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LAT Portfolio
- 1. Laser Ablation Tomography AMeso-Scale Volumetric Visualization and Analysis Tool
- 2. Cover Image: BurningBranch © 2014 by L4iS Current Page: Seed with Internal Network © 2014 by L4iS All images are the copyright and sole property of Lasers for Innovative Solutions LLC (L4iS) Laser Ablation Tomography (LAT) is a TM of L4iS All Right Reserved January 2015 Lasers for Innovative Solutions LLC | L4iS 200 Innovation Blvd. Suite 214 State College, PA 16803 (814) 531-5447 L4iS.com
- 3. CONTENTS ABOUT L4iS - Rootsat Penn State University & Founding Lasers for Innovative Solutions LLC - Who We Are ABOUT LASER ABLATION TOMOGRAPHY - New Discoveries Begin with LATTM - Laser Ablation Tomography Investigation Programs - Laser Ablation Tomography Case Studies Two & Three Dimensional Image Analysis for LAT ScanTM Cross-Sectional Images and Tomographic Models Hyperspectral Imaging – Processing Raw Images to Acquire Compositional Distributions: Visualizing Cellulose and Lignin Distributions PORTFOLIO - Our Approach to the Scientific Community & How Laser Ablation Tomography Assists in World-Class Research Opaque Plant & Insect Tissues
- 4. Laser Ablation Tomography Newdiscoveries begin with LAT TM Laser Ablation Tomography (LAT Scan™) is capable of rapidly acquiring structural and compositional data in three dimensions with micron resolution. LAT Scan™ uses laser ablation micro-sectioning and multispectral fluorescent imaging to obtain highly contrasted, colorful stacks of images in opaque specimens. An ultraviolet laser-sheet is created with a 355 nanometer q-switched, pulsed laser and a rapidly-swept galvanometer scanner. A camera focused on the face of the laser-sheet captures images of the sample as it is incremented into the sheet from the opposite side. Due to the Gaussian distribution of the laser-sheet radiation, the actively irradiated surface exhibits high levels of auto-fluorescence before being ablated. This process is continuously repeated until the desired sample volume is acquired. The high intensity of the laser-sheet induces fluorescent emission in the sample’s surface layer, producing distinct spectral characteristics depending on molecular composition. After imaging a complete sample, the stack of images is processed and reconstructed into a high-resolution volume rendering that can be analyzed, quantified, segmented and virtually dissected. Although dependent on sample size and composition, acquisition speeds exceed that of conventional techniques like x-ray CT and confocal microscopy. This method allows for data acquisition at unprecedented speed with little to no need for staining or complex preparatory procedures. LAT™ Process - (1) Cross-sectional images produced of Maize root. (2) Successive images are stacked together. (3) A 3D rendering of the sample is created. (4 & 5) 3D rendering can be segmented to quantify features of interest. Maize Root with Aerenchyma Volume Visualization: Laser Ablation Tomography of a Maize Crown Root. 1 2 3 4 5
- 5. Laser Ablation Tomography Newdiscoveries begin with LAT TM Primary Areas of Interest for Meso-Scale Volumetric Visualization and Analysis Biological Sciences: Opaque Tissue Analysis Biologists are tasked with some of the world’s most pressing questions. How does weath- er influence crop production? What are the effects of genetic modifications? How can we learn from ecosystems to inspire better designs of engineered structures? LAT™ technology can provide key insight into important contemporary scientific and en- gineering applications, such as designing bio-inspired materials, next generation crops, or studying pathogens. By acquiring data at an unprecedented rate with virtually no sample preparation requirements, LAT™ can characterize specimens in minutes rather than hours or days, facilitating more rapid scientific discovery. Additionally, the multispectral fluores- cent imaging enables queries into composition as well as structure. Natural Resource Exploration Geologists are often charged with finding new natural resource deposits. Geologists re- quire precision tools to determine composition, mineralogy, and total organic content. LAT™ is a new tool that can be used to characterize samples for natural resource explo- ration. With fluorescent imaging capabilities, material composition, porosity, permeability, and microstructure can be differentiated easily. Determining total organic content, thermal maturity, and mineralogy are possible applications of LAT™. With the desire to characterize these properties on-site and with the ability to determine organic content in drill cuttings, LAT™ Scans can provide near real-time data for fracking operation. Advanced Material Analysis & MEMS Device Investigation Aided by recent advancements in material fabrication technologies, scientists are design- ing next-generation materials and electronic devices for new high-tech applications in virtu- ally all industries. Fabricating these complex three dimensional structures is now possible, facilitating the creation of materials with exotic properties. Understanding the nature of these structures is critically important to research and development efforts, underscoring the need for rapid, robust feedback. LAT™ presents a novel approach to quickly gather and model complex structural data. Whether for additive manufacturing, improving design or assessing fabrication quality, LAT™ can provide comprehensive datasets on demand. Laser Ablation Tomography Features: • Geometric and Microanatomic Structure Visualization • Compositional Visualization • Morphometric Analysis • Multi / Hyperspectral & Fluorescent Imaging • Tomographic Models & Animations • Micron Resolution • Wide Range of Scales: < 1 mm to 25 mm • Fast Turn-around Time • Custom Structural and Compositional Screening
- 6. Laser Ablation Tomography Newdiscoveries begin with LAT TM Simple, quantitative, and rapid visual analysis technologies greatly assist in the understand- ing of plant growth and development. The ability to screen and perform morphometric anal- ysis of developing plant tissues is an indispensable tool in the quest to find unique, advan- tageous traits and insight into complex regulatory pathways. Laser Ablation Tomography facilitates these discoveries. LAT™ enables high-throughput anatomical imaging of phenotypic traits in a wide variety of materials. Thousands of sequential, cross-sectional images are acquired by incremental- ly ablating a specimen using an ultra-high power, picosecond pulsed duration laser. CUDA enabled libraries quickly access specimen features from captured images. Anatomical fea- tures can be measured from the two-dimensional cross-sectional images or combined to access three-dimensional parameters. The image below demonstrates a few features iden- tified in a Maize root. Analysis is completed for hundreds to thousands of samples. Each specimen has an asso- ciated LAT™ Data-set and quantification spreadsheet, organized on a deliverable hard drive. Total specimen data is accumulated into a project summary report. This enables easy as- sessment of parameter distributions as well as easy navigation to individual data-sets for either visual inspection or further analysis. Example of Measured Parameters for a Maize Root Preserved in a 70% EtOH solution: (1) Root perimeter. (2) Cortical cells, aerenchyma, protoxylem, and xylem vessels. (3) Three dimensional rendering of identified cortical cells superimposed on Maize root. Continued on next page. Two & Three Dimensional Image Analysis for LAT ScanTM Cross-Sectional Images and Tomographic Models
- 7. Sample graphs todemonstrate measurement results: Specific graphs can be re- quested to overview the most relevant parameters. Example spread sheet with selected root parameters: Each sample ID number is hyperlinked to its subfolder for easy reference. Intuitive, User Friendly Analysis Software Acquiring image data is only the first step in getting to answers. All LAT SCAN TM 1000 β Systems come equipped with data logging & analysis tools that are easy to use and enable quicker, more intuitive correlations. Avizo ® Fire – Automated summary report of your LAT Scan™. Logs record your LAT SCAN TM system parameters for quick reference and repeatability. Avizo ® Fire delivers advanced 3D imaging workflows for scientists who require insight into the details of materials properties on full 3D structures. From straightforward visualization and measurement to advanced image processing and quantification, Avizo® Fire provides a comprehensive, multimodality digital lab for advanced 2D and 3D visualization, materials characterization, 3D model generation and calculation of physical properties. Custom quantification plugins for Avizo® Fire are available for raw image processing of select plant anatomies. LAT SCAN TM Log – Quantification Plugins –
- 8. Laser Ablation Tomography Newdiscoveries begin with LAT TM Processing Raw Images to Acquire Compositional Distributions: Visualizing Cellulose and Lignin Distributions in Maize Roots Raw lignin and cellulose were illuminated with the 355 nm laser source and their spectrum characterized with a monochrome CCD camera and a liquid crystal tunable filter. Images were captured from 420-700 nm. The average spectrum for both cellulose and lignin were calculated by manually subdividing the image according to the regions in Figure 1. Each pixel in the image has an associated spectrum which was averaged to generate the curves in Figure 2. Once the average curves were known, a cubic polynomial was fit to the curve for later comparison. Using the same procedure for illuminating the raw materials, a maize root was imaged after following a complete removal of a surface layer via our laser. The auto-fluorescent spectrum was characterized using the hyperspectral camera. A representation of the maize root cross section is shown in Figure 3. Figure 1: Hyperspectral image of (1) cellulose and (2) lignin. Boxes represent subsections used to identify average spectrum. Figure 2: Representative spectrum identified in the raw material. The cubic polynomial is shown for the case of lignin. Figure 3: Represented Maize Root cross-sectional image. Continued on next page. 1 2
- 9. Figure 4: MaizeRoot spectral data points plotted against reference samples’ polynomials By assuming that the Maize root is only composed of two compositions (lignin and cellulose) it becomes possible to back calculate the relative concentrations in the root even though in some places the two spectrum will superimpose on one another. We assume that each pixel is an approximate representation of a volume of material containing a percent of material a (µa ) and a percent of material b (µb ). We make the following assumptions: (1) the light incident on a single pixel is only composed of the light from an individual, discrete sub-volume, (2) the light coming from the sub-volume is only composed of fluorescent light, & (3) Only the constituents of interest contribute to the fluoresced light. µa + µb = 1 Second, we assume that the light emitted by the pixel sub-volume is the superposition of the light emitted from the constituents. Therefore, if we know the spectrum for each of the constituents fa (λ) and fb (λ), we can back calculate the relative concentrations of materials a and b. That is: µa fa (λ)+ µb ffa (λ)= g(λ) Here g(λ) is the measured spectrum for that pixel. The concentration of each composition can then be measured by the following equations. Where µa and µb are measured in a calibration experiment with materials composed of 100% of each composition and is the spectrum measured in the specimen of interest. µb = g(λ) - fa (λ) / fb (λ) - fa (λ) µa = g(λ) - fb (λ) / fa (λ) - fb (λ) Where fa (λ) and fb (λ) are measured in a calibration experiment with materials composed of 100% of each composition and g(λ) is the spectrum measured in the specimen of interest.
- 10. Boll Weevil Blue ChannelMap OPAQUE TISSUES PORTFOLIO
- 11. Maize Brace Root Cross-SectionalImage Thistle Flower Cross-Sectional Image Black Ant Cross-Sectional Image
- 12. Dried Thistle Flower Cross-SectionalImage of Reproductive Organs
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- 14. Image #1 Image #4,849 Seriesof Cross-Sectional Images of Developing Sorghum Inflorescence
- 15. Series of Cross-SectionalImages of Cicada Killing Wasp (Sphecius speciosus) Image #1 Image #2,951
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- 19. Seed with InternalNetwork Blue-Channel Rendering
- 20. Cicada Killing Wasp HighResolution Rendering
- 21. Sorghum Inflorescence Digitally DissectedDeveloping Flower Salix Branch Regions Rendering with Vessel Locators Vitis amurensis Dormant Bud Sub-Volume Rendering of Bud Hummingbird Claw High Resolution Sub-Volume Rendering
- 22. Robber Fly High ResolutionSub-Volume Rendering
- 23. Hummingbird High Resolution Model- Digitally Dissected
- 24. Extruding Branch Sub-Volume XylemRendering
- 25. Yellow Jacket Head TransparencyRendering Horse-fly Head High Resolution Sub-Volume Rendering
- 26. Bee Stinger High ResolutionSub-Volume Rendering
- 27. Wheat Root Digital Dissectionof Volume Rendering
- 28. Maize Root Volume Renderingwith Vessel Segmentation
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- 30. Lasers for InnovativeSolutions LLC | L4iS 200 Innovation Blvd. Suite 214 State College, PA 16803 (814) 531-5447 L4iS.com ©Lasers for Innovative Solutions, LLC, 2014. L4iS and the L4iS logo are trademarks of Lasers for Innovative Solutions LLC. Laser Ablation Tomography & LAT SCAN are trademarks of Lasers for Innovative Solutions LLC.