Subcellular Imag by Dynamic SIM Ion Microsco A
nalytical chemists currently face the challenge of making classified measurements in subcellular compartments under physi- into dynamic or static SIMS ological and pathological conditions to understand the role regimes based on of elements and molecules in cellular organelles. Conventhe primary beam flutional approaches rely on cell fractionation and gross meas13 2 ence (>1 3 10 ions/cm urements in subcellular fractions. However, these approaches can redistribute the analyte from its native physiological for dynamic; equal to or less location to intracellular sites with higher chemical affinity. than that for static SIMS). Dynamic Such measurements can, therefore, provide misleading SIMS instruments are more suited to subcelluinformation. lar studies of elemental and isotopic gradients and the Newer, evolving analytical techniques offer localization of isotopically labeled molecules. direct subcellular-scale chemical measureStatic SIMS instruments are being developed ments in cells and tissues. For example, for molecular imaging studies (7–9). optical and laser confocal techniques This article concentrates on applicahave been used for imaging intracelluSubhash Chandra tions of the dynamic SIMS instrument 2+ lar ionized species, such as Ca and referred to as the “ion microscope”. + Na , with ion-sensitive fluorescent Ion microscopy is a unique techDuane R. Smith molecules in subcellular compartnique for studying subcellular elements of live cells (1, 2), immunomental distribution, ion transport, George H. Morrison cytochemical studies of receptors and drug localization (10–16). The with fluorescent monoclonal antitechnique can detect isotopic distribodies, and intracellular imaging of bution with high sensitivity, produce certain molecules by native fluoressingle cell images with subcellular cence and multiphoton microscopy details, and analyze a cell in three (3, 4). Cornell Universitydimensions (providing sequential, multiThe electron probe has long been isotopic distributions at various cell depths, used to study elemental gradients in subcelwhich reveal intracellular locations of chemicals). Moreover, ion microscopy can be used in lular compartments as small as the endoplasmic applications with stable isotope tracers for localizing the reticulum (5, 6). Secondary ion MS (SIMS)-based transport of ionic or isotopically labeled molecules. All of imaging techniques are gradually becoming more valuable these features suggest that ion microscopy holds great potentools in biology and medicine because of the method’s high tial in biological and medical research. sensitivity and isotopic detection and molecular imaging The main challenge is sample preparation. Because of the capabilities. Analyses from these techniques are generally
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By imaging isotopes, the transport -of i cules, and therapeutic drugs can be stu single cells.
high vacuum requirements of SIMS, live cells cannot be analyzed. Cells, therefore, must be preserved in their native state so that the analysis reveals the chemical makeup of the living cell. A primary focus of this article is state-of-the-art techniques, including cryogenic sample preparations and novel applications of ion microscopy in biology and medicine.
Instrumentation The most commonly used magnetic-sector dynamic SIMS imaging instruments offer spatial resolution in the submicrometer range. Several of these instruments are newer versions of the original design by Castaing and Slodzian (10). Dynamic SIMS instruments capable of producing higher spatial resolution images have also been developed (17, 18). In these instruments, the high spatial resolution is primarily provided by ion beams that can be focused to a small spot size (~50 nm) and scanned over the sample surface. The suitability of a particular instrument for biological samples depends on the problem; there are ample biological studies suitable for high- and low-spatial resolution instruments. As a rule of thumb, instrument users should consider that the higher the spatial resolution, the lower the sensitivity. A schematic of a dynamic SIMS ion microscope is available in Supporting Information (http://pubs.acs.org/ac). A typical instrument is composed of primary and secondary ion optics and a detection system. The primary ion optics contain a primary ion source(s), a primary magnet, and an
electrostatic lens system. The primary ion beam, such as O+2, – + O , or Cs , is accelerated to a desired energy of 5–20 keV and mass filtered by the primary magnet. The beam is then focused, using the electrostatic lens system onto the specimen, which is held at ±4500 V in the instrument’s high vac–9 uum (10 Torr) sample chamber. Bombardment of the primary ion beam on the sample surface produces characteristic secondary ions, which are then focused by the instrument’s secondary ion optics system. This section is composed of the immersion lens/transfer optics assembly, which extracts and focuses the nonmass resolved secondary ions into the spectrometer section. The modified Nier–Johnson double-focusing mass spectrometer then disperses the ions on the basis of their energy (electrostatic sector) and momentum using two different sections. The secondary magnet section allows the passage of one ionic species at a time based on m/z. The energy and mass-filtered secondary ion beam then passes through the spectrometer exit slits and into the projector lens assembly. This beam can now be subjected to various modes of detection. In the depth profile mode of detection, the designated secondary ion intensities are recorded (with an electron multiplier or Faraday cup) versus time of primary beam bombardment. This mode is commonly used for materials characterization. The microscope mode of detection produces secondary ion images, revealing isotopic gradients with 0.5-µm spatial resolution. In this mode, the projector lens system focuses the mass-resolved secondary ion image onto the microchannel plate and provides variable image magnification. The microchannel plate acts as an ion-toelectron converter/amplifier. The cascades of electrons from the individual channels of the microchannel plate impact on
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a phosphor screen to form a visible ion image. The ion optics of the instrument preserve the spatial distribution of emitted secondary ions through the mass spectrometer, so that a one-to-one correspondence is maintained between the position of a sputtered ion leaving the sample surface and its position in the final mass-resolved ion image. The ion image
(20). Freeze-substitution procedures that remove water from the frozen cell interior at low temperatures (~–80 °C) using solvents such as ether, acrolein, or acetone, may preserve the native distribution of some diffusible ionic and molecular species, but impregnating plastic into the cell for embedment completely alters the cell matrix. Such a major alteration of the cell matrix is not desirable for dynamic SIMS, and these methods may have limited use, especially for quantitative imagWith our sandwich frac ture ing. Although frozen hydrated and frozen method, ~15 s are needed freeze-dried sample preparations do provide optimal samples for SIMS analysis, neither approach preserves the living-state cell to prepare frozen hydrated matrix. In frozen hydrated samples, the physical form of the cellular water is completely fractured cells and altered, and, in freeze-drying, the water is removed from the specimen. The choice membrane pieces. between frozen hydrated and frozen freezedried sample preparations may depend on the type of SIMS analysis desired. The frozen on the phosphor screen can be recorded with a charge-couhydrated analysis is preferred for static time-of-flight molecpled device (CCD) camera and stored on a computer for ular imaging (21). Static SIMS experiments have also pixel-by-pixel digital image processing for subcellular image imaged molecules in tissue sections after air drying (22). On quantification. the other hand, frozen freeze-dried sample preparations are In instruments with a microprobe mode of imaging, the preferred in dynamic SIMS. Analysis of frozen hydrated cells primary beam is focused to a spot size as small as 50 nm under a dynamic primary beam preferentially removes water and scanned over the sample. The image is assembled along the z-direction. This effect also enhances other analyte point-by-point. Each mode has advantages and disadvansignals and causes false image contrasts in ion images (23). tages. The choice of mode depends on the type of problem Additionally, frozen hydrated biological samples offer poor encountered. electrical conductivity, whereas conductivity is enhanced upon freeze-drying. Sample preparation It should be noted that freeze-drying may cause cell Different samples, such as soft tissues, hard tissues, and cell shrinkage and often damages some of the cellular morpholcultures, require different sample preparations for SIMS ogy. To minimize this damage, sample freezing and freezestudies. In addition, our instrument requires that the specidrying at low temperatures are desirable. For example, mormen conduct electricity because the specimen is held at phological evaluations of fractured cells and membrane ±4500 V. To preserve them in their native state under high pieces prepared with our “sandwich fracture” method (24), vacuum, cells require careful fixation to preserve their using electron and laser scanning confocal microscopic techhydrated cell matrix and the asymmetric distribution of difniques, revealed well-preserved membrane particles, mitofusible ions (or water-soluble molecules) within the cells. An chondria, lysosomes, Golgi apparatus, and cell cytoskeleton ideal sample fixation should preserve both the structural and (24–27). This is not surprising, because the method quickchemical integrity of the living cell. Sample preparation has freezes cell monolayers of
