Article pubs.acs.org/ac
Ambient Mass Spectrometry Imaging: Plasma Assisted Laser Desorption Ionization Mass Spectrometry Imaging and Its Applications Baosheng Feng,† Jialing Zhang,† Cuilan Chang,† Liping Li,† Min Li,† Xingchuang Xiong,§ Chengan Guo,‡ Fei Tang,‡ Yu Bai,*,† and Huwei Liu† †
Beijing National Laboratory for Molecular Sciences, the Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China § National Institute of Metrology, Beijing 100013, China ‡ Department of Precision Instrument, State Key Laboratory of Precision Measurement Technology and Instruments, Tsinghua University, Beijing 100084, China S Supporting Information *
ABSTRACT: Mass spectrometry imaging (MSI) has been widely used in many research areas for the advantages of providing informative molecular distribution with high specificity. Among the recent progress, ambient MSI has attracted increasing interests owing to its characteristics of ambient, in situ, and nonpretreatment analysis. Here, we are presenting the ambient MSI for traditional Chinese medicines (TCMs) and authentication of work of art and documents using plasma assisted laser desorption ionization mass spectrometry (PALDIMS). Compared with current ambient MSI methods, an excellent average resolution of 60 μm × 60 μm pixel size was achieved using this system. The feasibility of PALDI-based MSI was confirmed by seal imaging, and its authentication applications were demonstrated by imaging of printed Chinese characters. Imaging of the Radix Scutellariae slice showed that the two active components, baicalein and wogonin, mainly were distributed in the epidermis of the root, which proposed an approach for distinguishing TCMs’ origins and the distribution of active components of TCMs and exploring the environmental effects of plant growth. PALDI-MS imaging provides a strong complement for the MSI strategy with the enhanced spatial resolution, which is promising in many research fields, such as artwork identification, TCMs’ and botanic research, pharmaceutical applications, etc. “One picture exceeds a thousand words.” Nowadays, imaging technologies, such as atomic force microscopy (AFM), magnetic resonance imaging (MRI), fluorescence, etc., are widely used in many fields,1−5 but all those methods cannot provide the direct identification and distribution of specific chemicals in different samples. Through combining mass spectrometry (MS) with relevant imaging software, mass spectrometry imaging (MSI) has emerged as a powerful tool to provide molecular images with high specificity, high resolution, and abundant structural information. In addition, MSI is a label-free method, which allows the detection and identification of molecules from a complex sample surface, and is capable of providing abundant information from small molecules to large biomolecules, from micrometer scale to millimeter scale, and from subcellular to multicellular scale.6 In the past decades, as an exploration-discovery method, MSI has been improved significantly with the development of the mass spectrometer. Classification of MSI is usually based on the ion source utilized in MSI. Secondary ion mass spectrometry © 2014 American Chemical Society
(SIMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are two types of commercial MS for MSI,7,8 which provide specific molecule distribution information with high spatial resolution and high sensitivity. However, complicated sample preparation steps may affect the natural molecular distribution of samples, and the ionization process is accomplished in a vacuum environment.6 Meanwhile, the MSI applications of SIMS and MALDI-MS are also limited by the type of equipment. With the emergence of ambient ion sources, this new technology, represented by desorption electrospray ionization (DESI),9 attracts more and more attention and brings a promising direction to MSI research. DESI imaging allows direct and in situ analysis with little sample preparation and the spatial resolution of about 100−200 μm.10,11 Following the success of DESI-based MSI, various Received: October 14, 2013 Accepted: March 26, 2014 Published: March 26, 2014 4164
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Scheme 1. PALDI-Based MSI Strategya
a
Blue dots: metastable plasma. Green dots: desorbed analytes. Red dots: ionized analytes; d1: the distance between DART and the inlet of the ceramic transport tube; d2: the distance between laser spot and the inlet of the ceramic transport tube. Logo used with the permission of Peking University.
complement for the MSI strategy with the enhanced spatial resolution, which is promising in many research fields, such as identification of modern printed documents, distinguishing TCMs’ origins and the distribution of active components of TCMs, and exploring the environmental effects of plant growth, pharmaceutical applications, and small-molecule biomarkers.
ambient ion sources, such as low-temperature plasma (LTP),12,13 air flow-assisted ionization (AFAI),14 laser ablation electrospray ionization (LAESI),15,16 infrared laser ablation metastable-induced chemical ionization (IR-LAMICI),17 and nanospray desorption ionization (nano-DESI),18−20 have been applied in MSI. On the premise of the rapid and in situ analysis, high spatial resolution, high sensitivity, and a wide scope of applications are still the limitations of MSI. PAMLDI,21,22 a novel ambient ion source constructed in our previous work, combines multiwavelength laser desorption and heated metastable plasma ionization of analytes. The ionization process takes place when the heated metastable plasma contacts analytes desorbed by the pulse laser, accompanied by a series of reactions such as Penning ionization, proton transfer, or charge exchange.23 Compared with DESI and other ion sources, such as AFAI and nano-DESI, both polar and nonpolar compounds can be ionized by PAMLDI,21 and no solvent is required which decreases ion suppression, reduces pH effect, and simplifies complicated spectra caused by adducts. Even the LTP ionizations are also carried out using plasma; with the introduction of laser desorption, higher resolution can be achieved in PAMLDI. Compared with LAESI with infrared lasers, the introduction of three different wavelength lasers in PAMLDI is applicable for a wider range of analysis. All of the above makes the PAMLDI-MS system more applicable in MSI applications. Here, the novel MSI system, plasma assisted laser desorption ionization mass spectrometry (PALDI-MS), was integrated on the basis of PAMLDI-MS with one wavelength of 532 nm. High spatial resolution of 60 μm × 60 μm was realized. The feasibility of PALDI-based MSI was confirmed by seal imaging. The authentication of the document was demonstrated by imaging printed Chinese characters, which may be expanded to authentication and analysis of paintings, manuscripts, calligraphy, and patterns. Furthermore, the imaging of Radix Scutellariae, a traditional Chinese medicine used to treat fever, allergies, inflammation, etc.,24 was accomplished, which proposed an approach for distinguishing traditional Chinese medicines’ (TCMs’) origins and exploring the environmental effects on plant growth. PALDI-MS imaging provides a strong
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EXPERIMENTAL SECTION Chemicals and Reagents. The Chinese seal of the Peking University badge was purchased from Chengxintong service center (Beijing, China). The vermilion inkpad was obtained from the Peking University cultural market (Beijing, China). Radix Scutellariae was purchased from Aomiao Chinese medicines Sales Co., Ltd. (Shanghai, China). PALDI-MS Imaging System. On the basis of our recently reported PAMLDI-MS,21,22 a new focusing lens was added to gain better resolution. Scheme 1 shows the PALDI-based MSI strategy. The equipped mass spectrometer was Agilent MSD TOF (Agilent Technologies, Santa Clara, CA, USA). The MSD TOF was tuned and calibrated with Tuning Mix (P/N: G196985000, Agilent) before experiments. Positive mode was chosen during imaging experiments, and the scan range of mass to charge ratio was 50−800 with the scan rate of 1.02 spectrum/s. The parameters of the mass spectrometer were as follows: drying gas temperature at 315 °C, drying gas flow rate at 3.5 L/ min, VCap at −3500 V, fragmentor at 175 V, skimmer at 65 V, and OCT 1 RF Vpp at 250 V. Plasma was produced by DART (Ionsense, Saugus, MA). High purity nitrogen was used as the discharge gas with temperature at 350 °C and the flow rate of 2.5 L/min. The pulsed Nd:YAG laser (Lai yin Optoelectronics Technology, Beijing, China) can provide a multiwavelength laser, 355, 532, 1064 nm, with a pulse length of 10 ns at 20 Hz. 532 nm was chosen in our experiment. The laser energy was regulated by the voltage and laser attenuation lens. The Qswitch of the laser was turned on during the imaging experiments. The angle between the laser beam and the automatic 3D platform was kept at 45°. The diameter of the laser beam spot was adjusted by the distance between the automatic 3D platform and laser focus lens. The accurate distance and moving speed of the automatic 3D platform was 4165
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Figure 1. Spatial resolution experiments of PALDI-based MSI. (a) Optical micrograph of the width of laser desorption notch. (b) Optical micrograph of gaps and vermilion inkpad regions in lateral resolution experiments. (c) Schematic illustration of lateral resolution experiments. Black rectangles represent the vermilion inkpad regions. (d) Extracted ion chromatogram of lateral experiments.
and a 1% attenuation lens. For the imaging of printed blue Chinese characters, “Peking University” was successfully obtained. The energy of the laser was set to 1.8 mJ with a 10% attenuation lens, and the distance d1 was 16 mm. The imaging strategy was a progressive scan, and each line was scanned from the same starting position. The speed of the automatic 3D platform was 40 μm/s along the x axis with a step size of 300 μm along the y axis. The step size was the moving distance of the platform between each scan line. The original MS data were processed by imgGenerator and MSI-view to obtain the final imaging results. Imaging of Radix Scutellariae. The purchased Radix Scutellariae was cut into about 800 μm thick slices for direct imaging without any other pretreatment. The energy of the laser was set to 1.8 mJ with a 50% attenuation lens. The other parameters were the same as the printed character imaging experiments. Safety Considerations. Laser protective goggles were necessary when using the UV, visible, and IR laser.
achieved by PSA100-11-X stepping motor and MC600 control box with control software (Zolix Instruments Co., Ltd., Beijing, China). MSI software, imgGenerator and MSI-view, was offered by National Institute of Metrology, P. R. China. The laser printer used was HP Color LaserJet CP1215 with a black print cartridge (CB540A), a cyan print cartridge (CB541A), a magenta print cartridge (CB543A), and a yellow print cartridge (CB542A). Micrographs were taken by Nikon Eclipse Ti-U (Nikon, Tokyo, Japan). Spatial Resolution Experiments. The spatial resolution experiments, measurements of the practical width of the laser desorption and lateral resolution, were accomplished using the vermilion inkpad and 532 nm laser. The distance between DART and the inlet of the ceramic transport tube was 16 mm (d1), and the distance between the laser spot and the inlet of the ceramic transport tube was 7 mm (d2) (Scheme 1). d1 was changed from 16 to 26 mm in the experiments depending on different sizes of the samples. The vermilion inkpad was uniformly distributed on white paper. With the movement of the paper, a 532 nm focused laser, whose energy was set at 1.8 mJ with a 1% attenuation lens, left its notch on the vermilion inkpad surface. The width of the notch was measured by microscopy. After the laser ablation, the generated trace on the vermilion inkpad was used for the calculation of spatial resolution. In the following experiments of lateral resolution, the movement of the automatic 3D platform was along the x axis with a 40 μm/s moving rate. Imaging of the Chinese Characters. The Chinese seal and printed characters were selected for the authentication experiments using MSI. The Chinese seal showed the Peking University badge which was composed of a transformation of the Chinese abbreviation of Peking University surrounded by a 16 mm diameter circle. The distance d1 was set as 26 mm. A 532 nm laser was chosen as the desorption laser with 1.8 mJ
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RESULTS AND DISCUSSION As discussed in the previous work,21,22 the introduction of the multiwavelength laser in PAMLDI-MS made it a potential approach for the MSI. Our work focused on the exploration of the feasibility of PAMLDI-MS imaging and its new applications. The parameters of the PAMLDI-MS system played an important role in the imaging experiments. The temperature of nitrogen working gas affected the signal intensity. The signal intensity increased while the temperature increased. Considering sufficient signal intensity and the appropriate temperature of plasma, 350 °C was chosen as the temperature for the imaging experiments (Supporting Information, Figure S1). Even though higher energy can desorb more analyte molecules, 4166
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Figure 2. MSI experiments of laser print blue Chinese characters. (a) Three different sizes of the electronic version of Chinese characters for printing. (b) MSI of three different sizes of printed Chinese characters using m/z 146.0488. (c) MSI of three different sizes of printed Chinese characters using m/z 354.2060. b-(L) and c-(L) are the imaging results of a-(L). b-(M) and c-(M) are the imaging results of a-(M). b-(R) and c-(R) are the imaging results of a-(R). Scale bar is in the lower left corner of each graph. Logo used with the permission of Peking University.
the unescapable drawback is the decrease of the spatial resolution. The width increased while the energy increased (Supporting Information, Figure S2). Keeping the sufficient signal intensity, minimum energy was conducive to improve spatial resolution. In resolution and imaging experiments, taking the sufficient signal intensity for imaging as premise, the energy was set to the corresponding minimum according to different surface properties of imaging objects. The design of the three wavelength laser in the PAMLDI-MS system offers multiple choices when imaging different targets. In the verification experiment of the feasibility of PAMLDI-based MSI, signals can be obtained using a three wavelength laser with the same energy. Higher signal intensity of vermilion inkpad ions at m/z 130.0656, m/z 158.0612, and m/z 316.2878 were obtained with a 532 nm laser. Considering the signal intensity and types of generated ions, a 532 nm laser was chosen. The mass spectra of the vermilion inkpad desorbed using PAMLDI-MS with a three wavelength laser were shown in the Supporting Information (Figure S3). After optimization of the experimental parameters, ambient PAMLDI-based MSI without sample pretreatment was systematically investigated. Spatial Resolution of PALDI-Based MSI. The word resolution contains two relevant meanings, “the smallest interval measured by instruments” and “the degree of detail visible in images”, in the Oxford English Dictionary.8 Accordingly, some articles used the size of the laser spot, the size of the ion beam, or the size of the droplet beam25 to define the pixel size as the resolution of the imaging methods, and others used the minimum distance that delivered adjacent features to determine the pixel size as the spatial resolution.13,19,26 Since the size of a pixel determines the quality of MSI, we provided a more detailed elaboration and study of the spatial resolution on both x and y size of a pixel. Here, we used the width of laser desorption notch as the pixel size along the y axis and minimum distance which provided adjacent features as the lateral resolution pixel size along the x axis. Figure 1 shows the spatial resolution experiments of PALDIbased MSI. First, the width of laser ablation and the size of the imaging pixel along the y axis were studied. The 532 nm laser ablated the vermilion inkpad regions (Figure 1a), and the average width of the obtained laser notch was 60 μm. Second, the lateral resolution was studied. Schematic illustration of lateral resolution experiment is shown in Figure 1c, and the micrograph of partial laser ablation is shown in Figure 1b. The gap made by the laser ablation was defined as the minimum distance, which divided the adjacent features. Figure 1d is the
extracted ion chromatogram of the inkpad regions when the laser vertically passed the inkpad regions obtained in Figure 1b. Variation of ion signal intensity was provided by the two most abundant ions at m/z 158.0612 and m/z 316.2878, in which the peak crest corresponded to the vermilion inkpad regions and the trough of the peak represented the gaps between them. The clear variation of signal intensity demonstrated that the adjacent features larger than the width of the gaps were clearly shown in MSI results. Average width of the gaps was 60 μm from the micrographs, which demonstrated that the lateral resolution as well as the size of the imaging pixel along the x axis was 60 μm, and a 60 μm × 60 μm pixel of spatial resolution was achieved in this PALDI-MS imaging system. Applications of PALDI-MS in Authentication of Printed Documents. Compared with traditional study and authentication methods of paintings, such as X-ray fluorescence (XRF),27 ambient MSI, for example LTP, has been reported as a new tool in artwork identification because of the direct evidence of the specific chemical distribution and the visual difference between the genuine and counterfeit seals in the open air.13 Here, for the research of both feasibility of MSI and the authentication of artwork using PALDI-MS, the imaging of the Chinese seal was performed. Three specific ions at m/z 130.0656, m/z 158.0612, and m/z 316.2878 (MS/MS spectra were shown in Supporting Information, Figures S8−S10) were selected to provide the imaging results (Supporting Information, Figure S7). In Figure S7d−f, Supporting Information, the outline of the Chinese seal and the distribution of three ions were clearly presented. These demonstrated the feasibility of PALDI-based MSI. In addition, the differences between various brands of vermilion inkpad were also studied (Supporting Information, Figure S5), which showed the feasibility of the authentication of the Chinese seal using PALDI-MS. Comparison of the seal before and after laser ablation (see Figure S7b,c, Supporting Information) showed that there is no obvious damage on the Chinese seal sample when using this nonsolvent and matrix-free PALDI MSI method. These provided a potential approach to the analysis and authentication of paintings and calligraphy. Modern printed documents are widely used in many fields such as data storage and business in our daily life. The identification of document authenticity and its changes over times is significant, especially in forensic analysis. Several methods such as Raman spectroscopy28 and Fourier transform infrared spectrometry29 have attracted great interest, but they cannot provide sufficient identification of compounds and their 4167
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Imaging of Radix Scutellariae. TCMs have a long history in China and play irreplaceable roles in the daily life. Their studies all along attract researchers’ attentions, which focused on several aspects, such as effective constituent analysis, fingerprint analysis, biochemistry research,30,31 etc. Scutellaria baicalensis Georgi is a genus of flowering plants in the mint family, Lamiaceae. Its root, Radix Scutellariae (Huang Qin), has long been used in antioxidant,32 anti-inflammatory,33 and antitumor research.34 Flavonoids, such as baicalein and wogonin, are the active components of Radix Scutellariae. There are many reports about the efficacy of the active components, but little research has been done for the distribution of the active components in Radix Scutellariae. Here, we take Radix Scutellariae as an example to study the distribution of active components in Radix Scutellariae using PALDI MSI. The optical image of Radix Scutellariae is shown in Figure 3a. Without any sample pretreatment, the thin slice was cut and imaged using PALDI-MS. Comparing Figure 3a,b, there is a slight color change before and after the desorption of the slice. The two active components of baicalein (m/z 271.0601) and wogonin (m/z 285.0757) were confirmed by the accurate mass to charge ratio. The imaging results are shown in Figure 3c−f, in which baicalein and wogonin showed similar distribution. Most of them were concentrated at the epidermis of the root while some were concentrated at the edge of the vascular cylinder. Besides, Figure 3e,f showed the distribution of ions at m/z 187.0728 and m/z 348.2131 in Radix Scutellariae. The distribution of active compounds is helpful to improve the understanding of the relationship between compound distribution and plant structure, which will also improve the efficiency of utilization in treatment and the extraction of active components in industrial production. In addition, molecule distribution provided by PALDI MSI can help us understand the differences of TCMs from different origins and the effects of different environment on the plants growth. Our results indicated that the distribution of active components in the Radix Scutellariae slice can be successfully achieved, which made PALDI-based MSI a potential method in TCM active component distribution and related research.
spatial distribution information. Modern printed documents could be authenticated using PALDI-based MSI. The assumption that different toners have their representative mass spectra makes this idea feasible in theory. The display of laser print is based on the toner which consists of dye, such as pigment yellow and phthalocyanine blue and polymers, etc. The mass spectra of different laser print colors using PALDIMS were provided (Supporting Information, Figure S6). Ions at m/z 354.2060 were detected in red, blue, green, and yellow while ions at m/z 146.0488 were detected only in blue and green. These results showed the differences of toners at the molecular level and laid the foundation for authentication, and for further research of the imaging feasibility, imaging of printed Chinese characters, Chinese characters of Peking University ( ), was studied. For evaluating the imaging capability of PALDI-MS, different sizes of fonts were tried. Three sizes of printed blue Chinese characters were used, including 30 mm × 9 mm, 15 mm × 4.5 mm, and 10 mm × 3 mm. The color of the Chinese characters was pure blue (RGB 0, 0, 252). Two specific ions, m/z 146.0488 and m/z 354.2060 (MS/MS spectra were shown in Supporting Information, Figures S11 and S12), were used to image the blue print Chinese characters (Figure 2b,c). For Figure 3 (L) and (M), MSI results exhibit the shape of the
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CONCLUSIONS The PALDI-based MSI was accomplished under ambient conditions without any sample pretreatments. Compared with other ambient MSI, the pixel size of 60 μm × 60 μm was greatly improved.13,17 Imaging results of the Chinese seal indicated the feasibility of PALDI-based MSI. Imaging of printed Chinese characters indicated the potential applications of PALDI-based MSI in analysis and authentication of modern printed documents. Furthermore, the distribution study of active components, baicalein and wogonin, in Radix Scutellariae using PALDI-MS demonstrated the practicability of the PALDI-based MSI method in Chinese medicine research. Imaging results showed that baicalein and wogonin were concentrated at the epidermis of the root and the edge of the vascular cylinder. PALDI MSI can help us understand the differences between TCMs from different origins and the effects of different environments on the plant growth. In future studies, quantitation, sensitivity, stability, application ranges, etc. will be the focus to improve the performance of the imaging strategy. In summary, the introduction of PALDI in MSI is likely to become an important complement for current methods in various fields of research, such as identification of
Figure 3. MSI experiments of Radix Scutellariae. (a) Optical image of Radix Scutellariae slice before imaging experiments. The black circle is the marker for the edge of the vascular cylinder. (b) Optical image of Radix Scutellariae slice after imaging experiments. PALDI-MS imaging of different selected ions at (c) m/z 271.0601, (d) m/z 285.0757, (e) m/z 187.0728, and (f) m/z 348.2131.
four Chinese characters clearly, and in Figure 3 (R), the image was not so clear but could still recognize the shape. Ten mm × 3 mm size is comparable to 9p fonts, and it may be the smallest shape of characters that can be imaged using PALDI-MS. These results presented potential applications of PALDI-based MSI in analysis and authentication of modern paper printed documents, no matter Chinese characters, English words, or patterns. In a word, the above results demonstrated that PALDI-based MSI is a flexible method with great potential in analysis and authentication of modern printed documents, manuscripts, calligraphy, and patterns. 4168
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modern printed documents, TCMs’ origins and distributions of active component studies, and pharmaceutical application and small-molecule biomarkers exploration.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +86-10-62758198. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21027012, 21275012, and 21322505) and the Fundamental Research Funds for the Central Universities. Agilent Technologies is thanked for providing us with the Agilent MSD TOF MS.
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