FluidFM ADD-ON for your JPK AFM system | Product Note

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1. 1 I ntegration of Cytosurge FluidFM ® functionality into BioAFM prod ucts Microfluidic techniques for s ingle c ell m ani pula - tion The use of microfluidic s for single cell manipulation applica - tions is a fast - growing field. Conventional techniques for cell manipulation that have led to valuable scientific findings fea - ture m icro - needle cell insertion [1] , pipette pulling [2] , canti le - ver - based cell scraping [3] , and micropipette cell aspiration [ 4, 5] . However, most of th ese techniques are either techni - cally challenging or not straightforward to interpret in terms of mechanical models . S ingle cell force spectroscopy appli - cations with functionalized cantilevers are an excellent al - ternative for measuring adhesion forces [6, 7] , but also re - quire labor - intensive protocols. In recent years , the Cyto surge FluidFM technology has emerged as an excellent solution that features the best of both microfluidic and force microscopy solutions [ 8 – 1 2 ] . FluidFM t echnology Th e FluidFM technology features FluidFM probes that have a force - sensing , micro - machined cantilever with an inner capillary channel and a fluidic pressure control system (Fig. 1) , which enables both suction and precise localized injec - tion [ 8, 11] . The FluidFM probes can be operated at a liquid flow rate down to < femto - lit er /sec and with a software - con - trolled applied pressure range of - 800 to 1000 mbar. Varying in size, stiffness, and type , the probes can be used in a range of applications such as single cell manipulat ion and adhesion measurements (FluidFM micropipettes), nano - printing , spotting , and bacterial cell adhesion (FluidFM na - nopipettes), as well as cell injection /extraction and basic im - aging applications ( FluidFM nanosyringes) (Fig. 2) . The Flu idFM ADD - ON (prob e, AFM specific probe holder, pres - sure control, pump unit, and software) is available as an add - on for all Bruker BioAFMs including BioScope® Re solve . A fully integrated solution , where the FluidFM func - tionality is completely embedded in JPK BioAFMs , is al so now available. Fig. 1: Setup. NanoWizard® 4 XP BioScience AFM with a Cyto surge FluidFM ADD - ON . FluidFM and JPK BioAFM family - a perfect match J PK BioAFM systems provi d e the perfect capabilities to lev - erage the FluidFM technology to a maximum . Thanks to the best closed loop sensors on the market, JPK BioAFMs pro - vide the most sensitive force control of the probe and high - e st accuracy for probe to sample positioning in xyz. With our leading probe - scanning design , where the sample stays stationary during operation of the AFM, the user has a n un restricted optical view o f the sample at all times . Fig. 2: Examples of FluidFM probe types . FluidFM micropipette with a 4 μm aperture (left), and FluidFM nanopipette equipped with a pyramidal tip with a 3 00 nm aperture (right). Image courtesy of Cytosurge AG .

5. 5 Benefits of FluidFM – JPK BioAFM combo ▪ Force sensitivity down to picoNewtons with Brukers low - est noise deflection detection ▪ Sub - nm positioning accuracy in xy and z as a result of market - leading closed - loop technology ▪ Combination with inverted optical microscope tech - niques , including p hase contrast, f luorescence, super - resolution microscopy ▪ Purpose - built probe - holder for JPK BioAFM s ▪ Lowest l iquid flow rate down to < f L /sec ▪ Pressure range - 800 to 1000 m b ar for highest flexibility in application s ▪ Two options: Either as add - on with Flow Control Software module OR f ull integration into JPK BioAFM systems via V7 software ▪ New DirectOverlay 2 feature for easy and precise optical navigation and combined information ▪ RampDesigner for synchronization of force, contact time , pressure and pressure duratio n Acknowledgements C ell samples were provided by Prof. A. Hermann, Humboldt University, Berlin , Germany References 1. Coman DR (1961) Adhesiveness and Stickiness: Two Inde - pendent Properties of the Cell Surface. Cancer Res 21:1436 – 1438 2. Colbert M - J, Raegen AN, Fradin C, Dalnoki - Veress K (2009) Adhesion and membrane tension of single vesicles and living cells using a micropipette - based technique. Eur Phys J E 30: . doi: 10.1140/epje/i2009 - 10514 - 7 3. Sagvolden G, Giaever I, Pettersen EO, Feder J (1999) Cell ad - hesion force microscopy. Proc Natl Acad Sci 96:471 – 476 . doi: 10.1073/pnas.96.2.471 4. Sung KL, Sung LA, Crimmins M, Burakoff SJ, Chien S (1986) Determination of junction avidity of cytolytic T cell and target cell. Science 234:1405 – 1408 . doi: 10.1126/science.3491426 5. Martinez - Rico C, Pincet F, Thiery J - P, Dufour S (2010) Integ - rins stimulate E - cadherin - mediated intercellular adhesion by regulating Src - kinase activation and actomyosin contractility. J Cell Sci 123:712 – 722 . doi: 10.1242/jcs.047878 6. Helenius J, He isenberg C - P, Gaub HE, Muller DJ (2008) Sin gle - cell force spectroscopy. J Cell Sci 121:1785 – 1791 . doi: 10.1242/jcs.030999 7. Friedrichs J, Legate KR, Schubert R, Bharadwaj M, Werner C, Müller DJ, Benoit M (2013) A practical guide to quantify cell ad - hesio n using single - cell force spectroscopy. Methods 60:169 – 178 . doi: 10.1016/j.ymeth.2013.01.006 8. Meister A, Gabi M, Behr P, Studer P, Vörös J, Niedermann P, Bitterli J, Polesel - Maris J, Liley M, Heinzelmann H, Zambelli T (2009) FluidFM: Combining Atomic F orce Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond. Nano Lett 9:2501 – 2507 . doi: 10.1021/nl901384x 9. Hirt L, Ihle S, Pan Z, Dorwling - Carter L, Reiser A, Wheeler JM, Spolenak R, Vörös J, Zambelli T (2016) Template - Free 3D Mi - croprinting of Metals Using a Force - Controlled Nanopipette for Layer - by - Layer Electrodeposition. Adv Mater 28:2311 – 2315 . doi: 10.1002/adma.201504967 10. Dehullu J, Vorholt JA, Lipke PN, Dufrêne YF (2019) Fluidic Force Microsc opy Captures Amyloid Bonds between Microbial Cells. Trends Microbiol 27:728 – 730 . doi: 10.1016/j.tim.2019.06.001 11. Dörig P, Stiefel P, Behr P, Sarajlic E, Bijl D, Gabi M, Vörös J, Vorholt JA, Zambelli T (2010) Force - controlled spatial manipu - lation of vi able mammalian cells and micro - organisms by means of FluidFM technology. Appl Phys Lett 97:023701 . doi: 10.1063/1.3462979 12. Guillaume - Gentil O, Rey T, Kiefer P, Ibanez AJ, Steinhoff R, Brönnimann R, Dorwling - Carter L, Zambelli T, Zenobi T, Vorholt JA ( 201 7 ) Single - Cell Mass Spectrometry of Metabo - lites Extracted from Live Cells by Fluidic Force Microscopy. Anal. Chem., 89, 5017−5023 , doi : 10.1021/acs.anal - chem.7b00367 JPK BioAFM Business Bruker Nano GmbH Am Studio 2D, 12489 Berlin, Germany [email protected] www.bruker.com/bioafm  www.jpk.com © 2019 Bruker Nano GmbH – all rights reserved. NanoWizard, QI , CellHesion, ForceRobot, HybridStage, HyperDrive , D i rectOverlay, ExperimentPlanner and RampDesigner are trademarks of Bruker Nano GmbH. All other trademarks are the property of their respective company.

4. 4 method can also be used for rapid cyclic manipulation of probe cells, where one cell after another can be picked up. This can dramatically shorten the duration of the experi - ment. FluidFM is also advantageous for objects where the tradi - tional chemical attachment is challenging, e.g. , bacteria, blood or yeast cells. Another possibility is aspiration of spherical particles, such as certain colloids, to the cantilever (see Fig. 5 ) . If the parti - cle diameter is known , the contact area can be defined and used to reveal mechanical properties via Force Spectros - copy, Force Mapping or QI ™ mode . Using the integrated fit models (e.g. Hertz or DMT) , precise elasticity maps can be obtained (see Fig 5 f). By applying an overpres sure, the sphere can be released, and the cantilever can immediately be re - used for the next experiment. Cell i njection A further interesting possibility is to fill the FluidFM probe with a solution, which can then be injected , for example , into a cell (see Fig. 6 ). Here, the fluorescence marker propidium iodide was injected into individual living cell s in order to stain the DNA and visualize the nucleus. It is also possible to use FluidFM for highly localized drug delivery. Individual cells can be specifically targeted . C hanges in the topography, elasticity and adhesion of the cells after injection can be determined using Force Mapping or QI ™ mode . Using RampDesigner, user - defin ed force dis - tance curves , including pressure changes, can be specified to perform high - precision injection experiments. Cell extraction The extraction of intracellular content from living cells is a n - other application which demonstrat es the benefits of the precise control of pressure, force and position provided by the FluidFM integration. O ptical integration with the BioAFM allows not only the precise positioning of the probe, but also direct observation of the process in real time. The sens itive force control of the BioAFM allows gentl e pen - etrat ion of the cell membrane , and the precise pressure con trol allows the extraction of predefined volumes down to < femto - lit e r [12] . Fig. 6 : Injection experiment (a) Phase contrast image (b) Fluores - cence image after injection of propidium iodide (c) Force distance curve during injection (d) Overlay of phase and fluorescence image to demonstrate nucleus position T his stands in stark contrast to traditional micropipette method s , wh ose use are limited to large cells , nanoliter as - piration and poor force control, all of which have a detri - mental impact on the cell survival rate . Conclusion The Cytosurge FluidFM ADD - ON is a highly flexible tool which can be used in a broad range of applications . It can be used for adhesion measurements or as a nano - syringe to extract cell material or inject specific target substances into cells or tissues. Precise sub - micron drug deposition and nanomanipulation are further ca pabilities of the technol - ogy . The combination of the FluidFM ADD - ON with JPK Bi oAFM s is the perfect solution, as it provides precise con - trol over force, position and fluidic pressure. The easy - to - use V7 software and perfect optical integration enable any user set - up quickly and perform high - throughput experi - ments. The large range of flexib le accessories for the Bi oAFM s allow further customiz ation of advanced experi - ments. FluidFM is available as a separate add - on (FluidFM ADD - ON Silver) or can be fully integrated into the systems of the JPK BioAFM family (FluidFM ADD - ON Gold and Platinum) . d c a b

3. 3 The bar code reader supplied is of great help and provides all the relevant information about the probe package being used , including a n SEM image of each FluidFM pr obe (Fig. 3) . This makes quality control easy. Complet e i m plementation of FluidFM functionality into the new JPK V7 software Users spend about 80% of their working - time in front of a screen. To improve the output, we developed with the new V7 version a workflow - based , user guidance software inter - face. The complete integration of the FluidFM functionality in to our V7 SPM control software guaranties m aximum productivity and enhanced performance . Equipped with modern user guidance and online help, users of all experi - ence levels can quickly set up an experiment and obtain high - quality data . The implementation enables the synchronisation of AFM and FluidFM operation and correlation with optical meas - urements. Using the JPK Advanced Force Spectroscopy module and its RampDesigner™ feature , force distance curves , including segments for pressure control , can be de - fined by th e users . This enables the synchronized control of force, contact time, pressure changes and travel distance s, etc. (Fig. 4 ). T he RampDesigner comes with predefined experiment schemes , e.g. , for cell injection , spotting or pick - up . In a ddi - tion, it provides experienced user s with the flexibility to fine - tune experimental setups to their specific needs. In combi - nation with the JPK ExperimentPla n ner , customized exper - iments can be performed in a highly automated and efficient way. With all these ingredi en ts, the new V7 software pro - vides the bas is for successful scientific work. Fig. 4 : Integration of the FluidFM functionality in the Ramp Designer . Synchronisation of AFM and pressure adjust - ments Fields of application With the FluidFM technology , positive and negative fluidic pressure can be precisely applied . Integrated into a JPK BioAFM with its ease - of - use, sensitive force control , and Fig. 5 : Pick - up of a bead for Force Mapping on living cells. (a,b,c): Phase contrast images of living vero cells, the FluidFM mi - cropipette and an 11 μm polystyrene bead in front of it (a), during (b) and after pickup (c). ( d) Overlay of optical image with Height map using the polystyrene bead. (e, f) Height and apparent stiffness map of two cells. accurate positioning , and combined with perfect o ptical in - tegration for observation and control, i t can be used for a large range of application s such as: ▪ Single cell, bacteria or microbe adhesion ▪ Colloidal force spectroscopy ▪ Nanos potting , Nanoprinting ▪ Single cell picking and isolation ▪ Single cell injection or extraction The accurate pressure control of the FluidFM system allows su bmicron precise deposition of an extremely small volume of a drug (< 1 f L ). This can be used to trigger local changes in the cell network , which can be characterized with nano - mechanical measurements using AF M. Single c ell / p article m anipulation A gentle suction can be applied to pick up an object, such as a cell, and to place it somewhere else. Alternatively, the suction can be maintained and the object itself can be used as a probe. This is a convenient way to reversibly attach a cell to the can tilever for cell adhesion measurements. This

2. 2 The systems are compatible with the broadest range of in - verted optical research microscopes . U sing optical micros - copy techniques, the user can identify a spot of interest on the sample where to apply FluidFM and ha s the FluidFM experiments under optical control. The unpar alleled optical design of our AFM head s with a free optical path , enables typical transmission - based contrast methods such as DIC and optical phase contrast using standard optical micro - scope condensers. Here , one of the biggest advantages comes to play. Compared to classical micropipettes , Flu idFM probe s delivers far better optical access to the sample spot and probe position. FluidFM can also be combined with fluorescence and ad - vanced optical microscopy tech niques (e.g. Confocal, FLIM, TIRF, FRET, FCS ). In particular, super - resolution micros - copy methods such as STED, SIM, and PALM/STORM can bring additional benefits to the FluidFM experiments and are , of course , perfect ly compatible with JPK s unique probe - sca nning BioAFM systems. O verlay ing AFM scanning space coordinates with the opti - cal image coordinates can be challenging , particularly for higher NA objectives with higher magnification. The optical images are stretched by non - perfect optical lense systems in the objectives , r esulting in a n imprecise overlay of AFM images and optical images. To overcome this restriction, we developed the DirectOverlay™ mode for perfect calibration of the o ptics and AFM. U sing the AFM probe as a ruler and the perfect AFM scanner motion (metrology - like) guided by the xyz sensor system, we can “de - stretch” the optical im - age and get a perfect ove rlay of the AFM and optical image. This works down to the single molecule level. Bruker ’ s newly developed and patented DirectOverlay 2 feature enables easy , optically - guided navigation to reach positions with highest precision , and maximizes the benefit o f simultaneous AFM and optical measurements. For en - hance d usability , DirectOverlay 2 has a “ 1 - click” calibration. The contact free calibration method , based on Sader , allows a gentle , quick , and tip - protecting cantilever calibration. This approach can be applied to all of the FluidFM probes . Other highlights are the HybridStage ™ and Motorized Stage, which enable automated access to a large sample area . Travel ranges of 2 0 x 2 0 m m² and the optical til ing feature allow the user perform experiments over a very large area . In addition, t he HybridStage has a long z - piezo scan - ner (>100 μm) , which is a significant advantage for c ell - a d - hesion measurements. Combined with the ExperimentPlan ner ™ software module , the user can seamlessly automate his experiments. JPK BioAFMs provide not only perfect optical integration, but also com e with their renown modularit y and a n exten - sive range of accessories, which fulfi l specific requirements for , e.g. , environmental control (temperature, atmosphere) , automation, large x , y travel and enhanced z - ranges . Compatibility The FluidFM ADD - ON can be used as an add - on or fully integrated with the new NanoWizard ® 4 XP AFM, a bench - mark in correlative, high - performance BioAFM combined with advanced optical microscopy (Fig.1). The FluidFM ADD - ON is also compatible with the entire JPK B io AFM family, such as CellHesion® 200, ForceRobot® 300, and the new NanoWizard Ul traSpeed 2 system. FluidFM ADD - ON The FluidFM ADD - ON can be controlled from the same PC as the JPK BioAFM . Th e newly developed JPK FlowControl software module allows the user to define and set constant pressure or apply pressure pulses of defined lengths (Fig. 3). T he intuitive handling of pressure control enables users to run complex experiments easily . Fig. 3 : FlowControl soft - ware module. (Top) The barcode reader provides information about the cantilever. (Right) Pressure on the FluidFM probe can be controlled by the Flow Control software.

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