High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue.

An atmospheric pressure laser desorption/ionization mass spectrometry imaging ion source has been developed that combines high spatial resolution and high mass resolution for the in situ analysis of biological tissue. The system is based on an infrared laser system working at 2.94 to 3.10 µm wavelength, employing a Nd:YAG laser-pumped optical parametrical oscillator. A Raman-shifted Nd:YAG laser system was also tested as an alternative irradiation source. A dedicated optical setup was used to focus the laser beam, coaxially with the ion optical axis and normal to the sample surface, to a spot size of 30 µm in diameter. No additional matrix was needed for laser desorption/ionization. A cooling stage was developed to reduce evaporation of physiological cell water. Ions were formed under atmospheric pressure and transferred by an extended heated capillary into the atmospheric pressure inlet of an orbital trapping mass spectrometer. Various phospholipid compounds were detected, identified, and imaged at a pixel resolution of up to 25 µm from mouse brain tissue sections. Mass accuracies of better than 2 ppm and a mass resolution of 30,000 at m/z = 400 were achieved for these measurements.

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer.

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.

Time-resolved imaging of the plume dynamics in infrared matrix-assisted laser desorption/ionization with a glycerol matrix.

The dynamics of the expanding material plume after irradiation of a matrix sample with two different infrared (IR) lasers, an Er:YAG laser of ca. 100 ns and an optical parametric oscillator (OPO) laser system of 6 ns pulse duration, were investigated by imaging the plumes with nanosecond time resolution. Both lasers emitted at an identical wavelength of 2.94 microm. Laser exposure parameters were typical for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS); glycerol was employed as a liquid matrix to provide a homogeneous sample and reproducible plume formation. A Nd:YAG laser (532 nm; 8 ns) was used as the illumination source and a CMOS camera with a ten-bit dynamic range served for recording of the images. Dark-field as well as scattered light illumination was employed to preferentially image the gaseous and particulate components of the plume, respectively. During the initial phase of its expansion (ca. 1 micros) the plume appears to consist of a continuous cloud of material of varying density. At later times after exposure, individual particles of several micrometers in size dominate the images. For both laser pulse durations material ejection was observed for times as long as 100 micros postexposure. Subtle but distinct differences in the plume dynamics are observed for the two different pulse durations. They are related to a transition between the regimes below and above acoustic confinement. The experimental findings are compared to results obtained in two previous studies by photoacoustic analysis of the desorption process and IR-laser postionization of the plume.

Infrared laser post-ionization of large biomolecules from an IR-MALD(I) plume.

A two-infrared laser desorption/ionization method is described. A first laser, which was either an Er:YAG laser or an optical parametric oscillator (OPO), served for ablation/vaporization of small volumes of analyte/matrix sample at fluences below the ion detection threshold for direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A second IR-laser, whose beam intersected the expanding ablation plume at a variable distance and time delay, was used to generate biomolecular ions out of the matrix-assisted laser desorption (MALD) plume. Either one of the two above lasers or an Er:YSGG laser was used for post-ionization. Glycerol was used as IR-MALDI matrix, and mass spectra of peptides, proteins, as well as nucleic acids, some of which in excess of 10(5) u in molecular weight, were recorded with a time-of-flight mass spectrometer. A mass spectrum of cytochrome c from a water ice matrix is also presented. The MALD plume expansion was investigated by varying the position of the post-ionization laser beam above the glycerol sample surface and its delay time relative to the desorption laser. Comparison between the OPO (pulse duration, tau(L) = 6 ns) and the Er:YAG laser (tau(L) approximately 120 ns) as primary excitation laser demonstrates a significant effect of the laser pulse duration on the MALD process.