IR-MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution
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High spatial resolution in mass spectrometry imaging (MSI) is crucial to understanding the biology dictated by molecular distributions in complex tissue systems. Here, we present MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) at 50 μm resolution. An adjustable iris, beam expander, and an aspherical focusing lens were used to reduce tissue ablation diameters for MSI at high resolution. The laser beam caustic was modeled using laser ablation paper to calculate relevant laser beam characteristics. The minimum laser spot diameter on the tissue was determined using tissue staining and microscopy. Finally, the newly constructed optical system was used to image hen ovarian tissue with and without oversampling, detailing tissue features at 50 μm resolution.
KeywordsMass spectrometry imaging IR-MALDESI Tissue imaging Spatial resolution Laser focusing
Mass spectrometry imaging (MSI) combines the molecular specificity of mass spectrometry with the spatially resolved analysis of an imaging technique . This can be performed in a label-free manner over a range of m/z values, allowing thousands of analytes to be analyzed simultaneously. The resulting data may be visualized using a variety of software  to depict the localization and abundance of each molecule. MSI has been routinely applied to the study of plants , proteins , lipids , and drug distributions . A common goal of nearly all applications is to achieve high spatial resolution without a loss in analyte detectability.
Laser-based MSI methods are the most commonly used methods to date, and an article reviewing the broad field of high resolution laser-based MSI across different technologies was recently published . The spatial resolution of all laser-based MSI methods is inherently tied to the laser spot size. Infrared (IR) laser desorption methods for improving spot size follow the same general principles as those in the UV regime ; however, IR lasers, which are the focus of this manuscript, are not as commonly used in the MSI field.
where D and f are as described above and k is a material constant. While these can be easily changed in custom sources, adjustments to commercial MSI instruments are more difficult . A short focal length lens presents a unique challenge in MSI because the ablated material must be proximal to and accessible for sampling by the mass spectrometer, often preventing the use of very short focal length lenses. To circumvent this issue, researchers may use transmission geometry IR laser ablation, where the laser is focused on the opposite side of the sample being analyzed . Alternatively, Römpp and coworkers used an optic with a central drilled hole to allow the ablated material to pass through . The laser beam propagation factor M2 is a defining parameter detailing the beam quality, with a perfect Gaussian beam having an M2 of 1. To reduce the detrimental effect of lasers with M2 > 1, a spatial filter may be used to remove the non-Gaussian and low laser energy edge . As seen from Equation 1, using the maximum diameter of the lens is beneficial to achieving a diffraction-limited spot size; however, the larger diameter increases the contribution of spherical aberration (Equation 2) if a plano-convex or meniscus lens is used, which do not correct for spherical aberration. Incorporating beam expanding lenses reduces beam divergence and the focal spot size of the IR laser. Alternatively, Hieta and coworkers have used the inherent beam divergence of their laser with a very long path length to completely fill the focusing lens and remove the beam edge .
Several methods have been used to achieve spatial resolutions lower than the IR laser spot size. Oversampling involves moving the sample by less than the laser ablation diameter with complete ablation at each position, effectively sampling only a small portion of the total laser spot size [14, 15]. This method results in irregular shapes of tissue ablated from a Gaussian profile laser beam. The coupling of IR laser ablation and a spatially resolved detector has been reported [16, 17] to achieve MSI resolution below the diffraction limit of the IR laser.
Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a technique combining infrared laser ablation and electrospray ionization [18, 19]. IR-MALDESI MSI has been used to analyze a variety of biospecimens . The laser ablation diameter on tissue has been previously reported as approximately 150 μm using a single focusing element [19, 21]. This method has been used and optimized for tissue imaging experiments of lipids, metabolites, and small molecule drugs .
This work details the utility of a multi-element optical system for IR-MALDESI MSI to simultaneously reduce spot size and improve spatial resolution. The multi-element optical system incorporates an adjustable iris, 3.75× beam expander, and aspheric focusing lens to improve the spatial resolution of IR-MALDESI MSI to 50 μm.
LC/MS grade methanol, ethanol, water, and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA). Laser burn paper was purchased from ZAP-IT (ZFC-23; ZAP-IT, Concord, NH, USA). Precleaned microscope slides were purchased from VWR (Radnor, PA, USA).
Mouse liver tissues were obtained from the College of Veterinary Medicine at North Carolina State University. Hen ovarian tissue was obtained from a C-Strain white leghorn commercial egg-laying hen. Animals were managed in accordance with the Institute for Laboratory Animal Research Guide, and all husbandry practices were approved by North Carolina State University Institutional Animal Care and Use Committee. Tissues were harvested and immediately flash-frozen with a dry ice in isopentane bath and stored at –80 °C until the time of the experiment. A Leica CM1950 cryomicrotome (Buffalo Grove, IL, USA) operated at –20 °C was used to prepare cryosections for analysis. Optimum cutting temperature (OCT) embedding medium (Fisher Scientific, Waltham, MA, USA) was used to adhere the tissue to a 40 mm specimen disc. The tissues were sectioned into 10-μm thick sections and thaw-mounted onto standard glass microscope slides immediately prior to analysis.
A custom acrylic enclosure housed the ionization source consisting of an X-Y translation stage, electrospray emitter, and a portion of the laser optics. Custom software developed in-house was used to drive the X-Y stage with the sample on a Peltier-cooled plate. The plate was cooled to –9 °C to form a thin ice matrix layer on the animal tissue sections to enhance energy absorption and improve ablation dynamics. The ice matrix layer was maintained by purging the enclosure with dry nitrogen (Arc3 Gases, Raleigh, NC, USA) to a relative humidity of approximately 10% throughout the imaging experiments. Two laser pulses at 20 Hz from a mid-IR laser (IR Opolette; Opotek, Carlsbad, CA, USA) tuned to 2940 nm were focused on the sample surface at each stage position. The ablated material was ejected normal to the target surface where it overlapped with an orthogonal electrospray plume.
Stable electrospray was maintained using a solvent composition of 1:1 methanol:water with 0.2% formic acid, a 2.0 μL/min flow rate, 4.0 kV potential, and a 75 μm i.d. silica taper tip emitter (New Objective, Woburn, MA, USA). Geometric parameters of the IR-MALDESI source remained constant through all experiments presented herein. The emitter tip was placed 5 mm above the sample, the laser ablation spot was 1 mm from the emitter tip and 5 mm from the customized extended MS inlet [21, 22]. The IR-MALDESI source was coupled to a Q Exactive Plus mass spectrometer (ThermoScientific, Bremen, Germany). Data were collected in positive ion mode with a scan range of 250–1000 m/z and resolving power was set to 140,000 (FWHM at 200 m/z). Automatic gain control (AGC) was turned off and the injection time was fixed based on the number of laser pulses. The total injection time (IT) was calculated as t = 25 + 50(n-1) ms, where n is the number of laser pulses (i.e., IT = 25 ms for 1 laser pulse, 75 ms for 2 laser pulses, etc.).
The MS.RAW data files were converted to the mzML format using MSConvert , and the mzML files were subsequently converted to the imzML format using the imzML converter software [24, 25]. The imzML files were then loaded into MSiReader for visualization and analysis of MSI data . Averaged mass spectra and S/N were obtained from Xcalibur ver. 2.2 (Thermo Fisher, San Jose, CA, USA).
IR laser safety glasses (pn 100-38-200; Laser Safety Industries, Minneapolis, MN, USA) were worn while the laser was on. Curved laser safety shields (TPS8; Thorlabs, Newton, NJ, USA) were installed on the optical breadboard around the mirrors used to steer the beam. All lenses were enclosed in a 1′′ diameter lens tube. A laser curtain (Laz-R-Shroud; Rockwell Laser Industries, Cincinnati, OH, USA) was erected around the IR-MALDESI laboratory space to isolate the IR laser from the rest of the laboratory.
Laser and Optics
Laser Beam Caustic Visualization
Fitting Real Data to Theoretical Real Laser Beam Focus
where W(z) is the real beam radius at position z along the beam optical axis, Wo is the real beam waist radius, M2 is the beam quality factor, and zo is the position of the position of the beam minimum radius with respect to a reference position . Parameters defining the laser beam were calculated by fitting the theoretical curve of a real laser beam in the XZ and YZ planes to the measured values. The fit was performed in Excel using generalized nonlinear regression. The residuals were calculated for each data point and plotted as a function of z height. The half angle divergence was calculated using a linear regression on the last three points in the curve.
Laser Ablation Diameter on Tissue
Mouse liver tissue was cryosectioned and mounted onto microscope slides. The number of laser pulses was varied from 1 to 5 and the diameter of the beam shaping iris was set to 2, 4, or 12 mm. For each combination tested, 20 positions on tissue were ablated. After laser ablation, the slides were removed from the stage, excess water was removed, and the tissues were stained with 100 μL of histogene staining solution (Applied Biosystems, Foster City, CA, USA) pipetted directly on top of the tissue and allowed to sit for approximately 2 min. The slide was then washed with 70% ethanol for 30 s followed by 100% ethanol for 30 s. Excess ethanol was removed and approximately 50 μL of Permount mounting medium (Fisher Scientific, Waltham, MA, USA) was pipetted onto the tissue and a coverslip was mounted on the slide. Digital images of the stained tissues with ablation spots were taken using an LMD7000 (Leica Microsystems, Buffalo Grove, IL, USA) with a 5× objective. The JPEG images were loaded into MATLAB 2016b (MathWorks) and circles were fitted to the ablation spots using the “imfindcircles” function in the Image Processing Toolbox.
MSI with High Spatial Resolution
Hen ovary tissue was analyzed using the beam expander optic design to perform high spatial resolution IR-MALDESI MSI. A region on the edge of a hen ovarian tissue section was analyzed using a 2 mm iris diameter and two laser pulses, corresponding to the smallest ablation diameter reliably achievable with this optical system. Five regions of interest (ROIs) 41 × 11 voxels in size were analyzed using decreasing step size of the stage in an oversampling mode with 100, 75, 50, 40, and 30 μm step sizes. After IR-MALDESI MSI, the tissue was stained using the tissue staining protocol described above.
Additionally, a hen ovary section was analyzed with three paired conditions: 2 mm iris diameter with 50 μm raster step size, 4 mm iris diameter with 75 μm raster step size, and 12 mm iris diameter with 100 μm raster step size, resulting in minimal under-sampling of the tissue. A custom birefringent optical attenuator was used to maintain a constant 1.2 J/cm2 fluence for each iris setting.
Results and Discussion
Multi-Lens Optical System
The optical systems presented in Figure 1 were easily installed and aligned using a 1′′ tube lens system (Thorlabs) to secure all lens components. The inclusion of two mirrors on kinematic mounts on the optical breadboard allowed precise adjustment of the laser path, which was needed to keep the laser beam centered throughout the multi-lens system. The total path length for both optical systems was approximately 1 m. The output beam profile of the OPO laser was measured using a Pyrocam III HR (Ophir-Spiricon, North Logan, UT, USA) beam profiling camera as seen in ESM 1. The beam profile of the laser output was not a perfect Gaussian, which affects the minimum spot size achievable due to the dependence on M2. To this end, a variable iris was incorporated into the multi-lens system to block the low-energy fringe of the laser output and thereby improve the minimum focal point. A 3.75× magnification Galilean beam expander was constructed from plano-concave (f = –40 mm) and plano-convex (f = +150 mm) lenses placed such that their focal points were coincident. The expanded beam (approximately 20 mm diameter) was incident on a 1′′ ZnSe aspherical lens (f = +50 mm), the shape of which corrects spherical aberration. A quartz coverslip was placed immediately after the last focusing lens to protect the lens from ablated sample debris.
Visualization of Laser Beam Caustic
The laser beam caustic was visualized by placing laser burn paper at various heights relative to the focal point. Although this limited the visualization to regions where there was sufficient fluence to ablate the laser burn paper, it was found to be acceptable to obtain a profile of the laser focus. It is likely that the ablation spots on the burn paper do not exactly reflect the laser beam width where the intensity drops to 1/e2, the common definition of laser beam width; however, the method is sufficient for comparison between two optical designs, as presented here.
The laser ablation for the single spherical focusing lens was recorded over a 10.16 mm distance shown in ESM 2. MATLAB was used to manually fit ellipses (blue) to the laser ablation spots, and major and minor axes were recorded with their corresponding z height. The laser ablation spots for the multi-lens system are shown in ESM 3. These ellipses were then plotted to give the three projections, XY, XZ, and YZ, seen in Figure 2. The two XY projections show the observed laser ablation spots. The beam expander design gave much more circular ablation spots than the single lens design, a desirable quality of a laser used for MSI. A circular laser ablation allows MS images to be acquired with the same spatial resolution in both the X and Y dimensions, even in the under-sampling mode. The reduced ablation area of the beam expander design resulted in a much higher fluence, as indicated by the heatmap. Laser fluence has a direct impact on IR-MALDESI MSI data quality. First, the laser system must have sufficiently high fluence to ablate or desorb the sample and matrix. Second, at energies above the ablation threshold, the fluence will have an impact on the ablation dynamics and overlap with the electrospray . Third, the ablation fluence determines the number of laser pulses required to completely ablate through the sample , which is a requirement for quantitative IR-MALDESI analyses . By the above points, the higher fluence of the multi-element design indicated it would successfully ablate tissue material, possibly with fewer pulses, than the single optic design. Optimizing the laser fluence to a wide variety of sample types for imaging or direct analysis could be accomplished by attenuation, using a polarization-based attenuation and an iris to balance the total energy and desired spot size.
To better visualize the laser beam caustic, a 3D surface was constructed from the stacked ellipses. The 3D surface is representative of the true laser focus as it is not composed of discrete sampling points, but rather a continuum of ablation spots based on interpolation of the data collected. To aid in comparing the two beam caustics, the axes and color maps are presented on the same scale. This emphasizes the reduction in the ablation diameter along with reduction in the depth of focus (Figure 2).
Using the model of the propagation of a real laser beam, important laser parameters describing the laser focus were defined to quantitatively compare the two optical systems.
Defining Beam Characteristics
M2, beam quality
Wo, beam waist (μm)
b, depth of focus (mm)
θ, half angle divergence
Ablation Diameter on Mouse Liver Tissue
High Spatial Resolution IR-MALDESI MSI
A multi-element optical design composed of an adjustable iris, beam expander, and an aspherical focusing lens was implemented for high resolution IR-MALDESI MSI. The design was compared with the previously used single lens design, and demonstrated improved beam quality factor (M2) and a smaller minimum waist, Wo, two of the most important laser ablation parameters in the context of MSI. A hen ovarian tissue was analyzed using the multi-element system, yielding high quality MS images with 50 micron spatial resolution.
The authors gratefully acknowledge financial support from the National Institutes of Health (R01GM087964), the W.M. Keck Foundation, and North Carolina State University.
- 2.Ràfols, P., Vilalta, D., Brezmes, J., Cañellas, N., del Castillo, E., Yanes, O., Ramírez, N., Correig, X.: Signal preprocessing, multivariate analysis, and software tools for MA(LDI)-TOF mass spectrometry imaging for biological applications. Mass Spectrom. Rev. (2016). doi: 10.1002/mas.21527
- 8.Riedl, M.J. Optical design fundamentals for infrared systems. SPIE Press (2001)Google Scholar
- 12.Römpp, A., Schäfer, K.C., Guenther, S., Wang, Z., Köstler, M., Leisner, A., Paschke, C., Schramm, T., Spengler, B.: High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue. Anal. Bioanal. Chem. 405, 6959–6968 (2013)CrossRefGoogle Scholar
- 13.Hieta, J.-P., Vaikkinen, A., Auno, S., Räikkönen, H., Haapala, M., Scotti, G., Kopra, J., Piepponen, P., Kauppila, T.J.: A simple method for improving the spatial resolution in infrared laser ablation mass spectrometry imaging. J. Am. Soc. Mass Spectrom. (2017). doi: 10.1007/s13361-016-1578-7 Google Scholar
- 18.Sampson, J.S., Hawkridge, A.M., Muddiman, D.C.: Generation and detection of multiply charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 1712–1716 (2006)CrossRefGoogle Scholar
- 24.Schramm, T., Hester, A., Klinkert, I., Both, J.P., Heeren, R.M., Brunelle, A., Laprevote, O., Desbenoit, N., Robbe, M.F., Stoeckli, M., Spengler, B., Rompp, A.: imzML–a common data format for the flexible exchange and processing of mass spectrometry imaging data. J. Proteom. 75, 5106–5110 (2012)CrossRefGoogle Scholar
- 27.Keicher, D.M.: Laser beam characterization results for a high-power cw Nd:YAG laser. SPIE Photonics West 95, 162–171 (1995)Google Scholar
- 28.Rosen, E.P., Bokhart, M.T., Nazari, M., Muddiman, D.C.: Influence of C-trap ion accumulation time on the detectability of analytes in IR-MALDESI MSI. Anal. Chem. 87, 10483–10490 (2015)Google Scholar