Products

LS AFM

Life Sciences AFM
For Correlative Microscopy

The LS AFM is used in life science applications when an inverted optical microscope is required for correlative imaging of cells or other biomaterials on a surface, dry or in liquid. The LS AFM can be retrofitted to almost any inverted optical microscope, or it can be purchased with the AFMWorkshop inverted optical microscope. $62,000 – $125,000

LS-AFM for correlative light and atomic force microscopy

Why an LS AFM?

Customers purchase the LS AFM to perform correlative light and atomic force microscopy measurements on fixed, dried or live biological systems in liquids. Mounted on an inverted light microscope, sample areas in correlative bright field or epifluorescence microscopy can be directly and simultaneously scanned by AFM. The LS AFM is also available as a standalone AFM that can be mounted on user-modified light microscopes or on other instrument platforms.

These images of epithelial cells from a cheek swab were collected on the LS-AFM. The cell was selected and captured in brightfield using a 20x objective. Apparently similar features could be differentiated by AFM into intracellular structures, bacilli, cocci, and unknown macromolecular components.

correlative microscopy of a cheek cell, 20x inverted microscope
atomic force microscopy topography scan of epithelial cell from a cheek swab

Overview

Available with AFMWorkshop inverted microscope Turnkey system with guaranteed results
Glass slides and petri dish sample holder No additional sample holding options required for most applications
Includes liquid scanner Readily scan samples in ambient air and liquids
Closed loop XY scanner  Zoom to feature with accurate positioning for F/D curves
LabVIEW software with USB communication Readily adaptable to new operating systems
Probe exchange tool included Reduce time for probe exchange (& use any manufacturer’s probes)
Includes top view video microscope Facilitates tip approach and laser alignment
Includes vibrating, non-vibrating, phase, LFM, and advanced F/D Most common scanning modes included for life sciences applications
Pricing starts at $59,000
Download: LS AFM Product Datasheet
3-D model of LS AFM
Measuring and Understanding Force-Distance Curves
The LS AFM can be purchased in two variations:

LS AFM-A

For customers who own an inverted light microscope, we fabricate a plate that pairs the LS AFM with your microscope.

LS AFM-B

This configuration of the LS AFM includes an inverted light microscope with standard optics and objectives.

Features of the LS AFM include:

  • Dry and Liquid Z Scanner
  • AFM Adapter Plate for Inverted Microscopes
  • Linearized XY Scanner
  • Advanced Force Distance Curves
  • Glass Slide and Petri Dish Sample Holder
  • Precision AFM Alignment System with Lock-Down
  • Included Modes: Vibrating, Non-Vibrating, Phase and LFM
  • Direct Drive Z Motor
  • Compatible With Standard AFM probes
  • Intuitive LabVIEW™ Software Interface
  • High Resolution Zoom Video Camera
  • High Resolution 24 Bit Scanning
  • USB EBox Interface
  • Available With AFMWorkshop’s Inverted Optical Microscope (or Without)

Stage

The AFM is secured on an Adapter Plate that is mounted on the inverted optical microscope.
The Sample XY Position Stage moves the sample over the inverted microscope objective. The XY Translator for AFM Stage moves the AFM into the optical axis of the inverted light microscope.

LS-AFM components and controls

Inverted Microscope (LS AFM-B Only)

The LS AFM may be purchased as an integrated AFM/Inverted Microscope. The Inverted Microscope includes all the options for Fluorescence, Phase Contrast, and standard Illumination imaging.

Back and side view of the LS AFM stage without the AFM/ video microscope. The feet at the bottom may be removed if the stage is rigidly mounted to a surface.

Included Items

  • Lamp Chamber for Florescence Microscopy
  • UV, V, B, G excitation Filters
  • Stage with 2″ X 3″ microscope slide translator

  • AFM Stage Adapter Plate (supplied by AFMWorkshop)

  • Objectives

    • Infinity LWD plan achromatic objective 10x/0.25 WD9.67
    • Infinity LWD plan achromatic objective 20x/0.40 WD7.97
    • Infinity LWD plan achromatic objective 40x/0.60 WD3.76
    • Infinity LWD plan phase contrast objective 20x/0.40 WD7.97
  • Centering Telescope
  • DIC Polarizer
  • Lambda Plate
  • Bulb Cover
  • Phase Slide
  • C – mount port
  • Main Body

Not Shown

  • Power supply for florescence lamp
  • Power supply for illumination lamp
  • Video Camera

Ebox

Electronics in the LS AFM are constructed around industry standard USB data acquisition electronics. The critical functions, such as XY scanning, are optimized with a 24-bit digital to analog converter. With the analog Z feedback loop, the highest fidelity scanning is possible. Vibrating mode scanning is possible with both phase and amplitude feedback using the high sensitivity phase detection electronics.

24 bit scan DAC

Scanning waveforms for generating precision motion in the X-Y axis with the piezo scanners are created with 24 bit DACS driven by a 32 bit micro controller. With 24 bit scanning, the highest resolution AFM images may be measured. Feedback control using the xy strain gauges assures accurate tracking of the probe over the surface.

Phase and Amplitude Detector Circuit

Phase and amplitude in the EBox are measured with highly stable phase and amplitude chips. The system can be configured to feed back on either phase or amplitude when scanning in vibrating mode.

Signal Accessible
At the rear of the EBox is a 50 pin ribbon cable that gives access to all of the primary electronic signals without having to open the EBox.
Precision Analog Feedback
Feedback from the light lever force sensor to the Z piezoceramic is made using a precision analog feedback circuit. The position of the probe may be fixed in the vertical direction with a sample-and-hold circuit.
Variable Gain High Voltage Piezo Drivers

An improved signal to noise ratio as well as extremely small scan ranges are possible with the variable gain high voltage piezo drivers.

Software

Software for acquiring images is designed with the industry standard LabVIEW™ programming visual interface instrument design environment. There are many standard functions, including setting scanning parameters, probe approach, frequency tuning, and displaying images in real time. LabVIEW™ facilitates rapid development for those users seeking to enhance the software with additional special features. LabVIEW also enables the LS AFM to be readily combined with any other instrument using LabVIEW.

All of the functions required before making a scan are on the pre-scan tab. This includes selecting the scan mode, red dot alignment, frequency scan, and automatic tip approach.

Images are acquired using the scanning tab. Parameters selected on the scanning tab include the scan size, scan rate, GPID parameters, and the color scale used for displaying images. Included with the scanning tab is an image buffer capability that facilitates in-zooming and out-zooming.

Software control for optional modes such as MFM, EFM, and advanced F/D are found in the modes tabs. The example shown here is of the advanced F/D mode tab. This allows fine control of all the parameters controlling acquisition of force-distance curves, as well as acquisition of F-D curve maps. Mapping of curve sin this way allows the user to locate and visualize regions of the sample with varying properties, such as presence of specific molecules, or mechanical properties.

Image Analysis Software

The Gwyddion open source SPM image analysis software is included with the LS AFM. This complete image analysis package has all the software functions necessary to process, analyze, and display SPM images.

  • Visualization: false color representation with different types of mapping
  • Shaded, logarithmic, gradient- and edge-detected, local contrast representation, and Canny lines
  • OpenGL 3D data display: false color or material representation
  • Easily editable color maps and OpenGL materials
  • Basic operations: rotation, flipping, inversion, data arithmetic, crop, and resampling
  • Leveling: plane leveling, profiles leveling, three-point leveling, facet leveling, polynomial background removal, leveling along user-defined lines
  • Value reading, distance, and angle measurement
  • Profiles: profile extraction, measuring distances in profile graph, and profile export
  • Filtering: mean, median, conservative denoise, Kuwahara, minimum, maximum, and checker pattern removal
  • General convolution filter with user-defined kernel
  • Statistical functions: Ra, RMS, projected and surface area, inclination, histograms, 1D and 2D correlation functions, PSDF, 1D and 2D angular distributions, Minkowski functionals, and facet orientation analysis
  • Statistical quantities calculated from area under arbitrary mask
  • Row/column statistical quantities plots
  • ISO roughness parameter evaluation
  • Grains: threshold marking and un-marking, and watershed marking
  • Grain statistics: overall and distributions of size, height, area, volume, boundary length, and bounding dimensions
  • Integral transforms: 2D FFT, 2D continuous wavelet transform (CWT), 2D discrete wavelet transform (DWT), and wavelet anisotropy detection
  • Fractal dimension analysis
  • Data correction: spot remove, outlier marking, scar marking, and several line correction methods (median, modus)
  • Removal of data under arbitrary mask using Laplace or fractal interpolation
  • Automatic XY plane rotation correction
  • Arbitrary polynomial deformation on XY plane
  • 1D and 2D FFT filtering
  • Fast scan axis drift correction
  • Mask editing: adding, removing or intersecting with rectangles and ellipses, inversion, extraction, expansion, and shrinking
  • Simple graph function fitting, critical dimension determination
  • Force-distance curve fitting
  • Axes scale calibration
  • Merging and immersion of images
  • Tip modeling, blind estimation, dilation and erosion

Optical Microscopes

Top View Video Microscope

The LS AFM includes a top view video microscope with a 45-400x mechanical zoom tube and coaxial illumination for aligning the laser in the light lever and rapidly locating cells and other biomaterials for AFM scanning.

The LS AFM presents three views of e. coli, from video optical microscope, inverted optical microscope, and atomic force microscope

Inverted Light Microscope

The AFM rests on a custom design plate that allows standard observation of the sample and probe with the inverted light microscope (bottom view). The inverted light microscope can be used for high resolution imaging of cells in coaxial bright field or epifluorescence. The inverted light microscope is operated and can be modified independently of the AFM to collect a wide range of correlative data. The pyramidal tip at the end of cantilever can also be visualized.

Probe Holder

The LS AFM utilizes a unique probe holder/exchange mechanism. Probes are held in place with a spring device and exchanged with a probe exchange tool. This combination makes changing probes fast and easy.

Gallery

The LS AFM is designed for the most widely used types of measurements made with an AFM, including measuring F/D curves and imaging cells in a liquid environment.

Measuring Forces in Biomaterials

Monitoring the deflection of a cantilever as it is pushed against a sample results in a force/distance curve. Several quantitative mechanical parameters can be extracted from the force distance curve, such as sample stiffness and probe-sample adhesion.

In biological samples, the most common applications are the measurement of intermolecular forces and cell stiffness in a liquid/live environment with nanometer precision. The more advanced experiments use chemical functionalization of the AFM tip and/or the substrate to attach biomolecules and entire cells. Examples include measuring binding forces between an antigen and an antibody, cell-cell adhesion forces, and cellular stiffness.

The screenshot on the right demonstrates Advanced Force Distance Curve software measuring an AFM image.

  • 1
    Force-Distance data display region
  • 2
    Slider indicates the extension of the Z piezoelectric ceramic
  • 3
    Control parameter selection options
  • 4
    AFM Image for selecting locations for force-distance measurements

The Force/Distance Curve Measurement Software Interface includes all the features required for making advanced measurements. F/D curves may be made on single or multiple points of a sample surface. Control parameters include extend/contract rate, turn around trigger, and number of measurements per selected region.

Correlative imaging of Cells

Images of cells are readily scanned in both a liquid and dry environment with the LS AFM. The combined optical microscopes facilitate direct placement of the probe on an area of interest for scanning with the AFM. The inverted light microscope is then operated independently to collect correlative data in bright filed or epifluorescence.

Inverted optical microscope image of neutrophil A cells. The dotted outline is the area scanned with the AFM.
Inverted optical microscope image of neutrophil A cells. The dotted outline is the area scanned with the AFM.
Light Shaded AFM image of the cells visualized in the optical microscope image.
Light Shaded AFM image of the cells visualized in the optical microscope image.
Inverted optical microscope image of Caco-2 cells in the LS AFM. Clearly visible is the AFM cantilever on the right side of the image. A box identifies the area for AFM scanning.
Inverted optical microscope image of Caco-2 cells in the LS AFM. Clearly visible is the AFM cantilever on the right side of the image. A box identifies the area for AFM scanning.
3-D color scale image of the Caco-2 cell. The scan range is 48 µm x 48 µm.
3-D color scale image of the Caco-2 cell. The scan range is 48 µm x 48 µm.
CACO-2 cell structure in the presence of low concentration of quantum dots.

Left: Epifluorescence, showing brightfield (red), DAPI (blue), 2.2nm quantum dot PL emission at 560nm (green).

Right: Topographic AFM image of the indicated area.

Modes

Modes included with the LS AFM

Non-Vibrating (contact)

In non-vibrating mode the deflection of the cantilever is held constant as the sample is scanned. This mode is used for hard samples, and for training purposes.

Vibrating (tapping)

A piezoelectric ceramic is used to vibrate the cantilever at resonance. The amplitude of vibration is held constant as the sample is scanned. Both soft and hard samples are scanned with vibrating mode.

Phase

While scanning in vibrating mode and holding the cantilever vibration amplitude constant, the phase shift between the drive signal and photo-detector are displayed. The phase image gives a map of the relative hardness at a sample’s surface.

Frictional Force

With the 4 segmant photodetector in the LS AFM the L-R signal can be captured and displayed while scanning.

Advanced F/D
The advanced force distance modes software allows force mapping, making curves from a non-feedback position, and controlling the inbound and outbound rates.
Dunk-n-Scan

With the dunk-n-scan liquid cell, images of samples submerged in liquids can be made (not heated).

Z Scanner for Liquid Imaging

Dunk-n-Scan cell for liquid imaging of samples

Optional LS AFM Modes:

  • Conductive AFM (C-AFM)
  • Magnetic Force Microscopy (MFM)
  • Lithography
  • Scanning Thermal Microscopy (SThM)
  • Scanning Tunneling Microscope (STM)
  • Electric Force Microscopy (EFM)

Options

XY Sample stage

The optional base with XY translator gives added flexibility to the LS AFM. By removing the inverted light microscope, noise floors as low as 100 picometers are achievable. The XY translator range is 12 X 12 mm with a resolution of 1 micron. At the top of the XY translator are magnets for holding standard AFM sample disks.

Vibration Solution

Performance of the LS AFM is greatly improved with the acostic enclosure combined with an active vibration table. The option includes:

  • Acoustic Cabinet
  • Active Vibration Table
  • Base

LS AFM Liquid Cell/Heater

The LS Heat option is a combination open liquid cell and heater that is compatible with the LS AFM. In operation, the liquid cell replaces the glass slide holder in the LS AFM.

Scanning Kelvin Probe Microscopy (SKPM)

SKPM measures the potential difference between a conductive probe and a conductive sample.

Specifications

50 Micron XY Scanner
Type Modified Tripod
xy Linearity < 1%
xy Range > 50 μm
xy Resolution < 3 nm closed loop
< 0.3 nm open loop
xy Actuator type Piezo
xy Sensor type Strain Gauge
16 Micron Z Scanner / Probe Holder
Noise < 0.2 nm
Strain Gauge Resolution 1 nm
Tip Angle 10 °
Z Linearity < 5%
Z Linearity-Sensor < 1%

Includes both Air, and Dunk And Scan

Z Motion
Type Direct Drive
Range 25 mm
Drive Type Stepper Motor
Min. Step Size 330 nm
Slew Rate 8 mm/minute
Limit Switch Top, Bottom
Control Software – Rate, Step Size
Light Lever AFM Force Sensor
Probe Types Industry-Standard
Probe Insertion Manual
Probe Exchange Tool
Probe Holding Mechanism Clip
Vibrating Mode Piezo
Electrical Connector to Probe
Laser/Detector Adjustment Range +/- 1.5 mm
Adjustment Resolution 1 µm
Minimum Probe to Objective 25 mm
Laser Type 670 nm Diode, < 3 mW
Laser Focus < 25 μm
Probe Sample Angle 10°
Control Parameters
PID Yes
Setpoint Yes
Range Yes
Scan Rate Yes
Image Rotate 0 and 90°
Laser Align Yes
Vibrating Freq. Display Yes
Force Distance Yes
Tip Approach Yes
Oscilloscope Yes
Image Store Format Industry Standard
Image Pixels 16 x 16 to 1024 x 1024
H.V. Gain Control XY and Z
Real Time Display Line Level, Light Shaded, Grey Color Palette
Calibration System Window
Probe Center
Analog Electronics >> Vibrating Mode
Freq. Range 2 kHz – 800 kHz
Output Voltage 10 Vpp
Demod. Freq. TBD
Analog Electronics >> Z Feedback
Type PID
Bandwidth > 3 kHz
Sample Hold Yes
Voltage 0-150 V
Analog Electronics >> XY Scan
Voltage 0 – 150 V
Bandwidth > 200 Hz
Pan & Zoom 22 Bits
Tip Approach Cutoff < 20 μm sec
Detector
Type 4 Quadrant
Bandwidth > 500 kHz
Signals Transmitted TL, BL, TR, BR
Gain Low, High Settings
Digital Data Input Output
Connection USB
Scanning DAC
Number 2
Bits 24
Frequency 7 kHz
Control DAC
Number 2
Bits 24
Frequency 2 kHz
ADC
Number 8
Bits 14
Frequency 48 kHz
Software
Environment LabVIEW
Operating System Windows
Image Acquisition Real Time Display (2 of 8 channels)
Video Microscope
Minimum Zoom Maximum Zoom
Field of view 2 x 2 mm 300 x 300 μm
Resolution 20 μm 2 μm
Working Distance 114 mm 114 mm
Magnification 45X 400X
Computer
  • Industry-standard Computer & Monitor (laptop available upon request)
  • Windows
  • AFMWorkshop LabVIEW.exe installed

Z Noise performance depends greatly on the environment in which the LS AFM operates. Best Z noise performance is obtained in a vibration-free environment. Contact AFMWorkshop for more information on our vibration isolation equipment and recommendations.

* Z Noise on the inverted microscope is <1nm.

** Every effort is made to present accurate specifications within this document. However, due to circumstances beyond the control of AFMWorkshop, specifications are subject to change without notice.