200 lines
15 KiB
Markdown
200 lines
15 KiB
Markdown
# APPI_art
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Markdown editable del articulo de APPI
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Basic backbone structure of APPI MSI
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Introduction
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- state of the art MSI. MALDI.
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- state of the art of AIMSI. LD-LTP, DESI, etc.
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- 3D printing in the AIMS world.
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- conventional troubles of imaging and AIMSI
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- APPI, definition, state of the art MS – biological samples.
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- Pros of APPI, very soft ionization, permits keeping molecules singly charged, only uses electricity, noble gases nor nitrogen cooling are needed.
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Experimental Methods
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Plants, how were they grown? (Biological material)
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Solanum lycopersicum, Micro-Tom variety, grown from seed, 3 seeds per pot, july 15th, imaging essays, october 7th,
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Substrate:
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20% perlite, 60% peatmoss 20% Quercus leaf litter,
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1L plastic pots.
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200 ml of water per pot, 3 times a week
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APPI Model, ASK NOVIONX HOW IT WAS DEVELOPED, krypton gas photodiode, volts provided(11 V), etc
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Laser desorption, ultra-violet (450?nanometers?) 12 v constant beam, lens used (Unknown camera model lens).
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LA-APPI-MSI system. Description.
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Data analysis, MALDI quant. Analysis using Julian’s software, description of the GUI and repository, JuliaMSI, normalization of masks for comparisons.
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Results
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Graphical abstract: Imaging process, muestra sobre placa, desorción, ionización, inlet, indicar que la placa es movible (OPEM LABBOT), image final a lado del setup como si fuera un "output", abajo del image poner algun análisis estadístico gráfico.
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Possible figures:
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fig 1: General physical aspects, a) RENDER, movilidad, etc b) Real photos, mounted in front of LCQ fleet.
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Fig 2: PEDIR A NOVIONX ESQUEMATICA/ RENDER DE LO INTERNO DEL APPI, IGUAL YA ESTÁ PATENTADO.
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fig 3: Dimensions of laser, resolution, A) microscope con escala chido, B) ejemplo de la medición casera.
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Fig 4. Imaging de jitomate bien, este peude ser del de daño mecanico, Side by side foto real vs imaging.
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fig 5. GRANDE. Información del experimento. Hojas distales, imaging de las hormonas especificamente y un empalme 50/50. barras despues de normalización y máscaras, comparación de hormonas. Como se ve montado en si la hoja en la placa. Espectro total. Fragmentation mirror plot. Show case mini de juliaMSI.
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Final system composition, photo of system. - Fusion 360 diagrams
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Distance optimization, graphs using MALDIquant
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Resolution under microscope - Orly
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Laser energy using 12 volts and amperage (how many?).
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Tomato mechanical damage experiment. Paper hole punch pliers, Show spectra, various important m/z. - pregunta biologica.
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Tomato plant volatiles, Fragmentation essay
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Jasmonic acid vs standard
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Salicylic acid vs standard
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REPEAT: fungi, take photo for 6-PP. FRAGMENT TOMATO PLANT VOLATILES VS STANDARDS LIKE JAZMONIC ACID.
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Possible experiment: Extracts vs standards on dried paper spots/TLC - matrix effect.
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Discussion
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Differences between LD-LTP system, things before that couldn’t be seen. Compare between abigail’s article and ours.
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Focus on that it's an all electrical system without the need for gases like nitrogen cooling or helium for LTP ionization.
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Introduction:
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Methods:
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**APPI case development**
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OMAR DESCRIBE EL DESARROLLO DEL CASE DE APPI. Sobre todo agrega las dimensiones reales del case y como se monta.
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The support components for the krypton lamp were designed using Autodesk Fusion 360° software. For a detailed visualization and real dimensions of the pieces, see the complementary section.
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The full assembly is 184 mm (depth) x 282.5 mm (height) x 150 mm (length) and consists of four main components:
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A rail that is attached to the underside of the aluminum profile that forms the body of the automated platform using screws and T-nuts.
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The laser bracket, whose upper part can be inserted into the rail, allowing adjustment of the horizontal distance of the laser beam relative to the ion transfer unit of the mass spectrometer. Once the adjustment is satisfactory, the screws can be tightened to lock this distance in place.
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The lens bracket, which is adjusted via a vertical rail on the side of the laser bracket, allowing adjustment of the laser's focal point distance. This distance can also be locked in place using screws.
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The krypton lamp bracket, which attaches via a horizontal rail, allowing adjustment of the lamp's distance relative to the ion transfer unit. The krypton lamp housing has a hinge-like joint for easy insertion and removal of the lamp.
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**3D Printing**
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All support components were printed using an Artillery 3D printer (Cite Model). The printing material used was PETG in black and gray (Cite brand and catalog) with an average melting point (Cite). The software used for slicing was Ultimaker Cura (Cite Version) at a 1:1 scale; it was not necessary to resize the STL model. The bed temperature was 85°C, and the extruder temperature (Cite extruder nozzle diameter) was 245°C. The approximate printing time varies from 1.5 hours for the Krypton lamp case to 10 hours for the Laser case. The printing parameters can be found in (Cite section, add table or screenshot of Ultimaker Cura printing parameters). 3 mm diameter hexagonal screws were used, and the corresponding holes were modeled with this diameter for the parts where it was necessary to join several pieces lock a certain position or allow for freedom of movement.
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**Instrumentation**
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The imaging experiments were performed on the Open LabBot developed by Ignacio Rosas-Roman (Ignacio Rosas-Roman, et al). The Mass Spectrometer used for all experiments was an LCQ-fleet ion trap by Thermo Scientific, which ran in positive mode and profile data acquisition mode. A long ion transfer was used for the mass spectrometer inlet (ADD LENGTH). An in-house bypass adapter (CITAR EL BYPASS) was coupled to the ion trap for the AIMS experiments.
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For the imaging experiments, we performed them on an APPI source (NovionX, https://www.novionx.com/) with a heraeus photoionization detector lamp (PID) (ahora conocido como excelitas) mounted to the Open LabBot using the 3D printed and developed in-lab APPI case (see section x). The APPI source was run on a Steren regulated DC power supply at 10.6 V (CITAR DONDE ENCONTRAR FUENTE DE PODER). The developed APPI case was placed perpendicularly at a 2 mm distance from the inlet.
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**VARIABLE REGULATED LASER POWER SOURCE (PEDIR A RAMON QUE EXPLIQUE COMO LO HIZO)**
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The word LASER stands for Light Amplification by Stimulated Emission of Radiation. It is a PN junction device based on semiconductors that converts electrical energy into luminous energy. It generates a coherent and monochromatic light of high intensity. The emitted radiations have the same frequency and phase or, in some cases, a very narrow bandwidth. A semiconductor laser diode serves as the gain medium of an external cavity (ECL). The laser diode can have wavelengths in the range of 375 to 2000 nm and output powers from 0.2 mW to 2 W. It is a semiconductor device approximately 250 to 500 μm long and about 60 μm thick, mounted on a copper or ceramic heat sink [https://doi.org/10.1016/B978-012222695-3/50009-X]. The current is injected through an upper ohmic contact. Photons are generated and guided by the epitaxial layers of the structure as shown in figure x1.
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Fig x1.- Parts of a laser diode.
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For laser desorption, a UV laser (λ = 450 nm) was used. The laser was powered by a variable and regulated power supply and operated at 12 V. The laser was mounted onto the Open LabBot using a custom, in-house–designed 3D-printed APPI housing, which positioned the laser perpendicular to the APPI source. The laser beam was focused using a lens, with the focal point located approximately 1 cm below the mass spectrometer ion transfer inlet and between the APPI focusing lens and the ion inlet. The output power of a laser diode is linearly related to the supplied current, so an increase in current results in a proportional increase in light intensity. Therefore, a voltage source with current regulation was designed to control the laser's power. This source can vary from 0 V to 30 V and features a current adjustment to regulate the output power. The current and voltage are monitored using a multimeter and a digital ammeter, and both voltage and current can be adjusted with a potentiometer.
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**Biological samples**
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Eighteen seeds of Solanum lycopersicum, variety Micro-Tom (CITAR QUIEN PROPORCIONÓ LAS SEMILLAS), were sown in six groups of three seeds each in 1L plastic pots with a substrate composed of 20% perlite, 60% peat moss, and 20% oak (Quercus) leaf litter. The plants were grown for 75 days, watered with 200 ml of water per pot three times a week, and placed in sunlight (approximately 5 hours of direct sunlight).
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**Software**
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For coordinate assignment to the robotic platform and synchronization of data acquisition, the LABI-Imaging software (part of the Open LabBot ecosystem) was utilized (CITAR A NACHO). MSI data were then initially acquired in proprietary RAW format and subsequently converted to the mzML open standard using msconvert (CITAR MS CONVERT). To leverage high-performance computational capabilities alongside extraction of meaningful metabolic information from the MSI data samples, we employed JuliaMSI for mzML file conversion to imzML (CITAR JuliaSMI). Regions of Interest (ROIs) were defined using binary masking via Otsu thresholding within the JuliaMSI graphical user interface to focus the analysis on biological tissue, enabling precise spatial segmentation before spectral processing. This approach allowed us to focus on biological tissue and minimize external sample noise.
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Using JuliaMSI's modular preprocessing pipeline, we processed all spectra within the defined ROIs of each sample's defined mask. The pipeline consisted of targeted peak picking for untargeted analysis, peak alignment synchronized across all spectra to a common m/z axis, and peak binning aggregated m/z intensities.
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For spectral feature extraction, we applied a local maxima peak detection algorithm with a Signal-to-Noise Ratio (SNR) threshold of 3.0 and a half-window size of 3. To distinguish metabolic signals from instrumental noise, we enforced a minimum peak prominence of 0.5 and a merge tolerance of 0.1 m/z. Additionally, we conducted LOWESS regression for peak alignment with an absolute m/z tolerance of 0.1 and a maximum shift constraint of 50 ppm. To ensure statistical robustness, we applied adaptive binning with an absolute tolerance of 0.1 m/z and a maximum bin width of 75 ppm. This method defines bin boundaries based on the local density of detected peaks across all spectra. A frequency threshold of 10% was also enforced to retain only those features present in at least 10% of the total spectra within the defined ROI.
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**Analysis of spatial distribution of metabolites with LD-APPI in Solanum lycopersicum plants** REVISAR ESTA PARTE CON OMAR, ESTÁ MAL PLANTEADO
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Plants grown to 75 days post germination were subjected to mechanical damage. Leaf samples were taken after 96 hours of exposure to mechanical damage. The first distal leafs proximal to the mechanically damaged leafs were analyzed at 0 hrs and at 96 hrs. First were cut from the stem and fixed using double sided tape on a sheet of glass (AGREGA DIMENSIONES). Glass plates containing leaf samples were placed on the Open LabBot imaging plate. Leaf samples were placed at the focal point of the laser (1 cm under the ion inlet; ESTA ERA LA LONGITUD?). APPI source was set to 10.6 V. UV laser was set to 12 V using the variable regulated power source.
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After 75 days from the germination of the first seed, two plants from the same pot were selected for imaging using the LA-APPI-MSI technique. In the first plant, the image was taken immediately after cutting a leaf from the third group that emerged after the cotyledons (control, 0 hours). In the second plant, the image was taken immediately after cutting a leaf from the sixth group that emerged after the cotyledons (control, 0 hours). hours, distal leaf).
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Two plants from a different pot were also selected to undergo mechanical damage using Paper hole punch pliers (CITE DIAMETER), making two perforations in a leaf of the 3rd group in both plants. The image was obtained in one plant immediately after making the perforation (Mechanical damage 0 hours), while in the other plant, the image was obtained immediately after cutting a leaf belonging to the 6th group, 96 hours after the lesion had been made in a leaf of the 3rd group that emerged after the cotyledons (mechanical damage, 96 hours, distal).
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**Results**
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**Ionization source and laser coupling assembly**
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**Dimensional resolution analysis**
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**Mass Spectrometry Imaging results* Daño mecanico original**
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**Detection of Pharmaceuticals**
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**Methyl Jasmonate production in response to mechanical damage in Solanum lycopersicum**
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**Discussion**
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Conclusion:
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References:
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INSERT FIGURE 1, Full Assembly main parts, Movement directions. APPI_MAIN_PARTS_MOVEMENT_1.png
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Fig 1. Diagram showing the complete assembly of the parts and the movement directions.
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A)General view of the main components, the rail that attaches to the structural frame of the automated platform, the laser case, the rail and lens support, and the hinged case for the krypton lamp.
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B) Visualization of the freedom of movement of the components.
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INSERT FIGURE 2, Movement. APPI_MOVEMENT_2.png
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Fig 2. Diagram showing the movement directions.
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C) Movement Directions
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D) Close-up view of the Krypton lamp hinged case movement
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INSERT FIGURE 3. Krypton lamp hinged case close-up. APPI_KRYPTON_LAMP_CASE_CLOSE_UP.png
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E) and F) Close-up of the opening movement of the krypton lamp case, closed and open position, respectively.
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G) Close-up of the open case, showing the location of the krypton lamp.
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H) Close up view of the case, showing the cone-shaped zone where the ionization occurs.
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INSERT FIGURE 4, Real dimensions of the APPI support structure. APPI_ASSEMBLY_DIMENSIONS.png
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I) General dimensions of the full assembly.
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INSERT FIGURE 4, APPI Heraeus Lamp Picture, Diode, APPI Case, Electronics.
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Fig 4. Heraeus Krypton Lamp Developed by NovionX.
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INSERT FIGURE 5, Variable Power Source, Developed by (CITAR A ROMAN)
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Fig 5. Variable Power Source conected to the laser.
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INSERT FIGURE 6, Variable Power Source Used with the Heraeus Kripton Lamp.
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Fig 6. Variable Power Source Conected to the Heraeus Kripton Lamp.
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Open LabBot and RmsiGUI: Community development kit for sampling automation and ambient imaging. https://doi.org/10.1016/j.microc.2019.104343
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Sierra-Álvarez, J. J., Camargo-Escalante, M. O., Sierra-Álvarez, C. D., Hernández-Caricio, C., Moreno-Luna, J. F., Buendía-Corona, I., & Winkler, R. (2025). JuliaMSI: A high-performance graphical platform for mass spectrometry imaging data analysis. Analytica Chimica Acta, 344613.
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