Laser Ablation Tomography. A Meso-Scale Volumetric Visualization and Analysis Tool

Transcription

1 Laser Ablation Tomography A Meso-Scale Volumetric Visualization and Analysis Tool

2 March 2015 Cover Image: Burning Branch 2015 by L4iS Current Page: Seed with Internal Network 2015 by L4iS All images are the copyright and sole property of Lasers for Innovative Solutions LLC (L4iS) Laser Ablation Tomography (LAT, LAT SCAN) is a TM of L4iS. All Rights Reserved. Lasers for Innovative Solutions LLC L4iS 200 Innovation Blvd. Suite 261 State College, PA (814) L4iS.com

3 CONTENTS ABOUT LASER ABLATION TOMOGRAPHY - New Discoveries Begin with LAT - Primary Areas of Application for LAT Scan Volumetric Visualization and Analysis - Laser Ablation Tomography Case Studies: Two & Three Dimensional Image Analysis for LAT Scan 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 New discoveries begin with LAT TM Laser Ablation Tomography (LAT Scan ) is capable of rapidly acquiring three-dimensional structural and compositional data with micron resolution in opaque specimens. The LAT Scan process uses an ultra fast UV pulsed laser and a rapidly-swept galvanometer scanner to continuously vaporize a thin surface layer of a sample. The emitted light is directed through an optical system for filtering and imaging, creating highly contrasted, colorful stacks of images. Stacks of images are then 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 micro 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, facilitating more rapid scientific discovery. Three dimensional analysis technologies that are simple, quantitative and rapid have greatly assisted in the understanding of plant growth and development. The ability to visually screen and perform morphometric analysis of developing plants is an indispensable tool in the quest to find unique, advantageous traits and insight into complex regulatory pathways. Laser Ablation Tomography was developed to facilitate these discoveries. LAT Process: (1) Cross-sectional image is produced from Maize root. (2) Successive images are stacked together. (3) A 3D rendering of the sample is created. (4) 3D rendering can be segmented to quantify features of interest. Maize Root with Aerenchyma Volume Visualization: Laser Ablation Tomography of a Maize Crown Root.

5 Primary Areas of Application for Volumetric Visualization and Analysis Biological Sciences: Opaque Tissue Analysis Biologists are tasked with some of the world s most pressing questions. How does weather 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 engineering 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 fluorescent imaging enables queries into composition as well as structure. Natural Resource Exploration Geologists are often charged with finding new natural resource deposits. Geologists require 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 exploration. 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 designing next-generation materials and electronic devices for new high-tech applications in virtually 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 Morphometric Analysis Tomographic Models & Animations Multi / Hyperspectral Fluorescent Imaging Compositional Visualization Micron Resolution Wide Range of Scales: < 1 mm to 25 mm Fast Turn-around Time Custom Structural and Compositional Screening

6 Laser Ablation Tomography New discoveries begin with LAT TM Two & Three Dimensional Image Analysis for LAT Scan TM Cross-Sectional Images and Tomographic Models Simple, quantitative, and rapid visual analysis technologies greatly assist in the understanding of plant growth and development. The ability to screen and perform morphometric analysis of developing plant tissues is an indispensable tool in the quest to find unique, advantageous 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 incrementally ablating a specimen using an ultra-high power, picosecond pulsed duration laser. CUDA enabled libraries quickly access specimen features from captured images. Anatomical features can be measured from the two-dimensional cross-sectional images or combined to access three-dimensional parameters. The image below demonstrates a few features identified in a Maize root. Analysis is completed for hundreds to thousands of samples. Each specimen has an associated 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 assessment 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.

7 Intuitive, User Friendly Analysis Software Acquiring image data is only the first step in getting to answers. All LAT SCAN TM Systems are equipped with data logging & analysis tools that are easy to use and enable quicker, more intuitive correlations. LAT SCAN TM Log 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. Quantification Plugins Custom quantification plugins for Avizo processing of select plant anatomies. Fire are available for raw image Sample graphs to demonstrate measurement results: Specific graphs can be requested 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.

8 Laser Ablation Tomography New discoveries 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 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 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.

9 Figure 4: Maize Root 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 (µ a ) and a percent of material b (µ b ). 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. µ a + µ b = 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 f a (λ) and f b (λ), we can back calculate the relative concentrations of materials a and b. That is: µ a f a (λ) + µ ff (λ)= g(λ) b a Here g(λ) is the measured spectrum for that pixel. The concentration of each composition can then be measured by the following equations. Where µ a and µ b are measured in a calibration experiment with materials composed of 100% of each composition and is the spectrum measured in the specimen of interest. µ b = g(λ) - f a (λ) / f b (λ) - f a (λ) µ a = g(λ) - f b (λ) / f a (λ) - f b (λ) Where f a (λ) and f b (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 Channel Map OPAQUE TISSUES PORTFOLIO

11 Maize Brace Root Cross-Sectional Image Thistle Flower Cross-Sectional Image Black Ant Cross-Sectional Image

12 Dried Thistle Flower Cross-Sectional Image of Reproductive Organs

13 Maize Stalk Cross-Sectional Image

14 Series of Cross-Sectional Images of Developing Sorghum Inflorescence Image #1 Image #4,849

15 Image #1 Image #2,951 Series of Cross-Sectional Images of Cicada Killing Wasp (Sphecius speciosus)

16 Stacked Maize Leaves Cross-Sectional Image

17 Maize Seed Cross-Sectional Image

18 Silicon Infused Branch Cross-Sectional Image

19 Seed with Internal Network Blue-Channel Rendering

20 Cicada Killing Wasp High Resolution Rendering

21 Sorghum Inflorescence Digitally Dissected Developing Flower Salix Branch Regions Rendering with Vessel Locators Solanum Graft Union Digitally Dissected Hummingbird Claw High Resolution Sub-Volume Rendering

22 Robber Fly High Resolution Sub-Volume Rendering

23 Hummingbird High Resolution Model - Digitally Dissected

24 Extruding Branch Sub-Volume Xylem Rendering

25 Yellow Jacket Head Transparency Rendering Horse-fly Head High Resolution Sub-Volume Rendering

26 Bee Stinger High Resolution Sub-Volume Rendering

27 Wheat Root Digital Dissection of Volume Rendering

28 Maize Root Volume Rendering with Vessel Segmentation

29 Wheat Root Vessel Segmentation

30 Chardonnay Bud Digital Dissection to reveal cluster primorida

31 Maize Seed with Laser Induced Damage Multi-Plane Orthoslice

32 Lasers for Innovative Solutions, LLC, 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. Lasers for Innovative Solutions LLC L4iS 200 Innovation Blvd. Suite 261 State College, PA (814) L4iS.com