UIUC Demo Reminder

Ian Armstrong

April 19, 2023

Samples and Prep

Celgard sample was run a few times as a “standard” sample.Most of the customer samples were various forms or DNA. As experienced during the demo, the most crucial part of taking good images of these is the sample preparation. Included in the next slide is sample preparation we use ourselves, but note there are a fair few different protocols to be found in the literature, and if the sample is substantially different from that studied before it might necessarily require a different procedure.“NanoDiscs” previously studied via an old MultiMode AFM were imaged.Noise tests were conducted on an MoS2 sample.Speed tests were conducted on a calibration grid.

• Celgard (Bruker sample)

• DNA

• “NanoDiscs”

• MoS2

• Customer Calibration Grid

DNA Sample Prep

Attached is the procedure we use to prepare DNA samples on mica in fluid. The recipe for the incubation/adsorption buffer is on the first page (attached .pdf). For adsorbing the chromatin you can jump to the second page to the section ‘On day of Experiment…’ (we have the steps in between as our DNA comes as a very concentrated solution that we dilute and store in the EDTA-containing buffer. The final dilution into the HEPES/NiCl2 buffer gives us a final concentration of 0.5 microgram/mL (others have used 1 microgram). You can replace the NiCl2 in this buffer with MgCl2.Briefly, You want to transfer/dilute your chromatin into your MgCl2 or NiCl2 containing buffer.Pipette an aliquot of the Chromatin in MgCl2/NiCl2 containing buffer onto a freshly cleaved mica surface (we use ~100 micro-L, depending on the size of your mica disk – you can use 50 micro-L if you need less).Allow it to adsorb for 10-15 minutesAt this point you can either image the surface directly – this will help you to see if your concentration is too high/low or if you need to incubate longer. However, chromatin will continue to adsorb to the mica over time – which can be messy. Alternatively, before you image you can rinse the sample with chromatin-free HEPES/NiCl2 or MgCl2 buffer (For rinsing I usually pipette most of the droplet off the mica and then pipette on fresh buffer – do this ~3 times) and then image in this buffer.If you want to image in air, you will need to rinse away the buffer with ultra-pure or milli-Q water (otherwise, once the buffer dries you’ll have beautiful salt xtals on your sample) and then gently remove the water from the mica surface (I usually tip off most of the droplet by tilting the sample over the sink. Then I’ll wick the remainder off the droplet slowly with the edge of a chem-wipe and allow the sample to fully dry on a benchtop. The AP-mica is a little better for keeping the chromatin attached on the surface for a dried sample. I find that sometimes, with the NiCl2 buffer when we dry the sample, removing the NiCl2 disrupts the interaction between the DNA and the mica and the DNA clumps up. This is why I try to dry as slowly as possible.I hope this sample preparation helps you. Please, feel free to call me if you have any questions. Good Luck!Andrea Slade, Ph.D. Life Science Applications Scientist andrea.slade@bruker-nano.com

Dimension Icon AFM

The Dimension Icon AFM is the latest generation of the worlds most popular tip scanning AFM. Keeping the successful design of previous generations which includes:• flexibility of sample size• easy customization• motorised and automated controls for ease of use• inherent stability and robust design• huge accessory array for multiuser experiment flexibility

On top of this the Dimension Icon has a new powerful controller (some details on next slide). The isolation platform and hood has been improved for even better stability and drift. The AFM has multiple upgrades including, heavier base, single Invar arm for increased space underneath tip, digital camera optics, LCD display, all new fully closed loop scanner with closed loop performance almost indistinguishable from open loop. The software has also been dramatically improved for this latest generation instrument. Neat experiment selectors with preloaded parameters and cues to help select appropriate probes. A probes database to load nominal values of probe parameters. Work flow models to aid novice and infrequent users. Context sensitive help menus, just hit F1 in realtime or offline software for comprehensive help.

AND: Peak Force Tapping, our latest scanning technology only available through Bruker. This technique makes scanning easier and more intuitive as you have the setpoint directly as a force in Newtons. No “tuning” as in tapping mode. We can scan at ultra low forces, even lock-in on the snap-in or negative force. We can vary force curve approach and modulation to suit sticky/ rough samples or for probe functionalisation experiments. Through this technique we can use ScanAsyst to automate gain/ scan rate/ setpoint for automated image optimisation. Now we are also fully leveraging this technique to look at mechanical properties by evaluating the force curves on the fly, or combining with existing techniques such as conductive AFM to be able to look at fragile or sticky conductive samples not possible to be imaged with standard CAFM. And more things to come…

Dimension Icon AFM

Nanocscope V Controller

• 8 Imaging Channels: 8 realtime channels capturing at up to 5Kx5K pixels each• 3 Built in Lock-In Amplifiers: 2 high speed (1 kHz to 5 MHz) 1 low speed (0.1 Hz to 50 kHz)

Generic Lock-In GUI: easily route reference signal and lock-in input

Monitor Cantilever Harmonics for: enhanced phase, mech properties,

dC/dZ from surface potential• 500kHz/ 50MHz sampling rate: 2us feedback response time = better tracking and tip wear• Thermal Tune: Identify resonant frequencies up to 2MHz, calculate spring constants• High Speed data Capture: Capture 64Mb of data.

2 Channels At 50 MHz (AC coupled) ~330 ms per channel2 Channels At 6.3 MHz2 Channels At 500 kHz (DC Coupled) ~2.5 seconds per channelWorks In Image, Ramp And Sweep Modes, Not Required To Be Engaged

Trigger on EOL, EOF, Edge or ManualData collection can start before or after trigger event

• Digital Q Control: Enhancements to 10x, damping to 10x, Q seekBenefits Include:

improved signal-to-noise ratio in phase-contrast imaging MFM, EFM etcfaster scanning in fluid with damped Qstay in attractive modebetter force control for soft samples

• 5k x 5k pixel images: Scan large areas at hi pixels for better statistics but same resolution• Digital Inputs/ Outputs: 2 Digital Outputs, end of line, end of frame

2 Digital Inputs, 3.3 Volts (5 Volt compatible) up to 25 MHz

e.g. Digital Photomultiplier, Photon-Counting CCDs, sync pulse

• Signal Access Front Panel: Access to lock-in amps, XYZ low voltage drive signals, input external dataUser Access To Data Signals, height, amplitude, phase etc…

StandardStandard1. ScanAsyst/ PFT2. TappingModeTM (air)3. Contact Mode4. Lateral Force Microscopy5. PhaseImagingTM

6. Lift Mode7. MFM8. Force Spectroscopy9. Force Volume10.EFM11.Surface Potential12.Piezoresponse Microscopy

OptionalOptional1. PeakForce-QNM2. HarmoniX3. Nanoindentation4. Nanomanipulation5. Nanolithograpy6. Force Modulation (air and fluid)7. TappingMode (fluid)8. Torsional Resonance Mode9. Dark Lift10. STM11. SCM12. C-AFM13. SSRM14. TUNA15. VITA

Dimension Icon Imaging Modes

• Dimension App Mods (electrical modes)– DM-SSRM– DM-CAFM– DM-SCM– DM-TUNAV-50/-60

• Heating/Cooling– Sample Heating and Cooling: -35C to 250C

– Sub-100 nm node heating (VITA)

• Software– Nanoacope V8 Realtime– Nanoscope Analysis Offline– Intuitive, comprehensive, context sensitive help

• Cantilever Holders and Probes to support all modes, manufactured by us

Application Modules and Accessories

Working in Liquid: Hardware

To work in a liquid environment the only accessory needed is the fluid cantilever holder (pictured left). This single holder allows working in fluid in contact mode, tapping mode, and Peak Force Mode and more…Loading a probe to the holder is identical to air i.e. place cantilever with tweezers under a spring loaded clip.

The fluid cantilever holder is then inverted and attached to the scan head just as for the air cantilever holder. The laser is aligned on the cantilever as normal. The final step is to place the sample on the sample holder and deposit ~50uL via pipette to the sample. This forms a bubble on the surface which the cantilever/ fluid holder is then lowered into. The bubble then forms a liquid tension seal around the circular portion of the cantilever holder. The user is then ready to start imaging. Pictured right is how it looks when actually scanning in liquid.

Working in Liquid: Software

As well as optimized and simple hardware we have dedicated fluid workspaces. This screen dump shows one example of that. The fluid workspaces have optimized engage routines/ scan parameters for liquid – also a very handy refractive index autocompensate checkbox (to account for different optical path when liquid is introduced – this makes it easier to find the focus position/ engage point). With our patented Peak Force Tapping mode there is also no need to tune the cantilever – a process inherently less trivial in liquid than air.

Sample Images: Celgard

Celgard is the tradename for a form of isotactic polypropylene that has been stretched such that it has drawn fibrillar sections at 90 degrees to crystalline lamella. It is used as a separator or filtration membrane. It is used as a somewhat standard AFM sample because you need:1. Good force control and resolution to clearly see the lamellae.2. Good force control and feedback to track the ~100nm deep freely suspended fibrils.3. And to be able to do this at reasonable scan rates.We routinely measure this sample in tapping and Peak Force Tapping. Peak Force Tapping is actually more beneficial for this kind of sample. The image right was taken on site directly after installing the instrument.

Sample Images: DNA

Shown right are 4 images from various groups that attended on the first day. The image top left shows a more standard DNA image. Here we pretty much had the prep technique good. There were a few large agglomerations that interfered with the imaging but we were nearly there. In spite of this you can still see the crisp DNA imaging. The other 3 DNA samples shown incorporated various differences in the sample such as chromatin. The affect we observed seemed to be to kink the DNA molecule and also at higher concentrations it seemed to preferentially form a network. Again, once sample prep had been optimised were able to scan at ~1um and obtain good images.

500nm

500nm

500nm

500nm

Sample Images: NanoDiscs

Mark McLean’s samples of Nanodiscs. Mark has been imaging these on an old Bruker MultiMode AFM. We got to the same or better results, quicker and he remarked with less drift affecting the measurements. The “discs” can be seen individually in these images. They are just 10nm wide. When they vary in height it can be indicative of a bound protein. Image taken with Peak Force Tapping at 30pN imaging force in liquid.http://sligarlab.life.uiuc.edu/nanodisc.html

200nm

200nm

Sample Images: MoS2 TAP RoughnessIcon Scanner

Here we are tracking multiple steps on the MoS2 sample. For roughness evaluation we first order flattened and plane fit to the region as indicated by the white dashed box. In the image it may be slightly unclear due to color contrast but I have highlighted in blue dash 3 single atomic steps ~6A in height (see original data for clear view). We analyzed the roughness in the white box area. Ra value ~16pm

Sample Images: MoS2 PFT RoughnessIcon Scanner

Same thing again, this time with Peak Force Tapping mode. In this instance we got 23pm Ra. It should be noted that in comparing one mode to the other and as a basis for instrument noise whilst imaging – a more statistical approach may help doing multiple areas on the sample. As we are right at the business end of the spectrum, very small insignificant things will affect the measurement. We do not believe PFT mode would actually affect the noise of the system.

Sample Images: MoS2 PFT Step HeightFastScanner

We repeated the process with the FastScanner. In this example we located a single step and imaged that. This shows the step height as ~6A

Sample Images: MoS2 PFT RoughnessFastScanner

Looking at the roughness over the region that the area was plane fit we get a value of just 14pm Ra.

Sample Images: MoS2 TAP Step HeightFastScanner

Looking in tapping mode we again found a single step.

Sample Images: MoS2 TAP RoughnessFastScanner

Taking the roughness measurement at this location we get 15pm Ra.

Instrument Noise Summary

From the four values we got on the day in PFT and TAP mode on both FastScanner and Icon Scanner:

Ra: 16pm, 23pm, 14pm, 15pm…

This is exceptional performance and we really didn’t have to try very hard. We could spend extra time making everything as “quiet as possible” and get these numbers a bit lower but these serve as a good example of what the instrument can produce in terms of low noise. I would add as well – these numbers are great for a sample scanning system…the fact we can do it with a tip scanning system with such a huge mechanical loop between tip and sample allowing for sample flexibility shows how sensitively engineered the AFM + acoustic enclosure really is.

Flexibility

Performance

Ra 14pm

Speed Test on Customer Calibration Grid

These 20um scan size images were taken with The FastScanner and Icon scanner respectively. We optimized imaging such that we were just attaining “acceptable” image quality in terms of tracking and gain noise etc…

Tip Velocity 233um per second Tip Velocity 80um per second

Full Summary

In one line: We see the Icon as the most fully featured, high performance, stable, flexible, expandable, robust AFM solution for multiuser environments.

Ra 14pm

19. April 2023 23© Copyright Bruker Corporation. All rights reserved