Full-Capability AFM in an Inverted Optical Microscope – MFP-3D-Bio™

The Asylum Research MFP-3D-BIO™ establishes the standard for cmbining AFM and optical microscopy for bioscience research. It is the only bioAFM that makes no compromises to AFM imaging resolution, application versatility or force measurement performance while seamlessly integrating with a complete range of optical techniques. This, together with unmatched support from Asylum experts, makes it the best, most productive bioAFM for attaining research objectives.

2.5 μm scan of supported lipid bilayers (5 nm tall) adsorbed onto mica and imaged in buffer.

The Performance and Versatility to Get the Desired Results

  • Supreme versatility to satisfy the varied needs of multiple users
  • Unrivaled support from bioAFM experts to get the desired results
  • Uncompromised AFM performance combined with optical microscopy
  • Simple, high-resolution imaging in liquid for soft biological samples
  • Performance leadership in force spectroscopy and force mechanics

Asylum Research MFP-3D-BIO™

We chose Asylum’s MFP-3D-BIO AFM because it has the most powerful AFM capabilities of the inverted optical integrated systems. It excels in all aspects, from optical integration to high-resolution imaging and dimensional measurements to force spectroscopy and elasticity measurements of soft tissue matrices. Asylum’s quality and reliability allow us to focus on the science.

Professor Dennis Discher, University of Pennsylvania

Dennis Discher, Florian Rehfeldt and Andre Brown.

From left: Dennis Discher, Florian Rehfeldt and Andre Brown.

Performance Matters in a BioAFM

“Performance matters” might sound like an obvious statement, but some companies make all sorts of compromises to performance in their bioAFMs. Some literally have a hole through the middle of their AFM head. While that allows the use of some standard optical condensers, it also interferes with quantitative force measurements and degrades imaging resolutions. In contrast, the stability of the MFP-3D-BIO allows it to measure piconewton forces and resolve the smallest features of samples.

The flexure-mounted optical lever mechanism moves as a single robust unit, almost eliminating non-linearities, interference fringes and off-axis motion. The custom-designed condenser optics offer unobstructed top-down sample viewing and allow standard optical microscopy modes, including phase contrast.

BioAFM

The MFP-3D-BIO includes powerful features for investigating cell mechanics. A) Here, a MRC-5 fibroblast cell was located using phase contrast optics, and a scan box was drawn to define a region for AFM imaging. The resulting AFM topography data was overlaid on the optical image using thresholding to remove the portion of the image corresponding to the bare petri dish. B) The same optical image was then used to guide the location of three individual force curves. C) The force curves clearly show the stiff petri dish substrate (red), the much softer cell body (black) - and a yet softer fiber (blue). D) Finally, a 32×32 point force map was measured over the entire area and built-in indentation model analysis was used to calculate the modulus at each point.

BioAFM

The unfolding of fibril amyloid beta-sheets in algal adhesive reveals a distinct sawtooth pattern. The force curve was fitted to the worm-like chain (WLC) model (dotted lines), and a persistence length of 0.22 nm was calculated. Data from Mostaert et al., J. Biol. Phys. 32(5):393 (2006).

Seamless Optical and AFM Integration

Light up your AFM without compromising performance

The MFP-3D-BIO sets the industry standard for uniting optical and atomic force microscopies in a single integrated tool specifically designed for biologists. Asylum's team of biologists and optical engineers have enhanced the MFP-3D AFM for use with the major inverted optical microscopes to guarantee that customers get the maximum benefit and productivity from the combination of these powerful techniques. Water immersion, high numerical aperture and TIRF objectives are all accommodated. All of the following optical techniques are supported:

  • TIRF
  • FRET
  • FCS
  • FRAP
  • Bright-Field Microscopy
  • Phase Contrast Microscopy
  • Epifluorescence
  • Confocal Microscopy
  • Ion Indicators (for example - Ca2+ response)

MFP-3D-BIO

The optical path through the head of the MFP-3D-BIO uses a high-quality objective that can be used to view the top of opaque samples and to align the laser onto the tip. The objective can also be used as a condenser for brightfield or phase-contrast illumination on transparent samples. High-quality illumination is achieved for observation with the inverted optical microscope objectives without undermining the fidelity of the optical lever or Z-flexure design of the AFM.

Multiply-labeled fibroblasts

Multiply-labeled fibroblasts imaged in buffer using contact mode AFM (A), and fluorescence microscopy (B). The MFP-3D’s standard overlay feature produced the composite image (C ). A proprietary IR filter blocks the AFM laser, enabling clean, full-spectrum fluorescence imaging - including far-red fluorophores.

The MFP-3D-BIO is a research instrument through and through and designed for the scientist. The optical integration is exceptional, and the flexibility of the platform offers almost endless possibilities.

Professor Jan Hoh, Johns Hopkins University

Professor Jan Hoh

Illuminate or View with Built-in Top-Down Optics

The MFP-3D-BIO extends the capability of the inverted optical microscope by allowing users to view transparent and opaque samples from above while scanning with the AFM. The integrated top-view optics allow in situ laser alignment and tip positioning without the need for removing the AFM head and without ancillary equipment. The unique design also enables the top-view objective lens to double as a superior-quality condenser for phase contrast illumination.

Real-Time Optical Navigation

Bottom or top-view optical images can be used for navigating the tip to any feature on the sample and then for scanning that area at the nanoscale with the AFM or select specific locations for force curves - seamlessly and easily.

Powerful Real-Time and Offline Rendering Options

Both AFM and optical images can be rendered and then viewed together in both real-time and offline. Optical images can be overlaid on AFM data in order to assist interpretation. Stunning 3D renderings combine AFM topography with the capabilities of light microscopy.

40X phase contrast image

40× phase contrast image of MRC-5 fibroblasts on a Petri dish. With a single click, you can direct the tip (red dot) to any point, or select a new scan area (green box) within the scan range (red box).

Top-view optical images

Top-view optical images: (top) HeLa cells on silicon and (bottom) pollen grains.

AFM topography

Locate a cell with phase contrast (gray) or fluorescence and examine features such as cytoskeletal structures (red) or the nucleus (purple), then zoom in for AFM high-resolution topography or force measurements (copper). Overlay optical data on AFM topography for 3D analysis and presentation (right).

Sequence of real-time 3D renderings

Sequence of real-time 3D renderings of E. coli on glass showing structure and fimbriae, 5 μm scan.

Full Capability and Ease of Use

A bioAFM that satisfies the requirements of experts and beginners alike

Not all AFM users are AFM experts, and even experts appreciate keeping operation as simple as possible. Asylum Research develops ease-of-use improvements that offer genuine benefits to both groups without compromising flexibility or capability.

ModeMaster™

  • Supports both advanced and basic imaging techniques
  • Makes getting started easy and quick
  • Automatically configures the software for the selected mode

ModeMaster enables one-click configuration

ModeMaster enables one-click configuration for more than 30 different modes.

GetReal™

  • Tip never touches the sample, so there’s no need for a hard surface
  • Process is fast, simple and accurate with no risk of damaging the tip
  • One click calibrates the cantilever sensitivity and spring constant

One click calibrates the cantilever

Automation? Advanced needs? No problem.

  • MacroBuilder™ allows users to effortlessly implement custom routines by just dragging “modules” together to form macros, no coding needed
  • Even greater customization potential is possible with the lower-level IGOR Pro scripting language

Our MFP-3D is a highly reliable and key tool in our NanoImaging Core Facility. In addition to ‘traditional’ imaging in air, it has outstanding capabilities for imaging in liquid and performing reliable and accurate force measurements. The simplicity of the software allows us to easily train students, yet the open setup of the instrument makes it flexible and powerful for very high-end projects, including nanolithography and nanomanipulation.

Professor Yuri Lyubchenko, University of Nebraska Medical Center

Yuri Lyubchenko with Luda Shlyakhtenko

Yuri Lyubchenko with Luda Shlyakhtenko

Leadership in Force Spectroscopy and Force Mechanics

  • Thermally-limited force resolution (<8 pm deflection noise typical, <20 pm guaranteed)
  • Large Z range (15 μm standard, 40 μm extended Z option) accommodates demanding applications such as cell-substrate and cell-cell adhesion measurements
  • Users can choose between open loop force curves with sensored Z for the ultimate in low noise performance or closed loop Z for the most accurate velocity control
  • Force Mapping measures force-distance curves at a grid of points with automated fitting of indentation models for estimation of elastic modulus and automated adhesion/rupture force analysis
  • Analysis software helps by proposing the most appropriate indentation model among many built-in options, including Hertz/Sneddon, Johnson-Kendall-Roberts (JKR), Derjaguin- Müller-Toporov (DMT), and Oliver-Pharr, or users may also freely enter their own customized models
  • Force Clamping for single-molecule and bond-rupture force spectroscopy measures unfolding or rupture kinetics under constant force, permitting direct comparison to theoretical models

Great for Biology, but Also Ready for Much More

The MFP-3D-BIO is based on the same powerful design as standalone MFP-3D AFMs and hence it is compatible with Asylum’s complete range of modes, accessories and options. This means it can be ready to successfully do its job for any application, whether in biology, photonics, biophysics or far outside the realm of ordinary bioAFMs in microelectronics, materials science and functional materials. It will continue to support your goals no matter what direction the research is taken.

Easy to Use, Effective Accessories for Biological Applications

Petri Dish Holder

Specifically designed sample plate for imaging cultured cells and other biological samples in petri dishes.

Petri Dish Heater

Petri Dish Holder supporting heating from ambient to 45 ˚C.

Closed Fluid Cell

Sealed chamber with outlet/inlet ports for gas or liquid media.

BioHeater™

Closed Fluid Cell for imaging in liquid between ambient and 80 ˚C.

Fluid Cell Lite

Economical, portless fluid cell. Perfect for individual users at multi-user facilities.

CoolerHeater

Heats and cools samples with a Peltier element. Nonstop temperature control from -30 to +120 ˚C.

Humidity Sensing Cell

Measures humidity within a sealed cell.

MicroFlow Cell

Small volume fluid exchange cell.

iDrive™

Simplifies imaging in liquid by enabling the cantilever to be auto-tuned with a single mouse click.

Specifications

High-Precision 3D Motion

Closed loop sensors on all three axes:

  • X and Y range 90 μm
  • X and Y sensors <0.5 nm noise, <0.5% non-linearity
  • Z range >15 μm
  • Z sensor <0.25 nm noise
  • DC height noise <50 pm
  • Optional Extended Z Head range >40 μm

Lowest Noise Single Molecule or Cellular Force Measurements

  • Cantilever deflection noise <15 pm (typical 8 pm)
  • Low coherence source Superluminescent diode (SLD) for ripple-free baseline
  • Cantilever spring constant calibration by the thermal noise and Sader methods or GetReal automated cantilever calibration
  • Flexible interface allows recording or triggering from any channel during a force curve, including amplitude/phase from AC or Dual AC™ mode; user-supplied input voltages; and photon count rate (with optional Digital Access Module)
  • Force mapping including automated adhesion and elastic modulus analysis

Optical Microscope Integration

  • Includes stage unit for mounting on inverted optical microscope: Olympus IX73, IX81/71/51, IX70/50; Nikon Ti-E/U/S, TE2000-U/S; or Zeiss Axio Observer Z1/D1/A1. Please inquire regarding other models.
  • All standard objective lenses including high NA oil- and water-immersion and TIRF objectives are supported.
  • Infrared source 860 nm SLD for compatibility with far-red fluorophores. Matched interference filters in AFM head and optical microscope totally block the SLD beam from fluorescence detectors and cameras.
  • Software overlay of optical images on AFM data in both 2D (variable alpha) and 3D (topographic rendering).
  • Optically guided region of interest (ROI) selection for imaging and force curves/maps.
  • Top view optics with 10× / 0.28 NA objective lens, for SLD spot alignment and positioning tip on opaque samples.
  • Phase contrast enabled with included condenser and annuli for Ph2, Ph1, and Ph0/C/L objectives (purchased separately).
  • Compatible optical techniques See list above and inquire regarding other techniques.

Superior Usability

  • Included modes Contact mode; Dual AC™; Dual AC Resonance Tracking (DART); DART™ PFM; Electrostatic force microscopy (EFM); Fluid imaging; Force mapping mode (force volume); Force modulation; Frequency modulation; Kelvin probe force microscopy (KPFM); Lateral force mode; Loss tangent imaging; Magnetic force microscopy (MFM); Nanolithography and nanomanipulation; Phase imaging; Piezoresponse force microscopy (PFM); Switching spectroscopy PFM; Tapping mode (AC mode); Tapping mode (AC mode) with Q control; Vector PFM.
  • Optional modes AM-FM Viscoelastic Mapping Mode; Band Excitation; Conductive AFM (CAFM) with Eclipse™ mode; Contact Resonance Viscoelastic Mapping Mode; Electrochemical Strain Microscopy (ESM); Force modulation; High Voltage PFM; iDrive™ (magnetically actuated AC mode in liquid); Scanning Thermal Microscopy (SThM); Scanning Tunneling Microscopy (STM); Ztherm™ Modulated Thermal Analysis.
  • Sample format 75×25 mm typical, maximum 80 mm diam. ×5 mm high. Up to 22 mm high with optional leg extenders.
  • Sample adapters included for coverslips (12 or 25 mm diam. or 22×22 mm) and petri dishes (plastic and coverglass-bottom).

Unmatched Support

  • Warranty Full two-year comprehensive warranty.
  • Support No-charge technical support and expert applications support for the lifespan of the AFM.

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