Scanning Microprobe Matrix-Assisted Laser Desorption ... · time-of-flight mass spectrometry or, after preparation with a suitable matrix, by matrix-assisted laser desorption ionization

Scanning Microprobe Matrix-AssistedLaser Desorption Ionization (SMALDI)Mass Spectrometry: Instrumentation forSub-Micrometer Resolved LDI and MALDISurface Analysis

Bernhard Spengler and Martin HubertInstitute of Inorganic and Analytical Chemistry, Justus-Liebig University Giessen, Giessen, Germany

A new instrument and method is described for laterally resolved mass spectrometric surfaceanalysis. Fields of application are in both the life sciences and the material sciences. Theinstrument provides for imaging of the distribution of selected sample components fromnatural and artificial surfaces. Samples are either analyzed by laser desorption ionization (LDI)time-of-flight mass spectrometry or, after preparation with a suitable matrix, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Areas of 100 � 100 �m arescanned with minimal increments of 0.25 �m, and between 10,000 and 160,000 mass spectra areacquired per image within 3 to 50 min (scan rate up to 50 pixels per s). The effective lateralresolution is in the range of 0.6 to 1.5 �m depending on sample properties, preparationmethods and laser wavelength. Optical investigation of the same sample area by UV confocalscanning laser microscopy was found to be very attractive in combination with scanningMALDI mass analysis because pixel-identical images can be created with both techniquesproviding for a strong increase in analytical information. This article describes the method andinstrumentation, including first applicational examples in elemental analysis, imaging of pinetree roots, and investigation of MALDI sample morphology in biomolecular analysis. (J AmSoc Mass Spectrom 2002, 13, 735–748) © 2002 American Society for Mass Spectrometry

Laser microprobe mass spectrometry was devel-oped more than 25 years ago as a method forlocalized chemical analysis of biological samples

or of, e.g., semiconductor surfaces [1, 2]. Several limita-tions (especially in sensitivity, mass range, and isotopediscrimination) of other microprobing techniqueswhich were well established at that time, such aselectron probe X-ray microanalysis, could be overcomeusing lasers for vaporization and ionization of micro-meter areas. Instruments were developed for transmis-sion mode analysis of thin sections and for reflectionmode analysis of bulk surfaces. Because of its attractivefeatures, the method found broad application over theyears. The various fields of research assisted by lasermicroprobing have been reviewed in several papers[3–6].

The main drawback of the classical laser microprobemass analysis (LAMMA) technique is its restriction toelemental analysis. Only few and specialized applica-tions were reported for molecular analysis of biological

samples with this technique [6–9]. The reason for thislimitation is the same as for the limited applicability of(non-highly focused) laser desorption ionization (LDI)in general. Before matrix-assisted laser desorption ion-ization (MALDI) was developed [10], analysis of or-ganic and inorganic material was possible by LDI, butanalytical success was strongly dependent on the indi-vidual sample parameters, such as spectral absorbance,volatility or ionization potential [11, 12]. Addition of amatrix to a sample of interest provided for a rathercommon behavior of almost any type of sample withrespect to ionizability, analytical sensitivity, molecularstability etc. [13, 14]. MALDI has nowadays become theionization method of choice for solid-state related bio-analytical mass spectrometry [15] while electrosprayionization (ESI) has reached a comparable level ofimportance for liquid-phase related mass analysis [16].The overall interest in MALDI is still growing in allfields where analytical information is coupled to surfaceimmobilization or surface separation techniques, espe-cially when structural information can be acquired fromsample surfaces by postsource decay (PSD) analysis[17].

During the recent years, another aspect of analyticalinformation came into focus in MALDI mass spectro-

Published online April 23, 2002Address reprint requests to Dr. B. Spengler, Institute of Inorganic andAnalytical Chemistry, Justus-Liebig University Giessen, Schubertstr. 60,D-35392 Giessen, Germany. E-mail: [email protected]

© 2002 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received August 16, 20011044-0305/02/$20.00 Revised February 14, 2002PII S1044-0305(02)00376-8 Accepted February 14, 2002

metry, which is the lateral or positional information, asis examined with the LAMMA technique for elementalanalysis. Highly sensitive investigation of single cells,neurons, or tissue areas is a field of dramatically grow-ing consideration [18–24]. Due to instrumental andmethodological limitations, MALDI imaging has so farbeen limited to a lateral resolution of approximately30 �m [18, 25–30], limiting its application to relativelylarge structures.

Secondary ion mass spectrometry (SIMS) is anotheranalytical technique capable of imaging inorganic ororganic matter. Both SIMS and MALDI imaging havebeen reviewed recently in reference [31]. Among themajor differences between these techniques are theaccessible mass range and the analytical depth. WhileSIMS provides analytical information on elements andsmall organic molecules, MALDI allows identificationof large biomolecules such as proteins. In the so-calledstatic SIMS mode [32], analytical information is ob-tained from the first monolayer only. Static SIMS istherefore known as a real surface analytical technique.MALDI and laser desorption in general, on the otherhand, are techniques which extract analytical informa-tion from a greater depth with each laser shot. Depend-ing on the focus diameter used, the ablation depth isbetween nanometers and micrometers [3, 4]. Spatialresolution in static SIMS can be as high as 100 nm to1 �m, but often has to be reduced to values in the rangeof 10 �m in order to increase the number of secondaryions accessible.

Scanning near field optical microscopy (SNOM) is awell-known method to increase lateral resolution abovethe classical optical limit of diffraction. Optical resolu-tion in the nanometer range is achievable with thismethod. Efforts to use SNOM for LDI mass spectro-metry were reported recently, but without success sofar in detecting larger molecular ions [33].

Combining LAMMA and MALDI appears to be anobvious and very promising goal. Its realization, how-ever, is not straightforward. First, the lateral resolutionnecessary for most biological applications has to beconsiderably higher than originally achieved with the(reflection mode) LAMMA 1000 instrument (which wasin the range of 10 �m). Second, application of the matrixto a sample of interest is not as straightforward as forgeneral MALDI mass analysis. MALDI preparation isknown to depend on formation of a homogeneousliquid-phase mixture of matrix and analyte before crys-tallization. For laterally resolved analysis of biologicalspecimen, on the other hand, it is crucial to avoidredistribution or migration of the substances of interestwithin the sample, meaning that one should not inducea laterally extended liquid-phase mixing of matrix andanalytes. Migration-free matrix preparation has so farbeen described only for lateral resolutions in the rangeof 30 �m [31] and not for lateral resolutions of 1 �m.Solving the problems and limitations of matrix applica-tion will probably become the major tasks for thecoming years in high-resolution scanning microprobe

MALDI mass spectrometry. The current status and firstachievements in this field will be described in a follow-ing publication. In this article the instrumental param-eters are described and first experimental resultsachieved with our dedicated instrument are presented.

Experimental

A scheme of the instrument is shown in Figure 1. Theimaging time-of-flight mass spectrometer is oriented inthe vertical direction, providing for a horizontal planefor easy sample mounting.

The vacuum chamber is pumped by two 360 l/sturbomolecular pumps (Leybold AG, Cologne, Ger-many) which are mounted horizontally at the back ofthe sample chamber and in the middle of the flight tube,respectively. The forevacuum is provided by a 16 m3/hoil rotary pump (Leybold AG) which is located in adistant place with respect to the microscopical part ofthe instrument to avoid degradation of lateral resolu-tion by vibrations. The working pressure of the instru-ment is about 5*10�7 mbar.

UV Lasers

Two different lasers can be used alternatively, both fordesorption ionization and for confocal scanning lasermicroscopy, a frequency quadrupled Nd:YLF laser with262 nm wavelength for optimized focusing conditionsand highest lateral resolution, and an N2 laser with 337nm wavelength for optimized MALDI conditions.

Figure 1. Scheme of the scanning LDI and MALDI reflectrontime-of-flight mass spectrometer.

736 SPENGLER AND HUBERT J Am Soc Mass Spectrom 2002, 13, 735–748

Nd:YLF laser. The pulsed Nd:YLF laser model 421 QD(ADLAS, Luebeck, Germany,) is pumped by laser di-odes. Laser light is frequency-doubled internally toreach output energies of 100 �J per 15 ns pulse at 524nm. Frequency quadrupling is performed externally bya temperature-controlled BBO crystal (barium beta bo-rate crystal for non-linear second harmonic generation).The final pulse energy is about 15 �J at 262 nm. Amongthe advantages of this diode pumped system are itshigh maximum repetition rate of 20 kHz (8 kHz atuseful UV pulse energies), its low performance varia-tions under change of repetition rates across the com-plete range of single shot to 8 kHz, and its smallphysical dimensions (360 � 90 mm laser head). Theoutput power of the laser can be controlled manually orby computer by adjusting the diode pump energy.

N2 laser. The nitrogen laser is a standard VSL 337 NDlaser (Laser Science Inc., Cambridge, MA) as used inmany commercial MALDI mass spectrometers. It pro-vides for maximum pulse repetition rates of 20 Hz atpulse durations of 3 ns. The output energy at 337 nm is300 �J per pulse.

Laser Focusing

Sub-micrometer focusing of the laser beam consists of aprefocusing part outside the vacuum and a final focus-ing part in the vacuum right above the sample manip-ulator. Both possible lasers are prefocused to about 10�m focus diameter by suprasil quartz lenses. While theprefocused beam profile of the N2 laser is almostcircular, the beam of the Nd:YLF laser is stronglyelliptical in shape after quadrupling by the BBO crystal.A special optical correction is required in this case, inorder to provide for a high numerical aperture at theentrance of the focusing objective lens. The ratio of themajor and minor diameters of the ellipse is about 1:8.The beam, after prefocusing with a spherical lens,would thus fill a small stripe only on the entrance lensof the focusing objective. For this reason a specialoptical unit was developed for circularization, whichprefocuses the two axes of the beam differently, asdemonstrated in Figure 2.

The x axis in Figure 2 (the minor axis) is focused bya short focal length ( f � 5 mm) cylindrical lens, whilethe y axis (the major axis) is focused by a long focallength ( f � 40 mm) cylindrical lens. Both one-dimen-sional foci are adjusted to the same focal plane. Thestrongly elliptical beam profile is transformed into acircular beam profile by this procedure. The cylindricallenses were bought from Melles Griot, Rochester, NY.

Final focusing is performed by a dedicated objectivelens specially designed and manufactured for thissetup. It consists of 5 suprasil quartz lenses (diametersbetween 35 and 50 mm) for minimized spot size at 262nm for axial laser beams. The objective has an o.d. of 90mm and has a numerical aperture (N.A.) of 0.6 at a freeworking distance of 16 mm. The long free working

distance is necessary for the mass spectrometrical setupto allow for placement of ion lens elements.

In order to allow ions to leave the ion source in adirection normal to the sample surface, the objectivelens has a central hole which is fitted with a stainlesssteel tube of 6 mm inner diameter.

The theoretical limit of lateral resolution is [34]

d � 1.22 ��

A(1)

where d is the diameter of the central disc of thediffraction pattern (� the “focus diameter”), � is thewavelength and A is the numerical aperture of the lens.With our setup at 262 nm laser wavelength and anumerical aperture of 0.6, the minimal achievable laserfocus diameter would thus be 0.53 �m. The massspectrometric lateral resolution, i.e., the diameter of asurface disc from which analytical information is ex-tracted, should be identical or very similar to the focusdiameter. Due to the strong non-linearity of the desorp-tion process, however, the practical lateral resolutioncan be higher or considerably lower than the focusdiameter, depending on the laser irradiance used andassuming a near-gaussian intensity profile [1, 3].

The practical optical lateral resolution can be higherthan described by eq 1, taking into account that struc-tures can still be optically resolved even if the diffrac-tion discs of two objects overlap to a certain extent. Thiseffect is usually described by the physiological factork � 1 [34], so that

d � 1.22 ��

k � A(2)

where d now is the smallest size of resolvable objects.The optical quality of the lens has been tested on an

optical bench outside the vacuum. Figure 3 shows theintensity distribution in the focus of the lens at 266 nm

Figure 2. Cylindrical lens system for prefocusing and circular-ization of the elliptical beam of the frequency-quadrupled Nd:YLFlaser.

737J Am Soc Mass Spectrom 2002, 13, 735–748 SCANNING MICROPROBE MALDI-MS

as measured by a diode array detector after 100�magnification by a microscopic objective lens (Leitzultrafluar 100, N.A. 0.85 in air). The diodes are 15 �mapart. The measured focus diameter at 262 nm (�diameter of the first minimum of the diffraction pattern)is thus 0.45 �m �/� 0.15 �m.

The optical setup for 337 nm, using the nitrogenlaser, was tested by microscopic investigation of burnpatterns of the focused laser beam on thin films of dyecoated by a felt tip pen onto a standard aluminumsample holder. Burn patterns were created under vac-uum conditions and under mass spectrometrical con-trol. At the irradiance threshold for detection of ions, noburn patterns could be observed optically. At twofoldto threefold threshold irradiance, circular holes of 0.6 to0.7 �m diameter could be observed after single-shotlaser desorption with a high-quality optical microscope(Olympus BX 40), as shown in Figure 4. As expectedfrom the near-gaussian intensity profile, the diameter ofthe burn holes increased markedly with increasingirradiance. It can therefore be assumed that at thresholdirradiance the analytical diameter of the desorptionregion is even lower than 0.6 �m with this setup at 337nm.

Sample Scanning

Since high optical resolution can be achieved only for anaxial beam path with the chosen setup, a fixed opticalsystem is mandatory. Sample scanning can thus not beperformed the usual way by scanning the laser beamacross the sample, but has to be performed by movingthe sample under the laser focus. This is done by a fastpiezo-driven nanostage (Graf Mikrotechnik, Wertingen,Germany), which is mounted on a stepper-motordriven x-y-z-stage for sample positioning. With thisarrangement samples can be positioned within an area of

20 � 20 � 20 mm, and areas of 100 � 100 �m can bescanned with a maximum resolution of 0.25 �m. In thehigh-speed mode of the piezo stage the sample load islimited to a maximum mass of 2 g, allowing us to scanthe 160,000 pixels of the target area within less than 2 s.The piezo stage is controlled by a Motorola 68000(Motorola Inc., Schaumburg, IL) microprocessor sys-tem. The stepper motors are located inside the vacuumand are controlled by a stepper motor controller via ajoystick or computer.

Confocal Microscopy

The same laser and beam path as for desorption ioniza-tion is used with decreased pulse intensities for optical,confocal laser scanning microscopy (CLSM). Pulsed UVlight at 262 nm is focused onto a certain pixel of thesample under investigation (see Figure 1). Reflectedlight from the sample pixel takes the identical beampath back through the lens and partly passes the 50%transmission beam splitter. At the same position as theprefocus of the incoming laser beam, on the one hand,and at a corresponding position behind the beam split-ter on the other hand, the reflected light from thesurface is focused. An aperture 10 �m in diameter islocated at this position to allow reflected light to reacha photomultiplier tube (PMT) behind the aperture. Anylight from sources other than the focused sample pixelis unable to pass the aperture with significant intensityand is thus not detected. The photomultiplier signal isprocessed by a box car averager and acquired by a200 kHz 12 bit A/D converter. The box car averagerintegrates the light intensity within a predefined timewindow. The actual position of the scanning stage isdetermined by a noncontacting displacement measur-ing system KDM-8200 (Kaman Instrumentation Corpo-ration, Colorado Springs, CO), which is used for feed-back control of the piezo system. The sensor signal is

Figure 3. Intensity distribution of the focused Nd:YLF laserbeam (frequency quadrupled to 262 nm wavelength) after 100�magnification as measured by a diode array with diode spacingsof 15 �m. Each data point corresponds to the light intensitymeasured by a single diode. The determined focus diameter is 0.45�m �/� 0.15 �m.

Figure 4. Laser burn patterns after single-shot irradiation attwofold to threefold threshold irrandiance, acquired at 337 nmnitrogen-laser wavelength on red felt tip dye.


acquired by a second channel of the A/D converter. Theintensity value is correlated with the pixel position andis used by the computer to form an optical two-dimensional b/w image of the sample. A completeimage of 100 � 100 �m acquired with a pixel resolutionof 0.25 �m (160,000 pixels) is formed on the computerscreen within 30 seconds.

Video Imaging

For overview observation the sample can be imaged bystandard light microscopy with a magnification ofabout 400. An area of about 500 � 400 �m is displayedon a video monitor. The sample is illuminated andobserved not from the side but through the objectivelens because of geometrical constraints. Since the lens isnot corrected for chromatic abberation, only monochro-matic light can be used for sample imaging. A He-Nelaser with an output energy of 15 mW is used forsample illumination. Coherent light is usually unusablefor illumination of surfaces having topological modula-tions in the range of the laser wavelength because of theformation of speckle interferences, considerably deteri-orating the visibility. The He-Ne laser beam can thusnot be used directly for microscopic illumination buthas to be processed first. This is done by feeding thebeam into a quartz fiber of 3 m length and shaking thefiber by a vibrating motor. This procedure leads tosufficient time-dependent distorsion of the mode struc-ture of the beam for a homogeneously averaged lightintensity distribution to be obtained on the sample.

Due to the longer wavelength of the He-Ne laser(633 nm) compared to the UV laser (262 nm or 337 nm,resp.) the focal length of the objective lens is consider-ably longer for the video imaging beam path. A correc-tion lens is thus used leading to a final distance betweenobjective lens and video camera of 800 mm.

The video image is observed on a b/w monitor ordisplayed on the computer screen by a video overlaycard, allowing acquisition and storage of video imageson the computer hard disk.

Mass Analysis

Ions formed by the desorption/ionization laser areaccelerated to 10 keV and collimated by electrostaticelements located in front of the objective lens. The ionbeam is steered through the stainless steel tube in thecenter of the lens by adjusting the potentials of theelements.

In the linear mode, ions pass the field free drift tubeand hit the microchannel plate detector at the top end ofthe instrument. The instrument is equipped with an ionreflector for flight time compensation of ions havingkinetic energy deviations from the ion source. In thereflectron mode ions are decelerated and reflected co-axially in a two-stage gridded reflector.

Ion Detection

Reflected ions are postaccelerated onto an annularcopper-beryllium conversion plate at �7 kV. Secondaryelectrons desorbed from the conversion surface areaccelerated to 7 keV and are deflected 90° by a magneticfield. After postacceleration to 9 keV they hit a plasticscintillator plate which is mounted on a vacuum win-dow. Outside the vacuum a photomultiplier tube de-tects the converted ion signal. This arrangement avoidshigh-cost annular channelplate detectors and takes ad-vantage of the stability of maintenance-free photomul-tipliers. Instead of the scintillator/photomultipliersetup, a microchannel plate detector was used alterna-tively with comparable performance for detection ofsecondary electrons after ion conversion.

Data Acquisition

The ion signal from the photomultiplier is split and fedinto a dual channel preamplifier connected to a dualchannel transient recorder card model PAD82 (Spec-trum Systementwicklung GmbH, Siek, Germany). Thesignal is digitized using two channels of 8 bit dynamicrange at different sensitivity settings. From the twochannels a combined spectrum with enhanced dynamicrange is composed by the data acquisition program. Foreach data point a virtual 16 bit memory area is filled byeither one of the two channels depending on the signalintensity at that data point. The system can be operatedalternatively with only 8 bit dynamic range by usingjust one channel.

Mass spectra can be processed and stored individu-ally, as in regular laser desorption mass spectrometry,or can be used to calculate two-dimensional ion imagesby transforming signal intensities of certain mass peaksinto gray-scale pixel values. Depending on the memorydepth of the acquired spectra (i.e., the acquired massrange) more than 50 spectra per s can be processed witha standard computer. This leads to acquisition times inthe range of 4 to 15 min for 100 � 100 �m ion images at1 �m pixel resolution. For each of the processed masspeaks an image file is stored to disk in TIF file format.These image files can be handled later by commonimage processing programs. Home-built software(ULISSES version 8.0, Bernhard Spengler copyright(1985–2002) was employed for all operations of instru-ment control, data acquisition and data evaluation.

Results and Discussion

The general system capabilities are demonstrated in thefollowing.

Confocal Microscopy

Figure 5 is a confocal image of a 100 � 100 �m area ofa lithographical object commonly used for testing elec-tron microscopes (PLANO W. Plannet GmbH, Mar-


burg, Germany). The sample is a 4.5 � 4.5 mm siliconwafer, covered with a 0.5 �m thick layer of SiO2 and a0.5 �m thick layer of aluminum on top. Structures arepatterned onto the chip by electron beam direct writingand dry etching. The image shows an area of 100 �100 �m of this sample scanned with steps of 0.25 �m at262 nm wavelength of the Nd:YLF laser. The areacontains elevated letters of 0.5 �m aluminum within anetched area of silicon. The lower part of the imageshows the rim of the non-etched (elevated) aluminumsurface. A strong surface contrast (shadow formation)and almost no absorption contrast is obtained. This isdue to the fact that the spectral absorption is similar foraluminum and silicon in UV light. Under visible lightobservation, on the other hand, aluminum areas appearwhite, while silicon areas appear almost black. Shadowformation was enhanced in Figure 5 by aligning thebeam path for optical confocal microscopy slightlyoff-axis. The (virtual) light source in this image is in thelower left corner.

The confocal image of Figure 5 was acquired with arather large photomultiplier aperture of 30 �m. Smallerapertures down to 10 �m have been used as well,enhancing the optical resolution in x, y, and z direction.With our current data system, however, the small-aperture images are more complicated to evaluate,because of the extremely low focal depth of less than

1 �m under these conditions. Since all off-focus areasappear black in confocal microscopy, a complete imageof a sample with surface topologies above the focaldepth can only be acquired by averaging (or three-dimensional processing) multiple scans at differentfocal positions. Three-dimensional processing has notbeen implemented in our data system yet. Using a30 �m aperture deteriorates lateral resolution to about2 �m but allows us to acquire realistical images withjust a single scan.

Ion Images

Figure 6a shows a confocal image acquired at 262 nm ofanother area of the test sample characterized by fouretched lines, a mechanically scratched area in the rightpart and a dark spot in the center. The aperture usedhad a diameter of 30 �m. As in Figure 5 the opticalbeam path was aligned off-axis resulting in a virtuallight source in the lower left corner. The small lines inFigure 6a are thus not elevations (non-etched alumi-num) but indentations (silicon substrate).

The corresponding pixel-identical ion image of thesame area is shown in Figure 6b for the m/z � 28 usignal of silicon. The image has been scanned with 0.5�m per pixel. It indicates a mean line width of 2 pixels(1 �m) which is in good agreement with the controlvalues given for the test object. In the area of the darkspot an irregular distribution of silicon is observed.

Surface Destruction

Laser desorption ionization is by nature a destructivetechnique. After laser irradiation and ion formation thesurface is deformed by ablation of a volume of about1 �m3 per pixel per laser pulse. This value of ablatedvolume has not been investigated systematically so farbut is approximated from earlier investigations on lasermicroprobe mass analysis, laser desorption ionization,and MALDI mass analysis [3, 11, 35]. In ordinaryMALDI or LDI mass spectrometry employing spot sizesin the range of 50 to 200 �m in diameter, this ablatedvolume is spread over a large desorption area leading to(unobservable) ablation depths in the low nanometerrange. In laser microprobing, however, the same abla-tion volume leads to ablation crater depths in themicrometer range. Since the confocal optical mode isvery sensitive with respect to surface topology, theablation effect can be visualized by confocal opticalimaging of the sample area before and after massspectrometric scanning. Optical scanning itself is, dueto the lower irradiance, non-destructive and can beperformed many times without causing any visiblesurface effect. Figure 7a and b show the same area of thetest sample before and after MS (high-irradiance) scan-ning at 262 nm. The surface is strongly affected byformation of desorption craters. Figure 7c shows thescanned area slightly shifted in order to demonstratethe effect at the edges of the scanned area.

Figure 5. UV confocal laser scanning microscopy (UV-CLSM)reflected light image of a lithographical test object acquired at 262nm laser wavelength. A large pinhole aperture of 30 �m indiameter was used in front of the photomultiplier in order toincrease the focal depth and thus reduce vertical (three-dimen-sional) discrimination.


As mentioned above, confocal optical imaging isvery sensitive to surface topology. Imaging of surface

modifications after MS scanning is therefore enhancedby confocal imaging. The destruction of analytical infor-mation by desorption scanning is much less pro-nounced, as demonstrated later in Figure 11. Severalimages from the same area can be obtained with onlyslight modification of the signal.

Surface Analysis

Imaging of the surface distribution of different sub-stances with high lateral resolution is demonstrated inFigure 8. The sample was prepared by depositing thinlayers of dye with two felt tip pens onto a flat alumi-num target. The target was first covered completelywith dye from the red felt tip pen. After drying, thecoating was partially overlayed with dye from thegreen felt tip pen. The edge of the green overlay wasimaged mass spectrometrically with 262 nm desorptionwavelength using the signals of m/z � 122 u, indicativeof the red dye, and m/z � 372 u, indicative of the greendye. An area of 100 � 100 �m was scanned with a pixel(step) size of 0.5 �m. The resulting two grey scaleimages for the red and the green dye were transformedinto one composite image by using the red channel ofan RGB color image for the red-dye image and thegreen channel for the green-dye image. The blue chan-nel was left empty. The resulting color image in Figure8 shows pure red intensities for all pixels, where onlythe signal of the red dye was observed (m/z � 122 u),and pure green intensities for all pixels, where only thesignal of the green dye was observed (m/z � 372 u).Only few pixels are colored in yellow, indicating thations from red and green dye were detected. Thisindicates that with the first scan of a multi-layer sampleonly the top layer is imaged (and desorbed), while thedeeper layers are still covered.

The image shown in Figure 8 demonstrates thatsurface analysis with effective lateral resolution in the0.5 �m range is possible with our setup, indicated bycrosstalk-free, sharp edges between red and green ar-eas.

The image was acquired with two laser irradiancesettings to demonstrate the influence of irradiance onimage characteristics. As can be seen the noise level ishigher at lower irradiance, but more information onsurface topology can be extracted from signal variationsin images acquired at lower irradiance.

Biological Samples

Figure 9 shows mass spectrometric images of a pine treeroot. The sample was provided by W. H. Schroeder atthe Forschungszentrum Juelich, Germany, and wasprepared by embedding the root in an epoxy resin. Aflat surface of the bulk sample was created by means ofa glass knife and investigated by MS scanning. Ionsignals of potassium and calcium were imaged (white �high, black � low intensity) in a cross section of theroot. The size of the total image is 100 � 100 �m. The

Figure 6. (a) UV confocal laser scanning microscopy (UV-CLSM)image of a lithographical test sample acquired at 262 nm laserwavelength and a pinhole aperture diameter of 10 �m. The sampleis a silicon wafer covered with 0.5 �m SiO2 and 0.5 �m Al. Theimaged area is 100 � 100 �m. The illuminating laser beam path isset slightly off-axis in order to enhance the surface (topological)contrast, resulting in a virtual light source positioned in the lowerleft corner of the image. (b) Scanning laser desorption ionizationmass spectrometrical image of the same area as imaged in (a) andacquired at 262 nm laser desorption wavelength. The mass signalof silicon (m � 28 u) was used for imaging. White pixels corre-spond to high signal intensities of silicon ions, dark pixels corre-spond to low signal intensities.


images were scanned with lateral increments of0.25 �m. Structures of the cell walls are clearly imagedand show the expected high concentration of potassiumand calcium in these areas [4].

SMALDI Mass Analysis of Peptide Samples

As a demonstration of Scanning Microprobe MALDIimaging on the micrometer scale (SMALDI mass spec-trometry), Figure 10 shows the distributions of thepeptide substance P ([M � H]� � 1348 u), of the matrix2,5-dihydroxybenzoic acid (DHB, m/z � 154 u used asmarker signal) and of potassium (m/z � 39 u) in aregular MALDI dried droplet preparation of an aque-ous solution of these compounds. The matrix wasprepared in high concentration (6*10�2 molar) while theanalyte (substance P) was added in 10�5 molar concen-tration. Potassium was present as contaminant only.The sample is typical of standard preparations inMALDI mass spectrometry of peptides in biochemicalanalysis. It was investigated by laser microprobing toclarify preparational mechanisms, which are known tobe important for successful analysis of biological mac-romolecules. The images were acquired from the rim ofthe dried droplet, known to be characterized by forma-tion of large matrix crystals in this preparation method[14]. The top left of the images is part of the flat innerarea of the dried droplet, formed by very small matrix

Figure 7. (a) UV-CLSM image of a selected area of the litho-graphical test sample acquired at 262 nm laser wavelength prior toLDI-MS imaging. The illuminating laser beam path is set slightlyoff-axis in order to enhance the surface (topological) contrast. (b)UV-CLSM image of the same area as in (a) after LDI-MS imaging.Ablation of material strongly distorts the surface topology result-ing in visible effects in the surface contrast image. (c) UV-CLSMimage of an area shifted versus the position used in (a) and (b)after LDI-MS imaging.

Figure 8. Scanning laser desorption ionization mass spectromet-ric image of an aluminum target covered with red felt tip pen dyeand partly overlayed with green felt tip pen dye. The image wasobserved at 262 nm laser wavelength with a scanning step size of0.5 �m and one laser shot per pixel. Mass signal intensities fromthe red dye (m/z � 122 u) are coded in red, those from the greendye (m/z � 372 u) are coded in green. Yellow pixels indicatedetection of ions from both dyes in the same spectrum. The totalscanned area is 100 � 100 �m (top image). The size of themagnified section is 10 � 10 �m (bottom image).


crystals. An overview of the sample is shown in themicroscopical image acquired with lower magnifica-tion. The inclusion of analyte molecules (substance P inthis case) into the growing crystals during the dryingprocess of the droplet, as well as the exclusion of alkali

ions has been discussed as a crucial step in successfulMALDI analysis [17, 36, 37]. The top row of Figure 10shows the analyte ion signal, which apparently closelyimages the physical structure of the matrix crystals.

In Figure 10 from left to right, the first four scans(one laser shot per pixel per scan) are displayed. After afew laser shots per pixel a hole is drilled into the matrixcrystal. Matrix ion signals (second row) are obtainedfrom the inner area and from the large crystals of thesample, but degrade considerably after the first scan.Loss of the matrix signal from the nanocrystalline innerarea is probably due to the fact that the sample isalready ablated after one highly focused laser pulse perpixel (see discussion of Figure 7). Loss of the matrixsignals from the larger matrix crystals, on the otherhand, is believed to result from charge exchange pro-cesses in the gas phase between (in this case) rareprotonated matrix molecular ions and relatively abun-dant neutral analyte molecules. Ions of matrix mole-cules are lost because of the formation of ions of analytemolecules which have a higher proton affinity than thematrix molecules. Such effects, including neutralizationand signal loss of matrix ions have been observed anddiscussed earlier [38]. Observation of low-intensity ma-trix ion signals is in accordance with optimized desorp-tion conditions in high ion transmission instruments.

The sequence of four consecutive scans supports theidea of inclusion of analyte into matrix crystals. Intenseanalyte ion signals are obtained not only from thesurface of matrix crystals (first shot) but also frominside the crystals (second to fourth shots). No consid-erable differences in ion intensities are observed be-tween the first scan and consecutive scans. Analytedistributions in MALDI samples were investigated ear-lier by confocal fluorescence microscopic imaging [39]and are confirmed by our investigations. With ourmethod, however, information not only on the analytedistribution in the sample but additionally on themicroscopically resolved origin of desorbed analyteions, i.e., the laterally and axially (depth) resolved ionyield, is accessible.

The bottom row (Figure 10) shows potassium ionintensities which obviously stem mainly from areasoutside the matrix crystals. The images of potassium arealmost perfectly inverted relative to the images of theanalyte, strongly supporting the idea that peptides (andproteins) are necessarily incorporated into the growingmatrix crystals upon sample preparation for a success-ful MALDI analysis, while salt contaminants are notincorporated and thus do not hinder the analyte ionformation process. The lateral distribution of potassiumand substance P in the ion images is shown in moredetail in another composite image (Figure 11a), codingthe potassium signal with the green RGB channel andthe analyte signal with the red channel. It is clearly seenthat only very few pixels have yellow color, indicatingthe detection of both potassium and analyte ions at thesame time. This behavior is even more pronounced afterseveral scans. Figure 11b is a composite image of the

Figure 9. Scanning laser desorption/ionization mass spectro-metrical image of a pine tree root, acquired at 262 nm wavelength.The image was scanned with a step size of 0.25 �m. High ionsignal intensities of potassium/calcium are coded in white, lowintensities are coded in black.


fifth scan, showing the strongly degraded matrix crystalwith far fewer yellow pixels, compared to the first scanin Figure 10. This observation can be explained thefollowing way. It can be assumed that the dried-droplet

prepared sample is characterized by formation of largermatrix crystals which are covered by a thin layer of verysmall nanocrystals formed from the drying solution.The first scan can therefore be taken as an overlay of

Figure 10. Scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spec-trometrical image of a regular MALDI sample preparation of the peptide substance P in 2,5-dihydroxybenzoic acid (DHB) matrix on a flat aluminum target. The image was scanned with 1 �mstep size at 262 nm laser wavelength and one laser shot per pixel. Laser irradiance was just abovethreshold for ion detection. See text for further explanation.


large-crystal and nano-crystal imaging, supported byobservation of a higher noise level in the first scan (datanot shown here). Even with highly resolved scanning at0.5 �m focus size one can, to some extent, still detections from different areas in parallel, analyte-dopedmatrix crystals and salt crystals. After the first scan thenano-crystal layer is ablated and the detection of saltand analyte in parallel is less pronounced. Segregation

of cationization agents from the matrix crystals wasobserved by TOF-SIMS earlier [40], but since analyteions are not detected in the SIMS experiments, theresults cannot be correlated to the actual analyte distri-bution and to analyte ion formation.

This strong mutual exclusion has been observed forseveral peptide samples investigated so far with sub-micrometer resolution. It is not observed after defocus-ing the laser beam, i.e., in a common MALDI massspectrometer. This is because a typical laser beam focusof, e.g., 50 to 100 �m diameter will always cover manydifferent topographical areas at the same time, leadingto detection of ions from salt contaminations and frompeptide ions within the same spectrum.

SMALDI Mass Analysis of Picospotted Samples

Spotting or “microdispensing” of nano- or picolitervolumes of sample, for example of HPLC or capillaryelectrophoresis eluents, onto MALDI targets is a topicof high interest [41]. It has been observed earlier thatsuch preparations behave rather differently in MALDImass spectrometers compared to regular dried-dropletpreparations or thin-film preparations. SMALDI massanalysis was found to be an ideal tool for investigatingthe microscopical desorption and ionization behavior ofpicospotted samples. Figure 12 shows, as an example,the ion images of a �-cyclodextrin picospot preparationobtained by SMALDI mass analysis with 1 �m resolu-tion at 262 nm laser wavelength. The investigatedrepresentative spot was prepared from a solution of2.5*10�4 M �-cyclodextrin in 2*10�2 M 2,5-dihydroxy-benzoic acid in ethanol/water. Microdroplets wereformed and deposited employing a modified inkjetprinter (HP deskjet 500C). These types of printers usethe so called bubble jet technique, were droplets areejected from microcavities by ultrafast heating. Theadvantage of the bubble jet technique over the morecommon piezo technique for microspotting is the con-siderably lower dead volume of the system. Negativeeffects of ultrafast heating on the integrity of biomol-ecules were not observed in our setup. Systematicinvestigations of this possible effect, however, are stilllacking. The prepared volume per droplet was 2 pL,estimated from the number of droplets formed from acertain reservoir volume. The total amount of analyte(�-cyclodextrin) per droplet therefore was in the rangeof 500 amol. The variation in this dimension wasapproximated to be less than 30% after microscopicexamination of the size homogeneity of the spotteddroplets.

Figure 12 shows the microscopically resolved ionabundances of different ion types. The signal of alumi-num (M � 70 u) in the lower right image resembles thetopology of the target surface and vanishes in the areaof the sample overlay. Matrix ions were formed fromthe complete picospot and were imaged using thesignal of the dehydrated DHB ion (m/z � 137 u). Unlikethe behavior of peptides (see Figure 10), the carbohy-

Figure 11. (a) Overlay image of lateral distributions of potassiumand substance P from Figure 11 (first scan). Potassium ion signalsare coded in green, substance P ion signals are coded in red.Yellow pixels indicate detection of ion signals from potassium andsubstance P in the same spectrum. (b) Overlay image as (a), butfrom the fifth scan (fifth laser shot per pixel). See text for furtherexplanation.


drate ions were obtained exclusively from the centralpart of the sample and not from the outer rim, expectedto be formed of larger matrix crystals. Since the cation-ized analyte was monitored here, it is expected from thecurrent understanding of the MALDI ion formationprocesses that carbohydrate molecules do not have tobe incorporated into matrix crystals to become ionizedby alkali attachment [42] and therefore should be ob-servable from the microcritalline, flat area inside therim. The images of MALDI picospots appear to confirmthis idea.

Sodium ions (m/z � 23 u) were observed from theouter rim only, not from the central part of the spot,most probably because they were used up for cation-ization in the central part and were observed only aspart of the detected quasi-molecular ions.

The sensitivity of the method is obviously very high.With a total sample amount of 500 amol per picospotteddroplet, the prepared sample amount per pixel is onlyin the zeptomol range, estimating that analyte ions weredetected from about 600 pixels. It is assumed that thedetection limit is still considerably lower, suggesting aSMALDI detection limit which is at least in the sub-attomol range per 1 �m pixel (as demonstrated) and ismaybe even in the lower zeptomol range per 1�m pixel.

The above examples show that MALDI imaging

(SMALDI) is possible on the micrometer scale. Transferof this instrumental capability to native samples (bio-logical cells) requires the development of preparationalprocedures which are compatible with the prerequisitesof matrix-assisted laser desorption ionization andwhich avoid migration of the imaged substituents dur-ing preparation. Application of SMALDI to biologicaland medical problems, as well as new matrix prepara-tion techniques for SMALDI will be described in sub-sequent papers. Detectability of high-mass analyte mol-ecules, such as larger proteins, is the topic of anotherinvestigation. It is not obvious that even if the MALDIprocess in general is compatible with 1 �m laser spotsizes, large proteins can be desorbed and ionized intactfrom such small laser spots. Systematic investigationswith variable focus conditions will document possiblelimitations and perspectives.

Conclusion

A couple of examples have shown the general capabil-ities of scanning laser microprobe mass spectrometryand of scanning microprobe matrix-assisted laser de-sorption ionization (SMALDI) mass spectrometry witha new instrument. They demonstrate the increase ofinformational content of two-dimensional concentra-

Figure 12. Scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spec-trometric image of a MALDI picospot preparation of �-cyclodextrin in 2,5-dihydroxybenzoic acid(DHB) matrix on a flat aluminum target. Approximately 2 pL of sample solution were spotted per dot,containing 500 amol of �-cyclodextrin. The image was scanned with 1 �m step size at 262 nm laserwavelength and one laser shot per pixel. Each pixel of the dot covered a total amount of preparedanalyte in the range of 800 zmol. See text for further explanation.


tion images versus zero-dimensional mass spectro-metry of selected spots. The extremely low focal depthleads to interesting possibilities for three-dimensionalmass spectrometry but requires the development ofdedicated software. Application of the SMALDI tech-nique to biological problems is the focus of currentinvestigations and will be reported soon.

AcknowledgmentsFinancial support by the Deutsche Forschungsgemeinschaft (Sp314/3-1) and instrumental support by the Medical Institutions ofthe University of Duesseldorf are gratefully acknowledged. Theauthors would like to express their gratitude to Raimund Kauf-mann (died 1997) who strongly supported the instrumental de-velopment project in its early phase. We would like to thank PeterIngram (Duke University, NC) for helpful discussions and W. H.Schroeder (FZ Juelich) for providing the pine tree root sample. TheInstitute of Plasma Physics of the University of Duesseldorf isgratefully acknowledged for drilling the quartz lenses. WernerBouschen and Volker Bokelmann helped to document the laserfocus characteristics.

References1. Hillenkamp, F.; Unsold, E.; Kaufmann, R.; Nitsche, R. A

High-Sensitivity Laser Microprobe Mass Analyzer. Appl. Phys.1975, 8, 341–348.

2. Hillenkamp, F.; Unsold, E.; Kaufmann, R.; Nitsche, R. LaserMicroprobe Mass Analysis of Organic Materials. Nature 1975,256, 119–120.

3. Van Vaeck, L.; Gijbels, R. Laser Microprobe Mass Spectro-metry: Potential and Limitations for Inorganic and OrganicMicro-Analysis. Part I. Technique and Inorganic Applications.Fresenius J. Anal. Chem. 1990, 337, 743–754.

4. Van Vaeck, L.; Gijbels, R. Laser Microprobe Mass Spectro-metry: Potential and Limitation for Inorganic and OrganicMicro-Analysis. Part II. Organic Applications. Fresenius J.Anal. Chem. 1990, 337, 755–765.

5. Odom, R. W.; Schueler, B. Laser Microprobe Mass Spectro-metry: Ion and Neutral Analysis. In Laser and Mass Spectro-metry; Lubman, D. M., Ed.; Oxford University Press: NewYork, Oxford, 1990; pp 103–137.

6. Spengler, B.; Karas, M.; Bahr, U.; Hillenkamp, F. Laser MassAnalysis in Biology. Ber. Bunsenges. Phys. Chem. 1989, 93,396–402.

7. Seydel, U.; Heinen, H. J. First Results on Fingerprinting ofSingle Mycobacteria Cells with LAMMA. Recent Dev. MassSpectrom. Biochem. Med. 1980, 6, 489.

8. Bohm, R. Sample Preparation Technique for the Analysis ofVegetative Bacteria Cells of the Genus Bacillus with the LaserMicroprobe Mass Analyzer (LAMMA). Fres. Z. Anal. Chem.1981, 308, 258–259.

9. Bohm, R.; Kapr, T.; Schmitt, H. U.; Albrecht, J.; Wieser, P.Application of the Laser Microprobe Mass Analyzer(LAMMA) to the Differentiation of Single Bacterial Cells. J.Anal. Appl. Pyrolysis 1985, 8, 449–461.

10. Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Com-pounds. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53–68.

11. Karas, M.; Bachmann, D.; Hillenkamp, F. Influence of theWavelength in High-Irradiance Ultraviolet Laser DesorptionMass Spectrometry of Organic Molecules. Anal. Chem. 1985,57, 2935–2939.

12. Spengler, B.; Bahr, U.; Karas, M.; Hillenkamp, F. Excimer LaserDesorption Mass Spectrometry of Biomolecules at 248 and193 nm. J. Phys. Chem. 1987, 91, 6502–6506.

13. Karas, M.; Hillenkamp, F. Laser Desorption Ionization ofProteins with Molecular Masses Exceeding 10,000 Daltons.Anal. Chem. 1988, 60, 2299–2301.

14. Karas, M.; Bahr, U.; Gießmann, U. Matrix-Assisted LaserDesorption Ionization Mass Spectrometry. Mass Spectrom. Rev.1991, 10, 335–357.

15. James, P. Proteom Research: Mass Spectrometry. Springer Verlag:Berlin, 2001, pp 1–8.

16. Cole, R. B. Electrospray Ionization Mass Spectrometry. John Wiley& Sons: New York, 1997, pp 383–570.

17. Spengler, B. Postsource Decay Analysis in Matrix-AssistedLaser Desorption Ionization Mass Spectrometry of Biomol-ecules. J. Mass Spectrom. 1997, 32, 1019–1036.

18. Li, L.; Garden, R. W.; Sweedler, J. V. Single-Cell MALDI: ANew Tool for Direct Peptide Profiling. Trends Biotechnol. 2000,18, 151–160.

19. van Veelen, P. A.; Jimenez, C. R.; Li, K.W.; Wildering, W. C.;Geraerts, W. P. M.; Tjaden, U. R.; van der Greef, J. DirectPeptide Profiling of Single Neurons by Matrix-Assisted LaserDesorption-Ionization Mass Spectrometry. Org. Mass Spec-trom. 1993, 28, 1542–1546.

20. Jespersen, S.; Chaurand, P.; van Strien, F. J. C.; Spengler, B.;van der Greef, J. Direct Sequencing of Neuropeptides inBiological Tissue by MALDI-PSD Mass Spectrometry. Anal.Chem. 1999, 71, 660–666.

21. Jimenez, C. R.; Burlingame, A. L. Ultramicroanalysis of Pep-tide Profiles in Biological Samples Using MALDI Mass Spec-trometry. Exp. Nephrol. 1998, 6, 421–428.

22. Jimenez, C. R.; Li, K. W.; Dreisewerd, K; Spijker, S.; Kingston,R.; Bateman, R. H.; Burlingame, A. L.; Smit, A. B.; van Minnen,J.; Geraerts, W. P. M. Direct Mass Spectrometric PeptideProfiling and Sequencing of Single Neurons Reveals Differen-tial Peptide Patterns in a Small Neuronal Network. Biochemis-try 1998, 37, 2070–2076.

23. Chaurand, P.; Stoeckli, M.; Caprioli, R. M. Direct Profiling ofProteins in Biological Tissue Sections by MALDI Mass Spec-trometry. Anal. Chem. 1999, 71, 5263–5270.

24. Li, L.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. In SituSequencing of Peptides from Biological Tissues and SingleCells Using MALDI-PSD/CID Analysis. Anal. Chem. 1999, 71,5451–5458.

25. Gusev, A. I.; Vasseur, O. J.; Proctor, A.; Sharkey, A. G.;Hercules, D. M. Imaging of thin-layer chromatograms usingmatrix-assisted laser desorption/ionization mass spectrome-try. Anal. Chem. 1995, 67, 4565–4570.

26. Caprioli, R. M.; Farmer, T. B.; Gile, J. Molecular Imaging ofBiological Samples: Localization of Peptides and ProteinsUsing MALDI-TOF MS. Anal. Chem. 1997, 69, 4751–4760.

27. Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. Automated MassSpectrometry Imaging with a Matrix-Assisted Laser Desorp-tion Ionization Time-of-Flight Instrument. J. Am. Soc. MassSpectrom. 1999, 10, 67–71.

28. Zhang, H.; Stoeckli, M.; Andren, P. E.; Caprioli, R. M. Com-bining Solid-Phase Preconcentration, Capillary Electrophore-sis and Off-Line Matrix-Assisted Laser Desorption/IonizationMass Spectrometry: Intracerebral Metabolic Processing of Pep-tide E in Vivo. J. Mass Spectrom. 1999, 34, 377–383.

29. Garden, R. W.; Sweedler, J. V. Heterogeneity within MALDISamples as Revealed by Mass Spectrometric Imaging. Anal.Chem. 2000, 72, 30–36.

30. Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M.Imaging Mass Spectrometry: A New Technology for theAnalysis of Protein Expression in Mammalian Tissues. Nat.Med. 2001, 7, 493–496.


31. Todd, P. J.; Schaaff, T. G.; Chaurand, P.; Caprioli, R. M.Organic Ion Imaging of Biological Tissue with Secondary IonMass Spectrometry and Matrix-Assisted Laser Desorption/Ionization. J. Mass Spectrom. 2001, 36, 355–369.

32. Niehuis, E.; van Velzen, P. N. T.; Lub, J.; Heller, T.; Benning-hoven, A. High Mass Resolution Time-of-Flight Secondary IonMass Spectrometry—Application to Peak Assignments. Surf.Interface Anal. 1989, 14, 135.

33. Stockle, R.; Setz, P.; Deckert, V.; Lippert, T.; Wokaun, A.;Zenobi, R. Nanoscale Atmospheric Pressure Laser Ablation-Mass Spectrometry. Anal. Chem. 2001, 73, 1399–1402.

34. Bergmann, L. Lehrbuch der Experimentalphysik/Bergmann;Schaefer Band 3. Optik, 8. de Gruyter: Berlin, New York, 1987;p 401.

35. Hillenkamp, F.; Bahr, U.; Karas, M.; Spengler, B. Mechanismsof Laser Ion Formation for Mass Spectrometric Analysis. Scan.Microscopy Suppl. 1 1987, 33–39.

36. Strupat, K.; Kampmeier, J.; Horneffer, V. Investigations of2,5-DHB and Succinic Acid as Matrices for UV and IR MALDI.Part II: Crystallographic and Mass Spectrometric Analysis. Int.J. Mass Spectrom. 1997, 169, 43–50.

37. Horneffer, V.; Dreisewerd, K.; Ludemann, H. C.; Hillenkamp,F.; Lage, M.; Strupat, K. Is the Incorporation of Analytes into

Matrix Crystals a Prerequisite for Matrix-Assisted Laser De-sorption/Iionization Mass Spectrometry? A study of FivePositional Isomers of Dihydroxybenzoic Acid. Int. J. MassSpectrom. 1999, 187, 859–870.

38. Bokelmann, V.; Spengler, B.; Kaufmann, R. Dynamical Param-eters of Ion Ejection and Ion Formation in Matrix-AssistedLaser Desorption/Ionization. Europ. Mass Spectrom. 1995, 1,81–93.

39. Dai, Y.; Whittal, R. M.; Li, L. Confocal Fluorescence Micro-scopic Imaging for Investigating the Analyte Distribution inMALDI Matrices. Anal. Chem. 1996, 68, 2494–2500.

40. Hanton, S. D.; Clark, P. A. C.; Owens, K. G. Investigations ofMatrix-Assisted Laser Desorption/Ionization Sample Prepa-ration by Time-of-Flight Secondary Ion Mass Spectrometry.J. Am. Soc. Mass Spectrom. 1999, 10, 104–111.

41. Miliotis, T.; Marko-Varga, G.; Nilsson, J.; Laurell, T. Develop-ment of Silicon Microstructures and Thin-Film MALDI TargetPlates for Automated Proteomics Sample Identifications.J. Neurosci. Methods 2001, 109, 41–46.

42. Karas, M.; Gluckmann, M.; Schafer, J. Ionization in Matrix-Assisted Laser Desorption/Ionization: Singly Charged Molec-ular Ions are the Lucky Survivors. J. Mass Spectrom. 2000, 35,1–12.


Documents

Scanning Microprobe Matrix-Assisted Laser Desorption ... · time-of-flight mass spectrometry or, after preparation with a suitable matrix, by matrix-assisted laser desorption ionization