The effect of laser profile, fluence, and spot size on sensitivity in orthogonal‐injection matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

The influence of incident laser parameters on sensitivity in matrix-assisted laser desorption/ionization (MALDI) has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N(2) laser and a Nd:YAG laser using 2-m long optical fibers. The N(2) laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N(2) laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. Thus, the optimum sensitivity is achieved at high fluence. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. The results have particular relevance for imaging MALDI, where sensitivity and spatial resolution are important figures of merit.

Probing the Relationship Between Detected Ion Intensity, Laser Fluence, and Beam Profile in Thin Film and Tissue in MALDI MSI

“…The I versus F curves shown within Figure 4 all have the similar general trends to those observed from peptides within the literature [2,7,9] whereby the low fluence data do not feature any detected ions, then as the fluence is increased past threshold, a steep rise in detected ion intensity is observed. For the single ion data, the curves then plateau soon after this initial increase and may even decrease again as fluence increases, whereas the TIC curves typically continue to increase to the maximum fluence, though the rate of increase is seen to drop off at the higher fluences, particularly for the 50 μm round fiber data (SI Figure S5).…”

“…A threshold fluence of ion detection is typically reported as being between~20 and 1700 J/m 2 . The threshold energy and the effect of fluence on the detected ion intensity are also observed to depend upon the size of the irradiated area [1][2][3]. Beyond threshold energy, the rapid conversion of photons into heat leads to the creation of sub-surface nucleation, pressure waves, and subsequent molecule cluster and particle ejection [4,5].…”

“…The effect of fluence on the detected ion intensity has been investigated by a number of groups for peptide and protein standards, e.g., substance P, with a range of matrices, e.g., 2,5-dihydroxybenzoic acid (2,5-DHB) or α-cyano-hydroxycin namic acid (CHCA) [2,3,7,9]. There have not, however, been any similar investigations employing other analyte classes, such as lipids or drugs.…”

“…This is not solely because it describes the area of the irradiated region but because the distribution of photons within the irradiated area will also influence the desorption and ablation process. The Ens group demonstrated that the different photon distribution, resulting from nitrogen and Nd:YAG lasers, give rise to different ion yields for substance P with CHCA [2]. This concept of some photon distributions being more optimal than others was then built upon by Holle et al in the creation and study of 'structured' laser beam profiles, demonstrating that lower total energy could be used where a more optimal photon distribution is employed, to give rise to equivalent quality data [13].…”

Direct Laser Desorption Ionization of Endogenous and Exogenous Compounds from Insect Cuticles: Practical and Methodologic Aspects

“…In experiments where the focal laser spot was reduced to a diameter of 100 μm by utilizing a second optical fiber with smaller core diameter, no noticeable loss of sensitivity was observed (data not shown). We assume that a spatial resolution in the low 10 μm range could be achieved if further modifications were made (e.g., by employing fibers with core diameters in the 10 μm-range [16]). This could allow spatiallyresolved probing of fine features of the insect body like glands.…”