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Park Systems Probiotics webinar
- 1. www.parksystems.com Probiotic bacteria morphologyand nanomechanical properties analysis Gabriela Mendoza, Ph.D., Applications Scientist, Park Systems, Inc. May 8th, 2019
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- 4. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 4 Park Systems supplies a comprehensive suite of AFMs that are suitable for various applications in the market Park AFM Product Line
- 5. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 5 Park AFM Product Line Research AFM Industrial AFM Full line from research to industrial AFM - Common platform: decoupled XY and Z - True Non-Contact - SmartScan automation
- 6. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 6 Park NX10 The most versatile AFM/STM/SICM system for general purpose: - Standard modes: Non-contact & tapping, contact, PinPoint - Available: SICM, STM The most versatile user coverage by SmartScan - Auto mode for ease of use (beginner level) - Manual mode for regular use (experienced users) - Scripting for programming and customized use (advanced users) Low noise Z detector based topography Standard EFM, PFM, KPFM, & MFM modes. EnviroChamber option for controlled pressure/humidity
- 7. Best Accuracy –Highest Resolution – Easiest to Use 7 Park AFM Scanner Decoupled XY and Z Scanners Park AFM Technology True Non-Contact™ Mode Park SmartScan™ Park AFM Operating Software
- 8. Learn more atwww.parksystems.com 8
- 9. Outline 9Confidential and Proprietary,Park Systems Introduction to Park Systems How AFM works Probiotics and AFM Experiment Concluding remarks
- 10. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 10 AFM Basic Principle Attractive force → Cantilever deflect towards the surface Repulsive Force Attractive Force Lenard Jones Plot AFM Schematic AFM uses a cantilever with a sharp tip to scan the surface Repulsive force → Cantilever deflect away from the surface An incident beam is reflected on the cantilever Cantilever deflection → PSPD Feedback loop → control the height of the tip and maintain constant laser position → AFM image Detection method
- 11. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 11 APPLICATIONS IN LIFE SCIENCE Confidential and Proprietary, Park Systems11
- 12. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 12 Probiotics However, probiotics must survive through the gastrointestinal tract (GIT) conditions, and stay attached to the human colon to provide the health benefits. Most common probiotic bacteria species include Bifidobacterium, Lactobacillus, Streptococcus, and nonpathogenic E. coli. Probiotics are live microorganisms that provide numerous health benefits to the host when supplied in adequate amounts, and improve the intestinal microflora of the host. Tripathi MK, Giri SK.. Journal of Functional Foods. 2014; 9: 225-241. FAO/WHO , 2002
- 13. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 13 Foods (or pharmafoods) with probiotics Probiotics are mainly administered to human in combination with food, widely used in functional food products.
- 14. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 14 Characteristics of Effective Probiotics Non pathogenic and non toxic. Able to maintain good viability. Anti-inflammatory, antimutagenic, immunostimulatory. Stability during processing, storage and transportation. Able to survive the passage through the digestive system. Able to utilize the nutrients and substrates in a normal diet. Able to attach to the intestinal epithelia and colonize. Aluko R E. Functional Foods and Nutraceuticals. Springer; 2012. 155p. Granato D, Branco G F, Cruz A G. 2010; 9: 455–470.
- 15. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 15 Probiotics and their influence on host health S. Vijayaram, S. Kannan. Probiotics: The Marvelous Factor and Health Benefits. 2018 Biomedical and Biotechnology Research Journal (BBRJ). Linares DM et al. Lactic acid bacteria and bifidobacteria with potential to design natural biofunctional health-promoting dairy foods. Front Microbiol 2017;8:846. Mechanism Host target Interference with pathogens SCFA production Bacteriocin production Nutrient competition Adhesion Intestinal lumen Mucus layer Gut microbiota Biological effect
- 16. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 16 Adhesion of probiotics Adhesion of probiotic strains to the intestinal surface and the colonization of the GIT are important prerequisites for probiotic action Bacterial adhesion is based on non-specific physical interactions between two surfaces. Adherence of bacterial cells is usually related to cell surface characteristics, which can be measured with AFM. S. Vijayaram, S. Kannan. Probiotics: The Marvelous Factor and Health Benefits. 2018 Biomedical and Biotechnology Research Journal (BBRJ). Khoder G, Al-Menhali AA, Al-Yassir F, Karam SM. Potential role of probiotics in the management of gastric ulcer. Exp Ther Med 2016;12:3-17. Probiotic bacteria Pathogen Intestinal epithelium Blocking of adhesion sites Nanomechanical property mapping PinPointTM Mode
- 17. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 17 Conventional Mechanical Characterization Force Modulation Microscopy • FMM operates in contact mode • Is used to detect variations in the mechanical properties of the sample surface (elasticity, adhesion, friction) → change in amplitude and phase • Height, FMM phase, FMM amplitude Topography FMM Phase Not Quantitative Force Distance (F-D) Spectroscopy Trace A B C E D • Indent a cantilever tip on sample surface → quantitative mechanical data • Trace → hardness/modulus • Retrace → stickiness/adhesion force • Height, modulus, adhesion force One Point at A Time = PinPointTM Mode
- 18. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 18 PinPointTM Mode Probe sample Electronics Controller Feedback control (A-B) d (Z displacement) F • The cantilever indents sample surface at each pixel → 256 × 256 pxl = 65536 FD curves • Cantilever deflection reaches threshold → Z scanner height recorded • Topography, stiffness and adhesion force maps are acquired simultaneously in real-time from high speed force-distance curves at each pixel. Force Threshold
- 19. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 19 PinPointTM Mode B. Stiffness & Modulus image: Calculated from selected range of FD curve A. Topography: Recording Z-scanner position once the threshold force is reached C. Adhesion Force image: Calculated from baseline of FD curve Force(nN) Z scanner travel distance (nm) approach retract Force threshold Stiffness calculating range Topography Δdistance (nm) Δforce(nN) Stiffness & Modulus Max. adhesion force Adhesion force
- 20. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 20 PinPoint Mode in action
- 21. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 21 PROBIOTICS BENEFFIC EFFECTS RESTRICTIONS TO PROVIDE HEALTH BENEFITS In vitro STUDY AFM SETUP PINPOINT MODE
- 22. Probiotic Bacteria Morphologyand Nanomechanical Properties Analysis Using AFM The instrument Park NX10 Evaluate how the morphology and nanomechanical properties of probiotic bacteria affect its adhesiveness to a host medium. This study used commercially available probiotics, and the PinPoint nanomechanical mode of a Park NX10. Confidential and Proprietary, Park Systems 22
- 23. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 23 Experimental setup 1 mL samples of commercial product were used to isolate bacteria Biomass pellet The wafer chip was put onto the AFM sample holder An optical microscope verified the bacteria concentration 0.1 mL drops were placed onto a silicon wafer chip Serial dilutions 2 3 4 Topo and PinPoint 1 5 Park XEI was used for image analysis
- 24. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 24 Results. Bacteria isolation AFM topography image from a 10-1 dilution Isolated bacteria 10-5 dilution
- 25. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 25 Results. Morphology images 3D image 2 um x 2 um Contact mode Topography images showed that the probiotic bacteria was attached onto the silicon wafer chip. The isolated bacteria shows a well-defined area and perimeter with a relatively flat surface. Isolated bacteria has similar morphology as Streptococcus thermophilus which is the isolated bacteria from the commercial product. Streptococcus chains are seen.
- 26. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 26 Line profile and grain analysis Dx=685 nm Line profile analysis topography image and line profile graphic. • Grain analysis show the image and statistics of isolated bacteria (i.e., area, volume, length, etc.) from the grain analysis. • The bacteria length (Δx) is ~0.7 µm from line profile analysis; which is the typical size from lactic bacteria. Grain analysis topography image and data from the selected grain
- 27. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 27 Nanomechanical properties Adhesion energy Adhesion force Height Surface 1 Surface 2 Surface 3 Bacterial adhesion is based on physical interaction between two or more surfaces
- 28. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 28 Nanomechanical properties Corresponding overlaid topography, adhesion energy and force line profiles. Adhesion energy Adhesion force Height Adhesion energy Adhesion force Height Adhesion energy and adhesion force behaved in a similar manner. Smaller values for the bacteria compared with substrate and clearly indicates that less force was required on the bacteria. The isolated bacteria in air is observed to be relatively less adhesive than that of the underlying Si substrate. 400 aJ 200 aJ 45 nN 20 nN Wafer chip Bacteria
- 29. Park Systems www.parksystems.com CopyrightPark Systems Corp., All rights reserved. 29 Concluding remarks We found from nanomechanical measurements that the adhesion force and adhesion energy are affected by the physicochemical characteristics from each substrate. This biological application addressed the importance of the morphology and nanomechanical properties of probiotics on its adhesiveness to a host medium. This application shows the usefulness of an AFM, to obtain information on probiotic bacteria including adhesion force and adhesion energy in addition to its morphology. Future work evaluate functionalized substrates, mechanical stress, different strains, measure in liquid environment.
- 30. Thank you Gabriela Mendoza gabriela@parksystems.com 30Confidentialand Proprietary, Park Systems
Editor's Notes
- #3 Before we get started, I will give you a brief introduction about Park Systems Park Systems is a world-leading manufacture of atomic force microscopes, and emerging nanoscale microscopy systems,
- #4 We develop solutions for many research and industrial applications, enabling advances in science for major universities, national labs, and a wide variety of national companies specialized in diverse fields.
- #5 Our product line covers just about every usage case with fully manual to fully automated cases. As you can see here, from XE7 to NX-Hivac for research uses on topo And from NX-HDM to NX-3DM in our industrial systems.
- #8 And our AFM technology offers the best accuracy, the highest resolution, and, is the easiest to use on market
- #9 If you want to learn more, please visit Parksystems.com for more information
- #10 Now, after the short introductory remarks about our company, let’s get started. Here is a brief outline for today’s presentation:
- #11 An AFM uses a cantilever with a very sharp tip to scan over a sample surface. As the tip approaches the surface, attractive force between the surface and the tip cause the cantilever to deflect towards the surface. However, as the cantilever is brought even closer to the surface, such that the tip makes contact with it, repulsive force takes over and causes the cantilever to deflect away from the surface. A Laser beam is used to detect CANTILEVER deflection. By reflecting an incident beam on the back of the cantilever, any cantilever deflection will cause slight change. A position-sensitive photo diode (PSPD) can be used to track these changes. By using a feedback loop to control the height of the tip above the surface and maintaining constant laser position, the AFM can generate an accurate topographic map of the surface features. In short, AFM works based on the interaction between the tip and the substrate. An AFM images the topography of a sample surface by scanning the cantilever over a region of interest.
- #12 As you may know, AFM has a wide variety of applications in diverse fields such as materials, electronics, manufacture, nanotechnology and last but not least life science. On the right hand side we can see AFM images from collagen fibers, topography of muscle fiber, cellular surface morphology
- #17 So now the question is, how can we measure bacteria adhesion using AFM ?
- #18 Two conventional methods First we have force modulation microscopy Force Modulation Microscopy (FMM) with Park AFM is an extension of AFM imaging that operates in Contact mode and is used to detect variations in the mechanical properties of the sample surface such as elasticity, adhesion, and friction. In FMM mode, the AFM tip is scanned in contact with the sample surface and the Z-feedback loop maintains a constant cantilever deflection as in constant-force mode AFM. In addition, a periodic signal known as the 'driving signal' is applied to the bimorph piezo and vibrates either the tip or the sample. The resulting tip motion is converted to an electrical signal. This electrical signal is separated into AC and DC components for analysis. The phase shift of the AC signal, called 'FMM Phase', also differs according to the elastic properties of the sample surface. Hence, FMM Phase can be used to generate an FMM image. Figure 2 shows the topography and FMM image of glass fiber-PP (poly-propylene) composite. One can observe the difference of stiffness between the glass and the PP regions. HOWEVER, FMM is NOT QUANTITATIVE. Secondly Fds aquire mechanical data quantitatively by indenting a cantilever tip on the sample surface ONE POINT AT A TIME To get a full picture of the mechanical properties of the sample, it is necessary to get both a distribution image and quantitative data simultaneously. Now, the question is: HOW DO WE DO IT? In making this practice possible, PinPoint mode provides the most effective method to acquire nanoscale mechanical maps with unprecedent speed and accuracy, This new mode was developed to be used on Park NX series AFM, allowing users to study mechanical properties at nanometer scale with accurate nN force control At this point, you might be wondering: HOW DOES PINPOINT MODE WORK?
- #19 During operation, while measuring sample morphology, the XY scanner stops at each acquisition, and during this pause, the Z scanner takes a rapid FD curve with finely controlled force, distance, and contact time between the tip and the sample. In brief, an AFM probe approaches and indents the sample surface at each pixel within the area to be imaged. Once the cantilever deflection reaches the force threshold, the Z scanner height is recorded, reconstruction of which will give the topography information of the sample In this way, pinpoint mode provides FD data in real time while reconstructing the surface topography. Let’s take a closer look at FD data for more detail about how the modulus, adhesion force data are obtained.
- #20 Here is a FD curve collected from one single data point. From the FD curve, stiffness, modulus, and adhesion force maps can be determined by an automatic calculation process in the system After the topographical data is collected, which is achieved by recording the z position once the threshold force is reached. Stiffness, and modulus can be calculated using a selected range of the FD curve. Finally, the adhesion force can be obtained after calculating the maximum from the baseline of the force distance curve. In PinPoint mode, these properties are acquired at each pixel in the entire imaging area, and displayed as image. As a result, topography, stiffness and adhesion force maps are acquired simultaneously in real-time from high speed force-distance curves at each pixel.
- #22 From probiotics in vivo and in vitro studies we begin to understand that this microorganisms provide health benefits, however they are sensitive to several factors during its processing and ingestion for example to the gastric acid, peristaltic movement. For this reason, it is important to maintain a high number of viable probiotics bacteria, and also to select strains with the ability to attach to the site of action and resilient to mechanical stress. This can be evaluated by AFM, specially by using PinPoint nanomechanical mode
- #24 To isolate probiotic bacteria, 1 mL samples of a commercial product were centrifuged to separate the biomass pellet. The supernatant was discarded and the biomass was recovered on a test tube. Then, serial dilutions were performed From the 5th dilutions, 0.1 mL were placed onto a silicon wafer chip and allowed to air dry. An optical microscope verified that probiotic bacteria were attached to the silicon wafer chip and that it was in an adequate concentration . The wafer chip was put onto the AFM (NX10, Park Systems) sample holder for further analysis. For bacteria characterization, a Park Systems NX10 AFM was used; measurements were performed in air Smart Scan and XEI software's were used for image acquisition and image analysis.
- #25 From the optical image on the left hand side, we can observe the isolated bacteria. This sample was placed in a wafer chip for its AFM analysis. 20 µm x 20 µm topography image from a 10-1 dilution where isolated Streptococcus chains are seen.
- #28 Adhesion energy and adhesion force images of a single bacteria as well as 3D topographical image were obtained at the same time on the NX-10 using PinPoint mode. The coloring of each pixel representing the elasticity and the adhesion map was determined from the calculation generated by PinPoint FD curve at each pixel. 3D topography shows exact positional match with the FD spectroscopy maps. Bacterial adhesion is based on physical interactions between two or more surfaces. If we consider the tip on the cantilever as surface 1, bacteria as surface 2, and the silicon wafer as surface 3. All three surfaces have different physicochemical properties: surface area, surface energy, surface roughness size and shape, which affect the adhesion force and energy.
- #29 The adhesion energy and adhesion force behaved in a similar manner; smaller values were observed for the bacteria compared with the substrate where they were attached. The isolated bacteria in air is observed to be relatively less adhesive than that of the underlying Si substrate. The test results show that the probiotic bacteria under study were less adhesive to the Si cantilever tip compared to the Si substrate. This phenomenon can be correlated with the adhesion of probiotic strains to the site of action, an important prerequisite for probiotic action.
- #31 With that I want to thank you for your attention. Richard?