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- 1. www.parksystems.com Análisis de morfología y propiedades nanomecánicas de bacterias probióticas Dra. Gabriela Mendoza. Cientifica en aplicaciones, Park Systems, Inc. Mayo 31, 2019
- 2. Park Systems es un fabricante líder mundial de Microscopios de Fuerza Atómica y Sistemas de Microscopía de Nanoescala Emergentes.
- 3. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 3 Park Systems permite grandes avances en la ciencia en instituciones de prestigio, laboratorios nacionales y diversas compañías a nivel mundial
- 4. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 4 Park Systems suministra una amplia gama de AFM que son adecuados para diversas aplicaciones en el mercado
- 5. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 5 Linea de productos Park AFM AFM Investigación AFM industrial - Plataforma común: escáner desacoplado - True Non-Contact - Automatización SmartScan
- 6. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 6 Park NX10 Sistema mas versátil de AFM - Modos estándar: Non-contact & tapping, contacto, PinPoint - Disponibles: SICM, STM SmartScan - Auto mode (principiante) - Manual mode (usuarios con experiencia) - Scripting (usuarios avanzados) Modos estándar EFM, PFM, KPFM & MFM. EnviroChamber, opción para control de humedad
- 7. Mayor precision – La más alta resolución – Fácil de usar 7 Escaner Park AFM Escáner XY y Z desacoplados Tecnologia Park AFM Modo: True Non-Contact™ Park SmartScan™
- 9. Contenido 9Confidential and Proprietary, Park Systems Introducción a Park Systems AFM: principio de operación Probióticos y AFM Experimento Conclusiones
- 10. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 10 AFM Principio de operación Repulsive Force Attractive Force • AFM usa un cantilever con una punta para escanear la superficie • Fuerza de repulsión → Cantilever desviar lejos de la superficie • Fuerza atractiva → Cantilever se desvía hacia la superficie • Deflexión del cantiléver → Deflexión del laser en el PSPD • Feedback loop → posición constante del laser → distancia constante entre cantilever-muestra → Registra posición Z scanner en cada pixel • Imagen de topografía AFM Sensor: cantilever Detección: laser y deflexión Deflexión: PSPD
- 11. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 11 Park AFM – Capacidad mas allá de topografía EFM MFM SICM Nano-indentacion Técnica multidisciplinaria
- 12. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 12 APLICACIONES
- 13. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 13 Probioticos Los probióticos deben sobrevivir las condiciones del tracto gastrointestinal (TGI) y adherirse al colon humano para proveer dichos beneficios. Las especies mas communes de microorganismos incluyen Bifidobacterium, Lactobacillus, Streptococcus, and E. coli no patógeno. Microorganismos vivos que cuando se administran en cantidades suficientes proveen beneficios a la salud del huésped. Tripathi MK, Giri SK.. Journal of Functional Foods. 2014; 9: 225-241. FAO/WHO , 2002
- 14. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 14 Alimentos (o nutraceuticos) con probioticos Se administran principalmente mediante alimentos funcionales
- 15. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 15 Características de los probióticos Seguro (no patógeno, ni toxico) Proliferación/colonización en el TGI Actividades deseables (inmunoestimulación) Resistente al pH del estómago, secreciones pancreáticas y biliares Efectos benéficos validados clínicamente para condiciones específicas Estabilidad y viabilidad durante la vida útil del producto en el que se administra Adhesión a mucus o células epiteliales Aluko R E. Functional Foods and Nutraceuticals. Springer; 2012. 155p. Granato D, Branco G F, Cruz A G. 2010; 9: 455–470.
- 16. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 16 Mecanismo de acción 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. Mecanismo Sitio de acción Interferencia con patogenos Producción AGCC Producción de bacteriocinas Competencia por nutrientes Adhesión Mucosa intestinal Efecto biologico Producción de sustancias antimicrobianas, H2O2, acido láctico, bacteriocinas Competencia por nutrientes y receptores Patógenos
- 17. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 17 Adhesión de probioticos La adhesion de bacterias probióticas a la superficie del epitelio intestinal y la colonización en el TGI son prerequisitos para la acción probiótica La adhesion de bacterias se basa en interacciones fisicas no especificas entre dos superficies . Se relaciona con las propiedades de la superficie que pueden ser medidas mediante 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. Bacteria probiótica Patógeno Epitelio intestinal Bloqueo de sitos de adhesión Mapeo de propiedades nanomecanicas PinPointTM
- 18. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 18 Caracterización mecánica convencional Microscopia de Modulación de Fuerza (FMM) • Cantilever accionado a una frecuencia preestablecida • Variación de propiedades mecánicas en la muestras → cambios en amplitud y fase • Altura, Fase FMM, amplitud FMM Topografía Fase FMM No cuantitativa Espectroscopia Fuerza-Distancia Trace A B C E D • Indenta un cantilever en la superficie de una muestras → datos mecánicos cuantitativos • Trace → durezamodulo • Retrace → rigidez/fuerza de adhesión Punto por punto = PinPointTM Mode
- 19. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 19 PinPoint Mode Probe sample Electronics Controller Feedback control (A-B) d (Z displacement) F • El cantilever indenta la superficie en cada pixel → 256 × 256 pxl = 65536 curvas FD • La deflexión del cantilever llega al limite → se registra la altura del escáner Z • Los mapas de topografía, rigidez y fuerza de adhesión se obtienen simultáneamente en tiempo real a partir de curvas de fuerza-distancia de alta velocidad en cada píxel Force Threshold
- 20. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 20 PinPoint Mode B. Rigidez & Modulo: Se calcula de un rango seleccionado de las curva FD A. Topografía: Movimiento del escáner Z capturado en el cantiléver con el límite de la fuerza C. Fuerza de Adhesión Calculado de la línea base de la curva FD 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
- 21. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 21 Modo Nanomecánico PinPoint
- 22. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 22 Modo Nanomecánico PinPointTM
- 23. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 23 PROBIOTICOS EFECTO BENEFICO RESTRICCIONES PARA EJERCER EFECTO BENEFICO In vitro AFM MODO PINPOINT
- 24. Probióticos: Evaluación de la morfología y propiedades nanomecánicas Park NX10 Evaluar la morfologia y propiedades nanomecánicas de bacterias probioticas y su influencia en la adhesion a un medio. Para el experimento se utilizaron probioticos disponibles comercialmente y el modo PinPoint en un AFM Park NX10. Confidential and Proprietary, Park Systems 24
- 25. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 25 Experimento Biomasa Diluciones 2 3 4 Topografia y PinPoint 1 5
- 26. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 26 Resultados Imagen AFM 10-110-5
- 27. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 27 Resultados 3D 2 um x 2 um Modo Contacto Imagen de altura muestra que la bacteria fue fijada al chip de silicio La bacteria aislada muestra un área y perímetro bien definidos con una superficie plana La bacteria aislada tiene morfología similar a Streptococcus thermophilus.
- 28. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 28 Perfil de linea y analisis de granos Dx=685 nm Imagen de topografía con análisis de línea y grafica correspondiente El análisis de granos muestra los datos de la bacteria aislada (área, volumen, longitud) La longitud de la bacteria es de (Δx) ~0.7 µm. Imagen de topografía con análisis de granos y datos del grano seleccionado
- 29. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 29 Propiedades nanomecánicas Energía de adhesión Fuerza de adhesion Altura Superficie 1 Superficie 2 Superficie 3 La adhesión de bacterias se basa en la interacción física entre dos o mas superficies
- 30. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 30 Propiedades nanomecánicas Energia de adhesion Fuerza de adhesion Altura Energía de adhesión y fuerza de adhesión tuvieron un comportamiento similar Los valores mas bajos se observaron para la bacteria comparados con el sustrato. Lo anterior indica que se requiere menos fuerza sobre la bacteria La bacteria aislada en aire es menos adhesiva que el sustrato. 400 aJ 200 aJ 45 nN 20 nN Chip de silicio Bacteria Energia de adhesion Fuerza de adhesion Altura
- 31. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 31 Conclusiones Se observó que la fuerza de adhesión y energía de adhesión están influenciadas por las propiedades de cada sustrato. Esta aplicación denota la importancia de la morfología y las propiedades nanomecánicas en bacterias y su adhesividad a un medio o superficie. Esta aplicación muestra el uso del AFM para obtener información cuantitativa incluyendo fuerza de adhesión y energía de adhesión, además de la morfología. Perspectivas Evaluar superficies funcionalizadas, estrés mecánico, diferentes cepas, mediciones en liquido.
- 32. Park Systems www.parksystems.com Copyright Park Systems Corp., All rights reserved. 32 Eventos próximos Workshop USP Campus Ribeirao Preto 4 de Junio Nano 2019 Stand #10 - Junio 5-7, Buenos Aires, AR XIX Encuentro de Superficies y materiales Nanoestucturados 2019 Workshop Colombia Julio 29 – Agosto 2 Bogotá: Pontificia Universidad Javeriana Medellín: Universidad Nacional de Colombia Medellin: Universidad EAFIT IMRC Cancun Stand #12, 18 de Agosto Nanoscientific Symposium, ciudad de Mexico 3-4 Octubre
- 33. Gracias Gabriela Mendoza gabriela@parksystems.com 33Confidential and Proprietary, Park Systems
Editor's Notes
- 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,
- 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.
- 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.
- And our AFM technology offers the best accuracy, the highest resolution, and, is the easiest to use on market
- If you want to learn more, please visit Parksystems.com for more information
- Now, after the short introductory remarks about our company, let’s get started. Here is a brief outline for today’s presentation:
- An AFM uses a cantilever with a very sharp tip to scan over a sample surface. As the tip approaches the surface, the close-range, 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, increasingly repulsive force takes over and causes the cantilever to deflect away from the surface. In short, AFM works based on the interaction between the tip and the substrate. - Detection Method A Laser beam is used to detect CANTILEVER deflection. By reflecting an incident beam off the flat top of the cantilever, any cantilever deflection will cause slight changes in the direction of the reflected beam. A position-sensitive photo diode (PSPD) can be used to track these changes. Thus, if an AFM tip passes over a raised surface feature, the resulting cantilever deflection, (and the subsequent change in direction of reflected beam) is recorded by the PSPD. - Imaging An AFM images the topography of a sample surface by scanning the cantilever over a region of interest. The raised and lowered features on the sample surface influence the deflection of the cantilever, which is monitored by the PSPD. By using a feedback loop to control the height of the tip above the surface—thus maintaining constant laser position—the AFM can generate an accurate topographic map of the surface features.
- The capability of Park AFM is far beyond topography imaging. HERE ARE SOME EXAMPLES… We have magnetic force microscopy, or MFM, which differentiate domains with different magnetic properties within a sample, here is a HDD media sample. Next, we have electrostatic force microsopy, or EFM, here are the topography and potential images of a solar cell surface. In addition, we also have a unique feature, as you can see here, termed as scanning ion conductance microscopy (SICM), which is another scanning probe microcopy technique that is ideal to image biological samples. Here is an SICM image of growth cone of neurons. Furthermore, we can do nano-indentation, which can provide LOCALIZED mechanical information about the sample understudy. I KNOW THAT MOST OF YOU ATTEND THIS SEMINAR BECAUSE YOU WANT TO LEARN MORE ABOUT NANOMECHANICAL PROP
- 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
- So now the question is, how can we measure bacteria adhesion using AFM ?
- 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?
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- With that I want to thank you for your attention. Richard?