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The sensitivity of a transient imaging technique based on laser-excited fluorescence is shown to be within the levels required for studying matrix-assisted laser-desorption (MALD) of large biomolecules. Results show that film morphology, particularly film thickness, has a major influence on plume dynamics. Fluorescent labeling of protein and of DNA is used to allow imaging in UV-MALD-generated plumes. Evidence that intact molecules are monitored is provided. Dye molecules, proteins and DNAs were found to behave very differently in the vaporization process. The spatial and temporal distributions are important for the design of secondary ionization schemes to enhance ion yields, for the optimization of ion collection, and for maximizing resolution in time-of-flight mass spectrometers.

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In the analysis of large biomolecules, one important aspect is the determination of molecular weight. An excellent tool for determining molecular weights is mass spectrometry (MS).One requirement for MS analysis is that the sample either be itselfin the gas phase or can be converted to the gas phase. However, biomolecules, by nature large and typically containing both polar and nonpolar regions, are not easily converted to the vapor phase. When vaporization does occur, a common problem for molecular weight determination is that the biomolecules fall apart into fragments. Several approaches that have been effective in producing intact biomolecules in the gas phase1 include field desorption,Z 252-Cf plasma desorption,3 SIMS,4 thermo-5 and electrospray,6 and laser desorption (LD).'LD of intact molecules requires that the laser pulse be short. In other words, the heating rate of the sample must be rapid enough to favor vaporization over fragmentation.The energy from fast pulsed lasers has been particularly effective in this regard. However, even with the use of a 10-ns pulse from a frequency-doubled Nd-YAG laser, significant sample destruction has been observed.8 A recent innovation9 is matrix-assisted LD (MALD). This approach involves combining a large excess of a matrix material with the sample species. Typical matrices are fairly volatile or sublime easily and are chosen to have significant absorption at the irradiation wavelength. An overview of the technique1 has recently been published. Current understanding of the process10 is that direct absorption by the sample molecules is minimal-rather, they are swept along with the vaporized matrix material. It is also possible that expansive cooling by the matrix minimizes fragmentation." The detailed dynamics of the process are not well understood, however.One interesting feature of MALD is that efficient generation of parent ions depends critically on the laser energy and the matrix-to-sample ratios1 Conditions that fall outside a very (1) Hillenkamp, F.; Karaa, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. (2) Beckey, H. D.narrow range have been found to be ineffective. A complication that exists in interpreting these MS results is that vaporization and ionization are not separately studied.Conditions that favor one process may not be identical to those required for the other. For example, fragmentation can occur as a result of additional excitation during the ionization step. There has been some indication that the bulk of the vaporized species is in the neutral form.12 If so, secondary ionization after vaporization, appropriately implemented, can conceivably increase the yield of the parent ions.Optical spectroscopic probes based on lasers have been successfully employed for monitoring laser-generated plumes previously, providing spatial and temporal information on atoms,13 diatomics,14 and large molecules.* These are based on absorption in the plume,8J3J4 reflection off the surface,lb and laser-enhanced ionization.16 The distinctive feature is that these probes can ...