111
optics [4, 5]. One reported approach was to drill a hole in the center of the final
focusing lens and position it between the mass analyzer and the sample [6]. The lens
is positioned very close above the sample while allowing ion transmission through
the lens hole. Another approach to achieve a small ablation diameter is to place a
high numerical aperture objective behind the sample. Thus, a sample placed on a
transparent substrate results in the generation of ions unimpeded by laser optics, and
this arrangement is termed transmission geometry (Fig. 7.1) [3, 7]. Transmission
geometry for high spatial resolution IMS has achieved an ablation diameter of 1 μm.
It has been subsequently used by other groups for high spatial resolution IMS [8, 9].
7.3 Increasing Ion Intensity for High Spatial Resolution IMS
High spatial resolution IMS brings multiple challenges including long acquisition
times, large data files, and relatively low ion intensities. As the sampling area
decreases, data size and length of acquisition increases as a square function. Also,
ion intensities are decreased. While MALDI is a relatively “soft” ionization technique, the ratio of neutral molecules to ions is considerable (estimated to be 1:10000)
[11]. While high spatial resolution increases the regiospecificity within an ion
image, ion intensities are decreased due to the smaller amount of material sampled.
This challenge is not unique to IMS –biological imaging using SIMS is routinely
performed at 1–2 μm spatial resolution even though the liquid metal ion beams can
be focused to the low nm range.
One approach to increase ion intensity for high spatial resolution IMS is to accumulate ions from a narrow mass-to-charge window. This approach is termed continuous accumulation of selected ions (CASI) and has been demonstrated with an
FT-ICR MS instrument. Under normal imaging conditions, a wide range of ions
introduced into to the ICR cell to be analyzed, but the presence of very abundant
species can quickly fill the cell before lower abundance ions can accumulate. CASI
can be implemented by selecting a specific m/z window to accumulate ions while
the sample is analyzed at different regions. In this way, the sensitivity is increased
for a selected m/z window.
A second instrumental approach to increase ion intensities is to introduce postionization strategies. Common post-ionization strategies include irradiation of the
ion plume with either plasma, or with a secondary, lower wavelength UV laser,
termed MALDI-2 [12–14]. In either case, post-ionization is delayed after the initial
MALDI event to irradiate the MALDI plume above the sample (Fig. 7.3). MALDI-2
was shown to provide a 100-fold increase in intensities of selected ions, and a 7-fold
increase in overall intensity from a spotted lipid homogenate [15]. The intensity
increase provided by MALDI-2 allows for higher spatial resolution imaging while
maintaining comparable signal-to-noise ratio with MALDI alone at a lower spatial
resolution. MALDI-2 has been used with high numerical aperture objectives
(NA = 0.95) in transmission geometry allowing for an ablation diameter of 1 μm
[15] (Fig. 7.2).
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…
optics [4, 5]. One reported approach was to drill a hole in the center of the final
focusing lens and position it between the mass analyzer and the sample [6]. The lens
is positioned very close above the sample while allowing ion transmission through
the lens hole. Another approach to achieve a small ablation diameter is to place a
high numerical aperture objective behind the sample. Thus, a sample placed on a
transparent substrate results in the generation of ions unimpeded by laser optics, and
this arrangement is termed transmission geometry (Fig. 7.1) [3, 7]. Transmission
geometry for high spatial resolution IMS has achieved an ablation diameter of 1 μm.
It has been subsequently used by other groups for high spatial resolution IMS [8, 9].
7.3 Increasing Ion Intensity for High Spatial Resolution IMS
High spatial resolution IMS brings multiple challenges including long acquisition
times, large data files, and relatively low ion intensities. As the sampling area
decreases, data size and length of acquisition increases as a square function. Also,
ion intensities are decreased. While MALDI is a relatively “soft” ionization technique, the ratio of neutral molecules to ions is considerable (estimated to be 1:10000)
[11]. While high spatial resolution increases the regiospecificity within an ion
image, ion intensities are decreased due to the smaller amount of material sampled.
This challenge is not unique to IMS –biological imaging using SIMS is routinely
performed at 1–2 μm spatial resolution even though the liquid metal ion beams can
be focused to the low nm range.
One approach to increase ion intensity for high spatial resolution IMS is to accumulate ions from a narrow mass-to-charge window. This approach is termed continuous accumulation of selected ions (CASI) and has been demonstrated with an
FT-ICR MS instrument. Under normal imaging conditions, a wide range of ions
introduced into to the ICR cell to be analyzed, but the presence of very abundant
species can quickly fill the cell before lower abundance ions can accumulate. CASI
can be implemented by selecting a specific m/z window to accumulate ions while
the sample is analyzed at different regions. In this way, the sensitivity is increased
for a selected m/z window.
A second instrumental approach to increase ion intensities is to introduce postionization strategies. Common post-ionization strategies include irradiation of the
ion plume with either plasma, or with a secondary, lower wavelength UV laser,
termed MALDI-2 [12–14]. In either case, post-ionization is delayed after the initial
MALDI event to irradiate the MALDI plume above the sample (Fig. 7.3). MALDI-2
was shown to provide a 100-fold increase in intensities of selected ions, and a 7-fold
increase in overall intensity from a spotted lipid homogenate [15]. The intensity
increase provided by MALDI-2 allows for higher spatial resolution imaging while
maintaining comparable signal-to-noise ratio with MALDI alone at a lower spatial
resolution. MALDI-2 has been used with high numerical aperture objectives
(NA = 0.95) in transmission geometry allowing for an ablation diameter of 1 μm
[15] (Fig. 7.2).
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…