High Spatial Resolution Imaging Mass Spectrometry

Commercial MALDI mass spectrometers are capable of routine imaging at resolutions greater than 25 µm and generally acquire molecular data at a spatial resolution of 50-200 µm. We are developing the means to reach spatial resolutions between 0.5 and 5.0 µm on a routine basis by modifying commercial instruments with custom optical and mechanical systems.

High Spatial Resolution Imaging with Conventional Instrument Geometry

Sub-cellular Imaging with Transmission Geometry 

Dual-mode IMS Instrument for High Spatial Resolution Imaging and High Magnification Microscopy

High Spatial Resolution Imaging with Conventional Instrument Geometry

We have developed and implemented two approaches that reduce laser spot size to 5 µm:

1. A Gaussian beam laser and an aspheric lens focused the laser beam with few optical aberrations, resulting in high quality protein MS images at 5 µm spatial resolution (Figure 1) [1].

2. Spatial filtration was successful for analytes that ablate at lower energies (due to a significant reduction in laser fluence with this method) such as lipids and metabolites (Figure 2) [2].

These approaches have been applied to several Driving Biology Projects (Table 1).

Figure 1. Protein imaging at 5 µm spatial resolution. J Am Soc Mass Spectrom 25 (6):1079-1082.

Figure 2. Lipid imaging at 5 µm spatial resolution. Modified from: J Am Soc Mass Spectrom 24 (7):1153-1156.

Table 1.  High spatial resolution imaging and its application to Driving Biology Projects

Sub-cellular Imaging with Transmission Geometry 

We developed a prototype transmission geometry vacuum ion source for two instrument platforms that allows the laser beam to irradiate the back side of a sample and achieves sensitivity sufficient to obtain molecular images down to 1 µm spatial resolution. We obtained high signal-to-noise mass spectra that differentiated between individual pancreatic cell types from mouse pancreas [3,4]. Experiments were also successful in obtaining a 1 µm beam, 2 µm step spatial resolution in human kidney, retina, and single cells (Figure 3 and Table 2).

Figure 3. Single cell IMS analysis. a) Optical microscopy image of laser ablated spot in a nucleus area of a single HEK-293 cell, using transmission geometry in point-and-shoot mode. (b) MS images of single HeLa cells, obtained at a raster step of 2 μm. MS images for ions m/z 617 and 885 are shown overlaid in green and red color, respectively. (c) Optical microscopy image of the HeLa cells before the MS imaging raster scan. (d) MS image of m/z 782 from HEK-293 cells (left) and bright field optical image (right) of the ablated area after MS imaging. (e) MS image of m/z 782 from RKO cells (left) and bright field optical image (right). Panels (d) and (e) have been modified from: J Am Soc Mass Spectrom 47 (11):1473-1481.

Table 2. High spatial resolution transmission geometry imaging and its application to Driving Biology Projects

Dual-mode IMS Instrument for High Spatial Resolution Imaging and High Magnification Microscopy

In collaboration with Marvin Vestal at SimulTOF Systems, Inc., the SimulTOF 200 Combo instrument was modified to enable transmission geometry experiments and simultaneous high magnification optical microscopy. This IMS instrument has features that have the potential to significantly advance the field:

1. A laser spot size of 1 µm (transmission geometry) and 50 µm (reflection geometry) provides sub-cellular and high spatial resolution imaging.

2. Simultaneous optical imaging by a video camera allows real-time monitoring of the laser focusing process.  Imaging with a 1 µm laser spot size and a 2.5 µm step size was achieved in several different tissues including brain (Figure 4), human kidney, retina, optical nerve, heart valve, and single cells in culture [5].

Figure 4.

References

1. Zavalin A, Yang J, Haase A, Holle A, Caprioli R (2014) Implementation of a Gaussian beam laser and aspheric optics for high spatial resolution MALDI imaging MS. J Am Soc Mass Spectrom 25 (6):1079-1082. PMCID: 4035219. Return to text.

2. Zavalin A, Yang J, Caprioli R (2013) Laser beam filtration for high spatial resolution MALDI imaging mass spectrometry. J Am Soc Mass Spectrom 24 (7):1153-1156. PMCID: 3690593. Return to text.

3. Zavalin A, Todd EM, Rawhouser PD, Yang J, Norris JL, Caprioli RM (2012) Direct imaging of single cells and tissue at sub-cellular spatial resolution using transmission geometry MALDI MS. J Mass Spectrom 47 (11):1473-1481. PMCID: Not Listed. Return to text.

4. Thiery-Lavenant G, Zavalin AI, Caprioli RM (2013) Targeted multiplex imaging mass spectrometry in transmission geometry for subcellular spatial resolution. J Am Soc Mass Spectrom 24 (4):609-614. PMCID: 3624063. Return to text.

5. Zavalin A, Yang J, Hayden K, Vestal M, Caprioli R (2015) Tissue Protein Imaging at 1 μm Laser Spot Diameter for High Spatial Resolution and High Imaging Speed using Transmission Geometry MALDI TOF MS. Analytical and Bioanalytical Chemistry Topical Collection: Mass Spectrometry Imaging (Accepted for publication). PMCID: Not Listed. Return to text.