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1 Molecular Cartography: Moving Towards Combined Topographical and Chemical Imaging Using AFM and Mass Spectrometry Olga S. Ovchinnikova Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory

2 What is Molecular Cartography? 2 Managed by UT-Battelle

3 Why do molecular chemical imaging? PET Scan of human brain RAMAN Imaging of pharmaceuticals 3 Managed by UT-Battelle MALDI MS Imaging of tissue

4 Need for spatially resolved information 4 Managed by UT-Battelle

5 Overview Goal: Conceive, implement, study, and advance an imaging platform that can combine measurements of spatially resolved topographic and other physical features of a surface, under real world conditions, with precise molecular characterization of the chemical species at those spatial locations a.k.a., Molecular Cartography Concept: Combined atmospheric pressure proximal probe atomic force microscopy (AFM) and thermal desorption/ionization mass spectrometry (TD/I-MS) Experimental Progression: Implement and study proximal probe TD/I-MS with probes from millimeter to low micrometer size Enable TD/I-MS using traditional AFM platform with proximal probe capable of nanometer scale TD 5 Managed by UT-Battelle

Established and Emerging Atmospheric Pressure Surface Sampling/Ionization Techniques 6 Managed by UT-Battelle Addressing the Challenges to Enable

6 Established and Emerging Atmospheric Pressure Surface Sampling/Ionization Techniques 6 Managed by UT-Battelle Addressing the Challenges to Enable Spatially Resolved Molecular Chemical Analysis of Interfaces Under Real World Conditions Van Berkel, Pasilis and Ovchinnikova, J. Mass Spectrom. 28, 43,

Auxiliary AP-Surface Methods: Sampling/Ionization Stirring/Heating Labile by Oscillatory Motion 7 Managed by UT-Battelle Atomic Molecular Molecules Reactive Separation Simplicity

7 Auxiliary AP-Surface Methods: Sampling/Ionization Stirring/Heating Labile by Oscillatory Motion 7 Managed by UT-Battelle Atomic Molecular Molecules Reactive Separation Simplicity Topographic Liquid/Gas Jet Liquid Extractive Thermal Laser Force Limits 1 Å 1 nm 1 nm nm 1 μm 1 μm μm 1 mm 1 mm Jet Based Probing Extractive Probing Thermal Probing Laser Probing mm AFM Probing

8 Proximal Probe Thermal Desorption with a Secondary Ionization: Experimental Set-Up 3. Thermally desorbed species are ionized by interaction with ionic species from ESI or APCI source 2. Desorbed molecules are vacuumed into the sampling cone 4. Molecular I.D. (Full scan, SIM, or SRM) 1. Spatially resolved thermal desorption with heated probe

Proximal Probe TD/I-MS: Experimental Set-Up

for different applications Probe adjustable

Camera to monitor probe to sample position

5mm 9 Managed by UT-Battelle (2) (3) (4)

9 Proximal Probe TD/I-MS: Experimental Set-Up Heated probe tips of various sizes and shapes for different applications Probe adjustable up to 35 o C Computer controlled sample stage Camera to monitor probe to sample position (1) Array of Heated Probe Tips Width=.5mm 9 Managed by UT-Battelle (2) (3) (4) Width=.2mm Width=1.6mm Thickness=.7mm Width=2.4mm Thickness=.8mm

10 Detection Limits for Compounds from TLC Plates Using TD/I-MS The Detection Limits of Compounds for the TD/I-MS technique are highly dependent of the compound s Vapor Pressure TNT 1 ng Pharmaceuticals Explosives Herbicides Acetaminophen ng Dyes Compound detection limits were done using thin layer chromatography (TLC) plates Sudan Red 7B 25 ng 1 Managed by UT-Battelle

11 Proximal Probe TD/I-MS: Analytes Separated on and Sampled from a NP-HPTLC Plate D 5 (a ) (b ) Developed Plate Total Ion Current 1.6 mm chisel point TD probe Relative Intensity Managed by UT-Battelle 5 (M +H ) + m/z 152 m/z (M +N a) + m/z 23 m/z m/z Distance (mm) (M +H )+ m/z 195 (c ) (d ) (e ) Caffeine (M+H) + = m/z 195 APCI + Acetaminophen (M+H) + = m/z 152 APCI + Aspirin (3) (M+Na) + = m/z 23 ESI + Ovchinnikova and Van Berkel. Rapid Commun. Mass Spectrom., 21, 43, ESI APCI Compounds (active ingredients in Excedrin tablet) illustrate versatility of the technique Alternating scan to scan ionization by ESI and APCI Simultaneous detection of compounds with disparate ionization properties

12 Micrometer Scale TD/SI-MS 12 Managed by UT-Battelle

Micrometer Resolved Spot Sampling Relative Intensity 5 5 5 5 Caffeine, APCI+ (M+H) + = m/z 195 Acetaminophen, APCI+ (M+H) + = m/z 152 Aspirin, ESI+ (M+Na) + = m/z 23 TIC 1.5 2. 2.5 3. 3.5 4.

13 Micrometer Resolved Spot Sampling Relative Intensity Caffeine, APCI+ (M+H) + = m/z 195 Acetaminophen, APCI+ (M+H) + = m/z 152 Aspirin, ESI+ (M+Na) + = m/z 23 TIC Time (min) Excedrin Tablet Thermal desorption sampling holes ~25 µm in diameter Spot to spot chemical variation consistent with other chemical imaging (i.e. Raman) Ability to use different ionizations simultaneously

14 Resolution Testing of MicroTD Imaging Optical Image Chemical Image 5 um (a) (b) 1 mm 45 um 5 μm 5 μm 5 um (c) (d) 5 μm 2 um 25 μm 25μm 14 Managed by UT-Battelle Ovchinnikova, Kertesz and Van Berkel. Anal. Chem, 21, Submitted

15 Influence of Scan Direction on Resolution Optical Image Chemical Image 5 um (a) (b) 5 μm 5 μm 5 um 8 um 2 um 25 μm 25 μm (c) 8 um 5 um 5 um 2 um (d) Current resolution limit with current set-up is 5 μm 25 μm 15 Managed by UT-Battelle 25 μm

16 Nanometer Scale TD/SI-MS 16 Managed by UT-Battelle

17 Marriage of Mass Spectrometry with Microscopy Chemical specificity Physical properties and nanometer resolution + =? New Tool for Studying Systems at a Sub-micron Level!!! 17 Managed by UT-Battelle

microscope A microscale cantilever with a sharp tip (probe)

When the tip is brought into proximity of a sample surface,

18 What is Atomic Force Microscopy-AFM? AFM is a very high-resolution type of scanning probe microscope A microscale cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever. 18 Managed by UT-Battelle

19 Local Nano-Thermal Analysis with an AFM Pre-Heating Scan Post-Heating Scan 4 C PMMA and SAN Polymer Blend 3 C 35 C 19 Managed by UT-Battelle Nano heating probes allow for rapid heating and cooling and precise temperature control.

20 Nanoscale Physical and Chemical Imaging via Proximal Probe Thermal Desorption/Mass Spectrometry 5. Combine AFM and MS information 4. Detect targeted molecules with a mass spectrometer 2. Image area with AFM to obtain topography and physical information 1. Locate area of interest on the tissue 3. Spatially Desorb areas of interest 2 Managed by UT-Battelle

Proximal Probe Thermal Desorption with a Secondary Ionization: Nanometer Scale Spot Sampling

surface 25 C 12 14 16 18 2 22 24 m/z Tip removed from surface 1 μm 5 35 C 12 14 16 18 2 22 24

fashion to 35 C at very rapid heating rates Surface is 1 μm caffeine thin film Caffeine (M+H)

21 Proximal Probe Thermal Desorption with a Secondary Ionization: Nanometer Scale Spot Sampling Atomic Force Microscope (AFM) cantilever used as thermal desorption proximal probe 5 Tip on surface 25 C m/z Tip removed from surface 1 μm 5 35 C m/z Tip on surface 35 C m/z Cantilever can be heated in a controlled fashion to 35 C at very rapid heating rates Surface is 1 μm caffeine thin film Caffeine (M+H) + = m/z 195 ESI + Caffeine only observed when the AFM probe is heating and engaged on the surface

22 Heated AFM tip lowered and held on caffeine thin film surface 1. Heater is turned on above surface 8 Relative Abundance Tip is engaged onto surface from 1 μm and heats the surface. Material desorbs Time (min) (SRM) of caffeine 195 m/z -> 138 m/z as tip is lowered onto surface and held there 22 Managed by UT-Battelle Single Reaction Monitoring (SRM) allows to only monitor the fragmentation transition of caffeine and therefore allows to significantly lower the noise

Size of Nano-thermal Holes Topography ~25 nm (a) (a) AFM topography of caffeine thin film surface after local thermal desorption with heated AFM tip (b) The holes are 25 nm wide

23 Size of Nano-thermal Holes Topography ~25 nm (a) (a) AFM topography of caffeine thin film surface after local thermal desorption with heated AFM tip (b) The holes are 25 nm wide x nm deep. This corresponds to approximately 8 Attomols. ~25 nm (b) 25 μm crater 2 μm crater ~ nm 2 μm crater 2 nm crater Region we are working in Attomoles of material sampled!

Proximal Probe Thermal Desorption with a Secondary Ionization: Nanometer Scale Spatial Resolution Array of holes created from locally heating surface Heater ON and lowered to surface Corresponding

24 Proximal Probe Thermal Desorption with a Secondary Ionization: Nanometer Scale Spatial Resolution Array of holes created from locally heating surface Heater ON and lowered to surface Corresponding chronogram from SRM of caffeine (m/z ) 8 Hot tip Cold tip 1. Heater is turned on at 1 μm 2. Hot tip is slowly lowered on surface 3. Hot tip is held on surface for 3 s 4. Heater is turned off and tip is removed from surface 5. Tip moves to next point (2 s programmed delay between engagement of surface of subsequent holes Relative Intensity Heater OFF and retracted from surface Time (min)

Nanomechanical Measurements of Plant Cell Walls with

x 5 nm area Nanomechanical studies of cells can

by measuring the response of the cells to excitations

Optical Image of Cells in Poplar Stem Cross section

3 μm x 4 μm 5 x 5 nm area of top of cell wall

25 Nanomechanical Measurements of Plant Cell Walls with AFM Map correlating Nanomechanical Properties of 5 nm x 5 nm area Nanomechanical studies of cells can reveal information about cell stiffness and structure by measuring the response of the cells to excitations supplied through the AFM tip. Optical Image of Cells in Poplar Stem Cross section in an AFM set-up AFM tip nm 2 μm Cell Wall dimensions 3 μm x 4 μm 5 x 5 nm area of top of cell wall Correlating 15 μm x 15 μm topography overlaid with AFM phase data of a plant cell wall

26 26 Managed by UT-Battelle Conclusion and Future Plans We have developed a platform for surface spot sampling chemical imaging via thermal desorption with a secondary ionization capable of millimeter to nanometer spatial resolution Spot or line sampling, or chemical imaging has been successfully demonstrated at different length scales Spot and line scans on HP-TLC plates at millimeter resolution Chemical imaging of printed inks on paper at micrometer resolution Spot sampling from thin-films on glass at nanometer resolution Combined TD spot sampling and topographic imaging of a surface at the 25 nm resolution Future Combined AFM and MS chemical imaging at higher resolution Image the distribution of small molecules in tissues and materials and correlate chemical information to physical properties

27 Acknowledgments Gary Van Berkel and Vilmos Kertesz of the Organic and Biological Mass Spectrometry Group, ORNL Maxim Nikiforov, Stephen Jesse, and Sergei Kalinin from the Center for Nanophase Material Sciences (CNMS) ORNL Michael Balogh from Waters Corporation for Beta-test TQD Instrument DOE Basic Energy Sciences 27 Managed by UT-Battelle For more information about TD/I-MS and the Mass Spectrometry Surface Sampling and Imaging Center

LECTURE 3. Ionization Techniques for Mass Spectrometry

LECTURE 3. Ionization Techniques for Mass Spectrometry LECTURE 3 Ionization Techniques for Mass Spectrometry Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology Cornell University Ithaca, NY 14850 Lecture 3, Page 1 Contents Electron ionization (EI)