Applications of Desorption, Ionization and Ablation

using a Mid-Infrared Free-Electron Laser*

Michelle Baltz-Knorr, David Ermer, Ken Schriver and Richard HaglundVanderbilt University, Nashville TN 37235

Danny Bubb, Jim Horwitz, John Callahan, Andy McGillNaval Research Laboratory, Washington, D.C.

Laser Processing Consortium - Newport News - January 18, 2002

�$$upported by U. S. Department of Energy, Air Force Office of Scientific Research and Office of Naval Research

Road map for the next ASBU*/2 ...

� Motivation and approach� Capitalize on rich vibrational mid-infrared spectrum� Try to avoid electronic excitation while energizing local modes

� Desorption and ionization of proteins� Basic elements of the IR-MALDI technique� Example: mass spectrometry of proteins in gels for proteomics� Application: mass spectrometric imaging

� Pulsed laser deposition of polymers� Motivation: digital control of films, high growth rates� A sampler of RIR-PLD examples, and ...� Some puzzling results

� Concluding thoughts: mechanisms and opportunities� Fundamental excitation and relaxation processes: an example� What kinds of laser tools are needed to make progress?

*ASBU=Academic Sound-Bite Unit

Why proteomics?

� Genome is static. Proteome is dynamic, varies withrespect to changes in surroundings.

� Large number of proteins requires a rapid analysistechnique.

� Small mass differences require technique with highmass accuracy.

� Small amounts of proteins require sensitivetechnique.

� Sequencing the genome has created a need forcharacterizing the proteins produced by genes(i.e. determining location, structure, function,etc).

� Characterization needed to understand how a celloperates in sickness and in health.

� Proteins are often truncated or modified (sugar,phosphate, methyl, etc. added) after production,so there may be many proteins per gene (withhumans having 70,000-100,000 genes).

Glycine AlanineLeucineIsoleucineValine ProlineTyrosinePhenylalanineTryptophan Cysteine

MethionineSerineThreonineHistidine LysineArginineAsparagineGlutamineAspartateGlutamate

Amino Acids

Figure from Modern Genetics, pg. 67.

AdenineUracilCytosineGuanine

RNA Bases

3 bases=codonCodon stands for aspecific amino acid.64 codons; 3 are stops;1 is start.

DNA RNA proteinTranscription Translation

Analyzing the Proteome

� Proteins separated based on their isoelectric points (IEF) or their masses� Known proteins are used as markers, and bands are detected by staining� Forecasts predict replacement by biochip technology in ~ 5 years

� Electrophoresis - motion ofcharged particles under theinfluence of an applied field

� Typical lane from an SDS-PAGE experiment

� Heavier proteins encountermore resistance as theymove through the gel

• Genome sequencing creates a need for the functional analysis of proteins• Separation by gel electrophoresis is not well-suited for:

� High throughput or sensitivity (small copy numbers, 100-1000/cell)� Studying proteins with post-translational modifications� Studying tryptic digests

Motivation for MALDI in proteomics research ...

� Resonant IR-MALDI enables novel approaches to proteomics� Have to understand mechanisms of ablation, desorption, ionization to� Rethink conceptual baggage from ns-MALDI with fixed-frequency lasers.

� “It warn’t so much what I didn’t know that hurt me, but what I knowed thatwarn’t so.” Huckleberry Finn, 19th century philosopher of science

Lyse the cell

Harvest the proteinsPrepare the proteins for electrophoresis(add detergents, buffers,denature, etc.)

Freeze and do MALDI

Identify protein ormodifications

Perform electrophoresis

Prepare the gel for mass spectrometry(add matrix, electroblot, elute, extract, etc.)

MALDI: A kinder, gentler way to ionize macromolecules ...

Ion yield is proportional to thespecific energy deposition: (E/V)~F•�(�,I)

Ionization rate is proportional to thek-photon cross section and intensity:

(dN/dt)~�N(t)·�(k)Ik

Matrix-AssistedLaser Desorption-Ionization

Matrix-ASSISTED desorption and ionization.

Ionization mechanism might be(depending on wavelength, etc.)

chemical thermal

photomechanical photolytic

photochemical

Advantages over gel electrophoresis:�- Rapid sample preparation and analysis�- Good mass accuracy (.01-1%)�- High sensitivity (femto-attomole)�- Large mass range (>105 Da)�- Ability to analyze mixtures�- Tolerant to sample impurities

Cramer, R.; Hillenkamp, F.; Haglund, R.F. J. Am Soc. Mass Spec. 1996, 7, 1187-1193.Cramer, R.; Haglund, R.F.; Hillenkamp, F. Intl. J. of Mass Spec. and Ion Proc. 1997, 169-170, 51-57.

��M � A � n ��� �� M � X� �� � A � X� ��

MassSpectrometry

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FTIR of a Polyacrylamide Gel

� 3 �m – O-H and N-H stretchingvibrations

� 6 �m – C=O and C-N stretchingvibrations; O-H bending mode; N-Hdeformation

� T=298K� Position and shape of bands

probably changes with temperature;not well characterized forpolyacrylamide gel, but known forwater

Wavenumber (cm-1)

Tran

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MALDI Mass Spectra on Polyacrylamide Gels at 5.9 �m

� Tricine polyacrylamide gel; Sum of 25shots; 100 ns pulse width; Energy ~ 240�J; Spot size ~ 200 �m diameter. Nomatrix added. Gel serves as the matrix.

� Native polyacrylamide gel; Sum of 100shots; 400 ns pulse width; Energy ~ 900 �J;Spot size ~ 200 �m diameter. No matrixadded. Gel serves as the matrix.

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Angiotensin II

Insulin

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Neurotensin

Transmission spectrum of polyacrylamide.The peak at 5.9 �m comprises C=O and C-Nstretching vibrations, the N-H deformationand the O-H symmetric bending mode.

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Mass Spectra from Gels at 6.9�m and 2.9�m

� Native polyacrylamide gel; Sum of 100shots; 800 ns pulse width; Energy ~ 600�J; Spot size ~ 200 �m diameter; Nomatrix added; No insulin signal visible.

� Native polyacrylamide gel; Sum of 100shots; 100 ns pulse width ; Energy ~ 200�J; Spot size ~ 200 �m diameter; Nomatrix added. No analyte signal visible.

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Transmission spectrum of nativepolyacrylamide. The peak at 3 �mcomprises the N-H and O-H stretchingvibrations. The peak at 6.9 �mcorresponds to the CH2 deformation.

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�Angiotensin II

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Wavelength Dependence of Mass Spectra from a Polyacrylamide Gel

� Angiotensin II and bovineinsulin

� 7.5%T Tris-HCl gel� 200��m diameter spot� 100 ns pulse width� 5.7 �m taken with 140 ns

pulse width� Average of 25 shots at one

spot� No signal at 2.9 �m under

any conditions

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70006000500040003000200010000

6.3 �m; E~210 �J

6.1 �m: E~190 �J

5.7 �m; E~540 �J

5.5 �m; E~295 �J

5.9 �m; E~150 �J

m/z

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Surface vs. Bulk Ablation from a Polyacrylamide Gel

� Angiotensin II and bovineinsulin

� 7.5%T Tris-HCl gel� 100 ns pulse width� Top – Sum of 25 at same spot� Bottom – Sum of 25 at

different spots

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MALDI Spectra of Angiotensin in Ice at 5.9 �m

� Angiotensin II in water with 0.1% TFA; Sumof 15 shots taken at the same spot; 120 nspulse width; Energy ~ 250 �J; Spot size ~ 200�m diameter. Note water clusters, no adduct.

� Angiotensin II in water with 0.1% TFA; Sum of15 shots taken at different locations; 120 nspulse width; Energy ~ 250 �J; Spot size ~ 200�m diameter. Inset shows Na and K adducts.

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Transmission spectrum of water.The band around 6 �m correspondsto the O-H symmetric bending modeand the first overtone of a librationalmode.

Water clustersAngiotensin II

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Angiotensin II

Wavelength (�m)

Wavelength (cm-1)

Plans for the future: high-speed MALDI molecular imaging

Mass spectrometry is essential to future ofproteomics, but … needs to be radicallyreconfigured to meet requirements of thelife sciences and biotechnology. Our goal:

HIGH-SPEED IR-MALDI IMAGINGDIRECTLY FROM GELS, WATERICES AND LIPID MEMBRANES

Proof of principle imaging demonstratedby Caprioli et al. (1998) using uv-MALDIwith matrix on negative microbead image.

WANTED: ps or fs, tunable mid-ir laserto use gels, water or lipids as matrices.

Requires picoliter sampling sensitivity,mesoscale volumes, control of energydeposition and relaxation processes andhigh-duty-cycle lasers.

Target plate

B+

m/z

A+

m/z

Spatial maps of molecules A and B

Resonant infrared pulsed laser deposition (RIR-PLD)

� Motivation and objectives:� Motivation: New parameter space for pulsed-laser deposition� Technical approach: Irradiation with ultrashort-pulse, tunable ir

lasers� Ansatz: laser ablation in materials with strong vibrational

resonances� Resonant infrared pulsed laser deposition of polymers

� Experimental geometry� Examples: poly(ethylene glycol), fluoropolyol, polystyrene, PLGA� Mechanistic studies

� Concluding thoughts: mechanisms and opportunities

FEL ablation of SiO2 vs wavelength and vibrational excitation density

Critical scaling factor is the ratio of the optical absorption depth 1/�compared to the thermal diffusion length Ldiff=[D�L]1/2,

STRONG confinement: 1/� << Ldiff�=9.6 µm, �L=4 µs, EL=15 mJVibrational excitation density HIGHExplosive vaporization (“phase explosion”).

WEAK confinement: 1/� >> Ldiff �=4.0 µm, �L=4 µs, EL=30 mJVibrational excitation density LOWSpallation at grain boundaries, defects.

500 µm� = 4.0 µm

MODERATE confinement: 1/� ~ Ldiff�=8.0 µm, �L=4 µs, EL=30 mJVibrational excitation density MODERATEVaporization front above molten layer.

Polymer Film Deposition by IR Irradiation*

Polyethylene glycol

We used n ˜ 34Applications include:

� Drug delivery coatings

�Chemical and bio-sensor chips

� Tissue engineering

� Spatial patterning of cells

Pulsed laser deposition

Startingmaterial

* “Resonant IR-Pulsed Laser Deposition of Polymer Films Using a Free-Electron Laser,”

D. M. Bubb, J.S. Horowitz, J. H. Callahan, R. A. McGill, E. J. Houser, D. B. Chrisey, M. R.Papantonakis, R. F. Haglund, Jr., M. C. Galicia and A. Vertes, J Vac Sci Tech A, in press.

Subs

trat

e

Targ

et

Proof of the pudding: Properties of RIR-PLD PEG films

� Polyethylene glycol IR spectra are virtually identical for resonant IRPLD films and starting materials - implying intact molecular structure

� Electrospray ionization mass spectra show very similar distributionfunctions, implying composition of film nearly unchanged from bulk

Electronic vs vibrational excitation: RIR-PLD of PEG

� Comparison of UV and IR PLD shows clearevidence of photodegradation by UV (above), andvirtually identidal starting material compared toRIR-PLD.

� In IR-PLD, PEG is deposited off resonance as well,but is clearly degraded in structure compared toRIR-PLD FTIR and difference spectra (at right).

Variations on vibrational excitation: RIR-PLD of PEG

� Comparison of IR PLD in PEG using gel permeationchromatography (above) shows bond scissionoccurs off resonance, consistent with FTIRevidence.

� In IR-PLD, PEG is deposited off resonance as well,but is clearly degraded in structure compared toRIR-PLD FTIR and difference spectra (at right).

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Wavenumber (cm-1)

Off Resonance (3.30 �m) On Resonance (3.40 �m)

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2.90 �m

PEG Starting Material

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.)

3.92 �m

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Starting Material 2.90 �m, 14.4 J/cm2 - ON 3.45 �m, 10.6 J/cm2 - ON 3.92 �m, 8.7 J/cm2 - OFF

Fluoropolyol - sorbent, chemoselective polymer

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Wavenumber (cm-1)

IR PLD Film

CF

CHOHFreeOH

Abso

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Starting Material

OH

O

F3C CF3

O

CF3F3C

OH

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F3C CF3

O

F3C CF3

n

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IR PLD Film Starting Material (Fluoropolyol)

�Sensors based on SAW or cantilever designs needto be coated with polymers in precise thicknesses

�Solubility and selectivity of the polymer controlledby pendant groups attached to polymer backbone

�Deposition carried out at 2.90��m resonant with thefree OH stretch of the molecule

Fluoropolyol

�Bubb, Horwitz, Papantonakis, McGill, Haglund, Applied Physics Letters (2001)

� Nearly identical FTIR spectra of startingmaterial and film deposited at 2.90 �mguarantee that chemical selectivity andsolubility are preserved during the laserablation process.

� Gel permeation chromatography showsthat polydispersity Mw/Mn is 1.24 for thenative material and 1.21 for the PLD film.

� Deposition rate is 0.3 nm/macropulse - or30x that of UV-MAPLE technique.

Example: a biodegradable polymer, PLGA

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Sample Mw (g/mol) Mn (g/mol) polydispersityNative Polymer 8225 6950 1.2222.90 Excitation 3125 1027 3.053.40 Excitation 3470 1322 2.63

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Molar Mass (g/mol)

Native Polymer 2.90 �m excitation 3.40 �m excitation

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Wavenumber (cm-1)

3.40 �m Excitation

HC C

O

O CH2 C

O

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CH3 n n'

n = n'

Native Polymer

�A biodegradable polymer, potential application in coating of drug particles�Solution coatings are too thick, UV-PLD destroys functionality of polymer�Laser parameters: 6-8 J/cm2, focal spot diameter ~ 300 �m�RIR-PLD at 3.40 �m preserves functionality, as shown in FTIR, but …�Mass distribution shows fragmentation and alteration of polydispersity.�Possible solution: Change macropulse length, raster ablation on target.

poly(DL-lactide-co-glycolide)

RIR-PLD of polystyrene at 3.3 �m and PLD at 2.9 �m

2.5 cm

Off

On3500 3000 2500 2000 1500 1000 500

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H2C CH

n

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3.42, 3.48 �m

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Wavenumber (cm-1)

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�Polystyrene is a useful model material because its properties are wellknown, but aromatic ring absorption makes UV-PLD problematic.

�Deposition at 2.90 (off-resonance), 3.30, 3.40 and 3.42 �m, depositionrates of 0.05-0.07 nm/pulse.

�Clear evidence of bond scission from gel permeation chromatography,but no deposition at all off resonance (see target photo).

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Sample A

Sample D

H2C CHn

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3.42, 3.48 �m

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Wavenumber (cm-1)

3.28 �m 3.48 �m PS STD

� Bubb et al., Chem. Phys. Lett., in press.

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Starting Material 2.90 �m, 14.4 J/cm2 - ON 3.45 �m, 10.6 J/cm2 - ON 3.92 �m, 8.7 J/cm2 - OFF

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1. Why does bond scission occur preferentiallyfor off-resonance irradiation?

2. What is the origin of the fluence dependence in the polydispersity Mw/Mn?3. If we have pure vibrational excitation, why is

there a highly luminescent ablation plume?

Important unresolved mechanistic questions about RIR-PLD

1. PEG

2. PEG3. PPR

Questions for RIR-PLD Studies

� Resonant ablation in the IR holds promise for materials processing� Absence of electronic excitation inhibits photodecomposition of polymers� Method seems to be generally applicable to many varieties of polymers� May also permit machining of dielectrics with strong vibrational modes

� Mechanistic understanding is still rudimentary and needs more work� Nonlinear absorption by vibrational modes appears to lead to phase

explosion� Studies of neutral molecule dynamics and plume dynamics are needed

� Near-term goals include� Studies of polydispersity as a function of laser intensity and fluence� Atomic force microscopy and ellipsometry of films vs wavelength� Detailed studies of mechanisms of ablation and plume dynamics

� Bothered by all the questions? “The only legitimate purpose of researchcan be, to make two questions grow where there was only one before.”(Thorsten Veblen)

Conclusions and outlook ...

� Fundamental mechanisms of bond-breaking, desorption, ionizationand ablation are the key to reproducible, robust protocols for bothmaterials analysis and materials processing.

� Keys to future progress in these fields:� Keep working on the mechanisms!� High average power for materials processing� High peak power and low repetition rate for analysis� Scalability needs to be demonstrated� Key issue may be repetition rate, rather than pulse duration

� “Research on the topic has proceeded at an ever increasing pace,and is rapidly enveloping the entire subject in thick darkness. Infact, so much progress is being made that in a very few years, wemay know nothing whatsoever about it.” (Mark Twain)