition	95:5 methanol water
Voltage	2–5 kV
MS scan time	1 s

2.

To determine if the system is operational and to obtain suitable starting point for the optimization using tissue samples, draw a large rectangle on a glass slide with a red Sharpie® that contains Rhodamine 6G dye (m/z 443) for positive mode analysis or black permanent marker (m/z 666) for negative mode. Following this preliminary procedure using the ink (see Note 6 ), the system needs to be further optimized using a tissue section, ideally one of the same origin as those intended for the study. Take the glass slide with the tissue section mounted from the −80 °C degrees freezer. Let it defrost at room temperature. No other sample preparation is required. Place the sample onto the DESI stage (see Note 7 ).

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