AFM: method and applications

Transcription

1 AFM: method and applications Ludmilla Morozova-Roche Umeå University February 16, 2017

2 AFM technology uses a sharp tip (in the range of few nanometers) at the free end of a cantilever and detects low forces (few piconewtons 1 pn = N) to provide high resolution information of the sample without its damage. AFM is capable of measuring nanometer scale images of various nano-object and surfaces with little or no sample preparation. AFM was invented by IBM Scientists in The precursor to the AFM - the scanning tunneling microscope (STM), was developed by Gerd Binning and Heinrich Rohrer in the early 1980s at IBM Research - Zurich, a development that earned them the Nobel Prize for Physics in Binnig invented the atomic-force microscope and the first experimental implementation was made by Binnig, Quate and Gerber in The first commercially available atomic-force microscope was introduced in 1989.

3 Bruker AFM Bioscope Cathalist and Nicon inverted fluorescence microscope

4 Bruker scanner AFM scanner

5 Agilent AFM

6 Agilent AFM scanner

7 AFM principle

8 We have the probes you need: Force Calibration Fluid Imaging Lithography Nanoindenting Force Spectroscopy (Biological) SCM TUNA SSRM EFM MFM LFM Phase Imaging Fast Scan Piezo Response STM imaging Polymers Scratching Nano particles And much more! AN AFM IS ONLY AS GOOD AS ITS PROBE which is why we manufacture our own AFM probes to provide the most comprehensive range (over 200!) for any experiment type. We also have expertise in guidance on probe selection. Please visit our web page to see our entire probe and accessory offerings. Also a worthwhile download is the recently released 2011 Probes and Accessories Guide.

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10 Interection of cantiliver with the surface: contact, tapping (intermitten contact) and non-contact modes

11 Tapping mode

12 Force Catalyst: technology strengths for force spectroscopy experiments Peak Force Quantitative Nanoscale Mechanical Characterization Peak Force Imaging Stiffness: DMT model Stiffness Fit Dissipation Adhesion Imaging Z

13 Peak Force QNM Mode PFQNM is a new mode that quantitatively maps out nanomechancial properties of a sample. It works my performing a fast force curve at every pixel of the image (like a fast, hi-res version of force volume). It then simultaneously analyses each force curve and calculates adhesion, stiffness, energy dissipation etc and plots the values in separate image channels to generate up to 8 distinct datasets. Axes in relevant calibrated quantities of Newtons, ev, Pa Example: Rubber in a polystyrene matrix

14 Force Curve Controls The generic controls of the force curve is probably the most important aspect of all. We have numerous feature that allow flexibility and control of your experiment including: Force curve measurement with control on approach and retract speed, maximum force, surface delay, retraction delay Z closed-loop operation to avoid hysteresis, nonlinearity and creep of Z piezo Force scripting to enable custom cycles and force clamp operation Control of force and piezo extension Configurable feedback parameters for each segment of a script User defined looping Fetch molecule option detects presence of interaction before starting routine of script

15 Point&Shoot Mode Point and Shoot mode allows an AFM image to be taken and then force curves can be targetted and acquired by simply clicking at a location in the image. This takes advantage of highly precise closed loop scanners. Force curves can be generated point by point, as lines with fixed spacing or as arrays with fixed row/ column spacing as shown in image (left)

16 10 μm scans of a multilayered polymer film Peak force mode Tapping mode height height adhesion phase image Topography (A) and corresponding modulus (B) images of living MDCK cells. Image size 32 um. Young s modulus Dark strips=100 Mpa Light strips=300 MPa

17 Catalyst: Software interface for logical experiment design and workflow

18 MIRO Mode MIRO (microscope image registration overlay) allows the same idea as Point & Shoot but using the IOM optical image. Now the brightfield/ darkfield/ DIC/ fluorescence image can be used to target force curves or target imaging as shown below.

19 Infected HELA Cells As discussed, full optical integration only requires teaching each IOM objective what the AFM scan range is within that objective field of view (takes 2 minutes and only needs to be done once). Secondly when inserting a new AFM probe into the AFM you need to tell the system where the tip is located (by pointing and clicking at it in the video screen takes 10 secs and needs to be done for each new tip). After this we can use the optic image to navigate the AFM scan as shown left. The ease of use for imaging cells etc is then fantastic and perfectly correlated.

20 Higher Harmonics imaging Imaging at different harmonics is suggested for clarification of contribution of various nonlinear (mechanical and adhesive) tip-sample interactions to the image contrast and the quantitative estimate of these properties. Page 20 Dr. Gerald Kada Nanotechnology Measurements Division

21 Higher Harmonics imaging (MAC mode in liquid) Bacterial S-Layer Topography (a-left) and 2 nd Harmonic image (a-right) (b) Averaged image of 55 unit cells 0.5 nm resolution on protein crystal in liquid (MAC) 3 pm amplitude sensitivity in 2 nd harmonics Preiner et al, PRL 2007 Page 21 Dr. Gerald Kada Nanotechnology Measurements Division

22 Live Human Rhinovirus (MAC mode in liquid) RNA release from virus by decreasing ph Kienberger, Hinterdorfer et al, J Virology (2004) & Structure (2005) Page 22 Dr. Gerald Kada Nanotechnology Measurements Division

23 Example Transport through nuclear pore membrane Topography imaging Page 23 Dr. Gerald Kada Nanotechnology Measurements Division

24 Topography and Recognition Imaging (TREC) Topography Recognition 50nm DNA-protein complexes as stored in the chromosomes H. Wang, ASU Page 24 Dr. Gerald Kada Nanotechnology Measurements Division

25 Single Molecule Studies of Antibody Antigen Interaction Strength Versus Intra-molecular Antigen Stability Sendai-loop Anti-Sendai Antibody on the Tip, Bacteriorhodopsin Surface ( Purple Membrane ) Unravelling single Bacteriorhodopsin Kienberger, Kada et al, JMB 2005 Preiner et al, BJ 2007 Sequence-dependent pulling pattern with and without Sendai-loop Page 25 Dr. Gerald Kada Nanotechnology Measurements Division

26 Our AFM applications

27 Amyloid structural changes Aggregation Foding From Morozova-Roche, Malisauskas, Curr.Med. Chem., 2007

28 Alzheimer's disease Anti-fibrillar antibodies S100A9 antibodies

29 Amyloid formation in aging prostate AFM and TEM imaging of ex vivo S100A8/A9 amyloid structures In vitro S100A8/A9 amyloids formed at ph 7.4 and 37 C with agitation In vitro S100A8/A9 amyloids formed at ph 2 and 57 C S100A8/A9 amyloids are formed in the presence of Ca and Zn, but not EDTA Yanamandra et al., PLoS One, 2009

30 Amyloid properties of S100A9 0.1 mm in PBS ph 7.4, 37 C, 500 rpm after 2h (A), 3 days (B) and 7 days (C) D E F 0.05 mm, 2 d, 500 rpm (D), 0.02 mm, 2 d, 700 rpm (E) and 0.05 mm, 3 d, 700 rpm Scale bars are 250 nm Wang et al, Acta Neuropath., 2014

31 Amyloid formation of Ab and S100A9 Ab, 1 d Ab:S100A9=1:1; 0.1 mm, 1 d Ab, 5 d Ab:S100A9=5:1; 0.02 mm S100A9, 1 d and 2d

32 Schematic summary of multiple amyloid pathways of S100A9, Aβ peptides and co-aggregated S100A9-Aβ. The aggregated structures of individual polypeptides are outlined in red; in co-aggregated complexes S100A9 is schematically denoted in yellow and Aβ in blue.

33 Aggregation and cytotoxicity of S100A9 and Ab 40 amyloid structures (a) Viability of SH-SY5Y cells measured by WST-1 assay after 24 h co-incubation with S100A9 amyloids. The viability of untreated cells is taken as 100%. White bars correspond to cells treated with S100A9 linear protofilaments, black bars with S100A9 rings. S100A9 concentration is shown along x axis. (b) Viability of SH-SY5Y cells measured after 24 h co-incubation with Aβ and S100A9-Aβ amyloids. Dashed bars correspond to cells treated with Aβ amyloids and grey bars with coaggregated S100A9-Aβ. Grey bar at zero concentration corresponds to the viability in the presence of 20 M S100A9 amyloids as in (a). Wang et al, Acta Neuropath., 2014

34 Cytotoxicity of S100A9 and Ab 42 amyloid structures Viability of SH-SY5Y cells measured by WST-1 assay after 24 h co-incubation with Aβ(1-42) and S100A9 amyloids. Aβ(1-42) concentration is shown along x-axis. S100A9 was at 20 M. Olive bars correspond to cells treated with coaggregated Aβ(1-42)-S100A9; bars with horizontal stripe pattern - with Aβ(1-42) oligomers and protofilaments formed after 7 h (a); bars with diagonal stripe pattern to cells treated with Aβ(1-42) mature fibrils (b). Wang et al, Acta Neuropath., 2014

35 Amyloid oligomers

36 Amyloid oligomers of equine lysozyme A Spherical cap model B C D Volume of the particles: V AFM =(πh/6)(3r 2 +h 2 ) E F G Volume of monomeric protein: V m =(M o /N o )(V 1 +dv 2 )

37 Population analysis of equine lysozyme amyloid oligomers Oligomers formed at ph 4.5 and 57 ºC Oligomers formed at ph 2.0 and 57 ºC 0 h 0 h 72 h 24 h 72h - 0h 24h - 0h 72h - 24h Malisauskas et. al., JBC., 2005

38 Cell viability (%) Stoichiometry of cytotoxic amyloid oligomers Amyloid oligomers imaged by AFM Height of structures, nm Number of monomers * Number of monomers ** ± ± ± ± ± 1 8 ± ± 4 25 ±7 Toxic Very toxic *was calculated by using spherical cap model **was determent by using grain analysis module of SPIP software controls Time of amyloid incubation, h Malisauskas et. al., JBC., 2005

39 Amyloid oligomers of Ab peptide Size distribution Gruden et al., J Neuroimmunology, 2011

40 Autoimmune responses to amyloid as biomarkers of Alzheimer's diseases Gruden et al., J Neuroimmunology, 2007

41 Amyloid destabilisation and removal

42 Insulin lability landscape in coordinates of ph and amyloid ageing Malisauskas et al., JMB, 2011

43 Degradation of insulin amyloid fibrils by protease K Decay of ThT fluorescence upon 50x dilution into ph 8.1 buffer containing protease K of insulin stock aged 24h (blue), 28h (green), 32h (yellow), and 44h (red). Rate constants from the exponential fits of the time decays during the protease K amyloid digestions Malisauskas et al., JMB, 2011

44 Degradation of insulin amyloid fibrils by protease K monitored by real time AFM in liquid. rate 4 nm/min 53min 1:24 1:46 2:09 2:37 2:56 3:14 3:32 Malisauskas et al., JMB, 2011

45 Cellular properties studied by AFM

46 Imaging of SH-SY5Y living cell by AFM Cell before adding S100A9 Cell with 100A9 46

47 Deformation, Young Modulus and Height Cross Sections of SH-SY5Y cells 47

48 Jagan Mohan, Björn Morén, Elin Larsson, Mikkel Holst and Richard Lundmark Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae, J Cell Sci, 2015, accepted.