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Hydra™ Bio-AFM

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Imaging Live Cells with Optical Integration

The HydraTM  BioAFM is a revolutionary BioAFM that enables complete optical integration allowing the researcher to use the traditional biology toolkit with advanced SPM functionality in both air and liquid.  Applicable integrations are: Integration with any upright or inverted microscope, confocal , fluorescence NSOM and Raman spectroscopy.

The HydraTM incorporates an extremely sensitive tuning fork feedback mechanism allowing for measuring single pN’s of force.  Finally with the largest Z range available and multiprobe capabilities the Hydra opens new directions in Bio AFM imaging and nano-manipulation.

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KEY FEATURES

Complete optical integration 

Systems compatible with all optical microscopes including upright (see right), inverted, and dual optical microscopes.  

The Hydra systems also provides a completely optically transparent axis from top and bottom with its unique 3D FlatScanTM  stage and NanoToolKit glass probes. This enables easy simultaneous viewing and positioning of your probe on your sample, as shown here. 

 

 
The Ultra Thin Transparent Glass Tip with a High Cantilever Structure prevents any optical interference even in DIC. DIC images show PC 12 Cells during AFM imaging In and Out of focus. The In-focus image shows a clear optical axis without optical interference.

 

 

Superior force sensitivity in air and liquids with critically acclaimed tuning fork feedback

Cantilevers designed for soft contact with sample

Optically free feedback ideal for fluorescence and Raman

Improved tip approach to sample

Liquid cell without optical and mechanical constraints or interference

Compatible with all biological sample preparation

High magnification objectives with large NA including water immersion objectives

Largest Commercial Z range of 170um

Perfectly suited for all cellular imaging applications and measurements requiring large z range

Independent and simultaneous multiprobe operation 

Up to 4 SPM probes 
Multifunctional probes for manipulation and imaging

NSOM capability - true reflection, transmisssion, and collection modes

A NanoTool KitTM of multifunctional probes for multiprobe operation

 Conductance & Patch Clamping 
 Apertured & Apertureless NSOM 
 Single Gold Ball Tipped Probes 
 BioMolecular Nanolithography 

Environmental Control

 Humidity/Temperature/Vacuum retaining full optical transparency

 

NSOM CAPABILITIES

All modes of NSOM:   Transmission, Collection, Reflection

True reflection mode NSOM in liquid environment

In true reflection mode,  light is introduced via the NSOM probe, and then collected by a detector above the probe.  Through the use of a water immersion objective and separation of excitation and collection paths,  True Reflection mode NSOM is possible on the Hydra.

This mode is particularly powerful for biological samples as it resolves the problems of bleaching and correlation present in other system designs.   The Hydra enables apertured NSOM imaging in reflection mode, which results in localized illumination under the probe where now 1) no bleaching of fluorescent molecules occurs outside the local spot of illumination 2) only molecules under the probe tip are excited 3) there is absolute correlation between the AFM and NSOM image since they are collected simultaneously.

Results collected with a Hydra system below show total correlation between AFM (right) and fluorescence (green excitation, top) and absorption (blue excitation, bottom) with no bleaching on Murin stem cells.

Transmission mode and True Collection mode in liquid environment

For an explanation of all modes of NSOM, please see the NSOM tutorial.

Completely flexible optical access designed for NSOM operation

This enables total flexibility in your NSOM setup, as the Hydra can be integrated with any optical microscope including upright microscopes.  Additionally, total optical access to sample and probe position from above is possible since the cantilever probe/scanner assembly does not obscure access

A summary of all NSOM features found in Nanonics systems can be found here.

RAMAN CAPABILITIES

Effortless AFM and Raman integration

With a Hydra, no modification of the Raman path is necessary to accomodate the SPM tip and head.  Transparent optical probes provide a completely transparent optical system with both reflection Raman and transmission Raman provided as standard modes.

Superior performance in liquids

The Hydra is compatible with a water immersion objective in the upright configuration.  The water immersion objective collects the optical signal in liquid more effectively and improves the imaging quality significantly. The example of the Raman imaging with (left) and without (right) water immersion objective of a silicon grating surface is shown below.

 

 

The Hydra provides a free optical axis from the top, even with a water immersion objective for superior imaging capabilities in fluids.  Such objectives have ultra-low working distances as small as 3.5 mm that provide high numerical apertures of up to 0.8.  On left is shown a side view of tuning fork immersed in liquid during a scan.

 

 

 

Modular Design and Open Architecture

The unique, modular design of the Hydra allows an upgrade from one probe to two probes. The Hydra offers a completely free optical axis from above the probe, below the sample and 270 degrees around the probe. The Hydra boasts a 4.5mm working distance from above the probe for ultrahigh resolution optical or electron/optical viewing probes on opaque samples.

Hydra with one SPM probe

Hydra with one SPM probe              Hydra with two SPM probes

Tuning fork feedback without Raman laser interference

The Hydra is equipped with a laser-free feedback method called normal force tuning fork feedback. For example, many SPM laser-based feedback systems employ red lasers, which would then interfere with the Raman emission in the redo from the sample. The Hydra gets rid of all problems associated with optical interference in your Raman experiments.

A summary of all Raman features found in Nanonics systems can be found here

COMPARE HYDRA/STANDARD BIOAFM

A Ground-Breaking Advance in AFM Bio-Imaging in Liquids

Comparison list between Nanonics Hydra and standard BioAFMs: 
 

Standard BioAFMs Nanonics HydraTM
Beam Bounce Feedback Tuning Fork Feedback
Poor Sensitivity due to Feedback Effects
- Soft Cantilever with low spring constant  
- Low Q-Factor
- Severe damping
- Jump-to-contact and ringing effects
Ultra-Sensitivity using Dynamic Methods of Frequency Modulation.
- High Force Constant 
- High Q-Factor
- No damping and No Jump-to-contact  in liquid
Optical Interference No Optical Interference

Strict requirements for optical purity of the feedback laser reflection.
- Imposes severe limitations on liquid cells and scanning stages

Flexible geometry with:
- Free optical axis from top and bottom
- Uses Water Immersion Objective
- Standard petri dish
- Tip and sample scanning
Single Probe AFM Systems On Line MultiProbe SPM Operation:
- Optically and Spatially friendly
- Uses Nanonics' NanoTool KitTM of multifunctional probes for multiprobe operation
Severe geometric obstruction for online integrations.  

Flexible Integration with online complementary techniques such as Raman, FTIR, DIC, Confocal, etc.

 

 

ONLINE INTEGRATIONS

 

 
 
 
 Hydra Integration with Dual Optical Microscope  Short Working Distance of the Hydra with Tuning Fork feedback allows for integration with Water Immersion Objective from above.

 

Humidity/Temperature/Vacuum  Environmental Control

    • Retains full optical transparency

 

   
 MultiProbe Hydra Environmental Chamber mounted on Dual optical microscope  

 

SPECIFICATIONS

Modes Of Operation

AFM   AC Mode
Contact Mode (Optional)
All AFM Modes of Operation with probe or sample scanning
Near-field 
Optical Imaging & Illumination
Transmission, Reflection, Collection, Illumination
Differential Interference Contrast Reflection and Transmission
Refractive-Index Profiling Reflection and Transmission
Thermal Conductivity and Spreading Resistance Profiling  Contact or AC mode
No Feedback Laser Induced Extraneous Carriers in Semiconductors with tuning fork feedback option
On-line Far-field Confocal with
Raman and Fluorescence
Spectral Imaging
Reflection and Transmission
Tip Enhanced Raman Scattering for Selective Raman Scattering of Ultrathin Layers such as Strained Silicon
NanoLithography  NanoFountainPen delivery of chemicals and gases; Near-field photolithography; and other conventional means of nanolithography such as electrical oxidation etc; with on-line analysis with an additional probe
NanoIndentation Application of MegaPascals of force, allowing exact positioning and controlled application of force with on-line analysis with an additional probe.
All the above modes of operation are provided fully integrated with on-line AFM imaging.

SPM Scan Head Specifications

Sample Scanner     Piezoelectric Based Flat Scanner (3D Flat Scanner™)
Height 7mm 
Suitable for Variety of Scanning Stages open and closed loop.
Probe Scanner 

Up to 4 independently controlled piezoelectric Flat Scanners (3D Flat ScannerTM) modules
Height 7mm

SPM Scan Range   30 microns (XYZ) for each probe scanning module 
100 microns (XYZ) sample scanning only
130 microns (XYZ) with sample and probe scanning
160 microns (XY) with sample and two probe scanning
Scanner Resoluton   Z : 0.002 nm  XY : 0.005 nm
Feedback Mechanism Ultra sensitive tuning fork feedback with high Q-Factor
Sample Geometries       Sample size:   Up to 16 mm standard
                     Up to 34 mm for upright microscope operation
                     Up to 55 mm without sample scanning
Custom sample sizes up to 200 mm also available
Liquid cell sample:
-        Standard petri dish
-        Modified liquid cells with different cover slips size
Unconventional Geometries: Hanging samples for edge profiling and other unconventional geometries possible.
Probes  All forms of cantilivered glass probes from Nanonics' exclusive NanoTool KitTM, NanoSensors including Akiyama tuning fork probes and Si probes.

Imaging Resolution

Far-field Diffraction Limited
Optical Optics providing 500 nm diffraction limited non-confocal operation
Confocal 200 nm
NSOM 100 nm on installation; 50 nm probes available
Topographic Z noise 0.05 nm rms.
X.Y lateral resolution: convolution of tip diameter & sample
Thermal From 100 nm
Resistance From 25 nm

Thermal & Resistance Imaging

Temperature 300 o C or greater, depending on sample to be investigated
Thermal  Unique exposed tip dual platinum nanowire probes fully insulated with glass coating:
Thermal Sensitivity 0.01 ºC
Measured Resistance Change per degree; 0.38 Ω/ºC
Resistance   Unique exposed tip platinum nanowire probes fully insulated with glass coating, allowing for coax geometry structures:
Ultra high electro potential resolution.
Few tens of ohms contact resistance for probes <100nm.
Electrically stable & free from oxidation.

Electronics & Software

Control System Integra Controller Specifications
Supports various imaging modes including AFM (contact and non-contact), phase, error signal and NSOM.
Up to 8 data channels can be read and imaged simultaneously.

All ADCs are 16 bit and DACs have16-bit resolution.
Image size continuously variable from 2x2 to 1024x1024
Inbuilt lock-in amplifier

There are two alternative software packages available:    

Quartz Software Package Specifications:
User-friendly 32-bit Windows application available for Windows 95/98, NT and XPIntuitive scan parameter setup
.
Image and line profiles displayed in real time

2-D and 3-D image rendering
Extensive image processing options
Comprehensive image analysis features including: cross section, particle analysis, fractal analysis and z-data histogram

Import data as Windows bitmaps and ACSII. 
Export data as TIFF and Windows bitmaps and ACSII.

LabView Software Package Specifications:
User-friendly LabVIEW SPM based software for PCI-7344 with the following specifications:
AUX Data acquisition
Image and line profiles displayed in real time
Intuitive scan parameter setup
Open Design enabling customization by user and interfacing with other LabVIEW modules. Nanonics Controller and software package based on Windows XP and Windows.

XP LabView based software package: Real time image display, image acquisition up to 8 channels. Full access to all signals and readily integrated with external signals from other sources. Analysis software including all standard image processing routines and 3D rendering including collages of multiple signals.
Software modules available for spectral acquisition and analysis including Raman and fluorescence spectra, nanoindentation, nanolithography including
NanoChemwrite™ Fountain Pen NanoChemistry™ software suite.

Data Acquisition From 2x2 to 1024x1024 and multiple Z acquisition
Analog Lock-in Provides quadrature output. Information is readily available on R/? and I/Q in
output bandwidths of 15kHz (depending on DT card in use; the controller can
give up to 100 kHz).
Frequency Synthesizer Direct Digital Synthesizer (DDS) system for frequency and phase adjustment with 32-bit frequency determination and 20-bit phase determination. This system uses three independent generators. Two of these generators provide quadrature for lock-in processing and the third generator is used for exciting with an autophase algorithm. The system uses a clock frequency of 20 MHz with a stability of 5 ppm and provides frequency resolution of <5 mHz.
Amplitude 0 to 5 V p-p and maximum resolution of up to 0.2 mV Amplitude, Phase and Frequency of the oscillator can be controlled with 100 kHz updates.
X, Y, and Z High Voltage Outputs -145V to +145V

On-line Optical and Electron/ Ion Optical Integration

Type Far-field, Confocal Optics, Near Field, micro-RamanScanning Electron Microscope (SEM) or Focused Ion Beam (FIB)
Integration                           Free optical axis from above and below the sample for on-line optical or electron/ion optical characterization. Integration with all forms of optical microscopes including upright microscopes and upright microscope probe stations. Integration with all standard microRaman 180 degree backscattering geometry configurations, inverted microscopes and state of the art dual (4Pi) microscopes, such as Nanonics unique dual microscope. All conventional far-field optical modes of operation are available, including phase imaging and differential interference contrast. NSOM with any optical microscope including upright, inverted and dual. The completely free optical axis from above and below in all Nanonics MultiView Systems also allows for integration with (4Pi) dual microscopes for non-linear optical techniques including second harmonic and sum frequency generation microscopes, third harmonic imaging, coherent anti-Stokes Raman microscopes and stimulated emission depletion microscopy.All Nanonics Systems and all Nanonics Multiple Probe Systems are unique
scanned probe microscopes with a completely free axis above the sample and
thus can be integrated transparently into scanning electron microscopes including field emission SEMs or focused ion beam systems.
Minimum Working Distance (WD) with High Numerical
Aperture (NA) Optical
Microscope Lenses
Upright Microscope or SEM or FIB:
   Optical Objective: 100 x 0.75NA
   Objective WD: 4.8mm Inverted Microscope:
   All available objectives including oil immersion optical objectives
Detectors Photomultiplier Tube, Avalanche Photo Diode or InGaAs Detectors
Lasers Variety of lasers can be used from deep UV to near-IR
Video system On Line CCD video imaging

 

 

 

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