Multimodal Imaging: Combining Optical
Microscopy with Atomic Force Microscopy
Dr. Peter Schön
Analytica 2008
Munich, April 1st
AFM of native photosynthetic membranes
100 nm
10 nm
wild type of purple bacteria Rhodobacter Sphaeroides
S. Bahatyrova et al. Nature, 430, 1058-1062 (2004)
©2006 Veeco Instruments Inc.
AFM provides topography: 3D data
Dog Epithelial Cell
Cell volume : 3500 µm3
180 x 180 µm2
Contact mode
©2006 Veeco Instruments Inc.
AFM. Principles
©2006 Veeco Instruments Inc.
How AFM can help biology ?
AFM
©2006 Veeco Instruments Inc.
AFM provides insight into…
Structure (nm resolution)/Dynamics
Forces (pN resolution)
Manipulation
©2006 Veeco Instruments Inc.
Pulling Single Molecules:
Force Spectroscopy
• simulation for pulling of single polymer molecule
•Jason Bemis, Pitt University
©2006 Veeco Instruments Inc.
Intramolecular Forces
Unfolding Bacteriorhodopsin
©2006 Veeco Instruments Inc.
Intermolecular Forces/ Affinities
Force Volume Measurements → Cell Affinity / Adhesion Maps
Layer of red blood cells from two different blood groups (A and O)
Tip functionalized with antibody specific to type A blood cells
Map lateral distribution of antibody binding sites
Affinity
Height
Grandbois, M., Dettmann, W., Benoit, M., and Gaub, H.E. Journal of Histochemistry & Cytochemistry
(2000) 48: 719-724.
©2006 Veeco Instruments Inc.
Cell Mechanics and Stiffness:
Effect of aldosterone on endothelium
Elongation of dorsal root ganglion neurites (A-C) and complex structure
of DRG growth cones (D-F): ridges and spines appear/disappear
©2006 Veeco Instruments Inc.
Oberleithner et al. (2005) Kidney International 67: 1680-1682
Identifying cancer in a tissue sample…cell stiffness capability
8
Analysis of a tissue sample
using a Veeco AFM
7
Counts
6
Cancerous Cells
5
4
3
Normal Cells
2
1
0
0
50
100
150
200
250
300
350
Cell Stiffness
Veeco collaboration
with UCLA
©2006 Veeco Instruments Inc.
400
Combining Optical Microscopy with AFM
AFM Cantilever
AFM Stage
Objective
©2006 Veeco Instruments Inc.
Seeing the AFM tip “in action”
©2006 Veeco Instruments Inc.
Nuclear Pore Complexes
©2006 Veeco Instruments Inc.
Nuclear Pore Complexes
©2006 Veeco Instruments Inc.
Combined AFM-Optical Microscopy:
Applications in Life Science
Navigation: Direct AFM tip to ROI
Optical identification + high-resolution AFM imaging.
Direct correlation of fluorescence with sample topography
= image correlation.
Optical identification + nanomechanical measurements
(elasticity, molecular unfolding, ligand-receptor
interactions, etc.).
Mechanical stimulation/ manipulation by AFM with
optical observation of an induced response.
©2006 Veeco Instruments Inc.
Optical identification + high-resolution
AFM imaging.
Combined AFM/Fluorescence Studies
of Filopodia of Macrophages
(Data Courtesy of P.Schoen, Veeco and W.Wittke, Leica.
Cells: Peter Hanley, Münster, Germany)
©2006 Veeco Instruments Inc.
Direct correlation of fluorescence with sample
topography = image correlation.
Epifluorescence
a
A
d
B
b
e
E
c
(Samples supplied by Dr. Frank Lafont, Pasteur Institute Lille)
a-c: Epifluorescence
a: Golgi green
b: F-Actin red
c: Nuclei blue
d: 100 µm AFM amplitude
image
e: Image overlay
Real Time Image Overlay
exact registration of the AFM tip
©2006 Veeco Instruments Inc.
Real Time Image Overlay
An AFM overlay scan, in the moment of generation.
©2006 Veeco Instruments Inc.
Real Time Image Overlay
The same AFM overlay scan, in the moment of finalization
©2006 Veeco Instruments Inc.
Living endothelial cells,
Contact mode in buffer, at 37 degress
10 µm
Living endothelial cells,
Contact mode in buffer, at 37 degress
10 µm
AFM & Epi-fluorescence Image Overlay
Optically Guided Force Spectroscopy
Eliminates need to acquire an
overview image of sample:
↓ time to data.
Non-disruptive to sensitive
samples (live cells).
Preserves functionalized
probes.
Courtesy C. Callies, H.
Oberleithner. Institute for
Physiology II, University of
Muenster, Germany
Optical identification + nanomechanical
measurements
inositol
glucose
mannose
ethanolamine
tip
GFP
PA
Proaerolysin (PA)
Force measurements on living Hela cells
©2006 Veeco Instruments Inc.
Force measurements on living Hela cells
A
A
B
B
A
B
endogeneous GPI-anchored proteins
Alexandre Berquand, Veeco Life Science Team
©2006 Veeco Instruments Inc.
Optical identification +
nanomechanical measurements
Mechancial Probing of Bacteria
incooperated into Dendritic Cells
©2006 Veeco Instruments Inc.
©2006 Veeco Instruments Inc.
Mechanical stimulation/ manipulation
Combined AFM/TIRF Studies of Cellular Elasticity
(Data courtesy of Andre Brown & Prof. Dennis Discher, University of Pennsylvania.)
©2006 Veeco Instruments Inc.
Compressing Casein Microdoplets
©2006 Veeco Instruments Inc.
Top-View Optics for opaque samples
Specifications:
Working Distance = 86mm
System Mag. = 1.16x to 14x
(Total Mag. = 14x to 168x)
Field of View = 0.45mm to 5mm
Depth of Field = 1.39mm to
0.05mm
Adjustable illumination angle.
Compatible with:
IOM-mounted configuration.
stand-alone configuration.
Currently, can be ordered as a
Special.
©2006 Veeco Instruments Inc.
Application Example:
Nanomechanical Properties of Mammary Gland Tissue
The thickness of the tissue sample prevents viewing of the surface with the IOM.
The BioScope II TVO allows us to clearly distinguish the area of the lymph node (left) from the
surrounding mammary gland tissue (right).
The resulting force curves are very different, and indicate the lymph node to have a higher stiffness
than the rest of the mammary tissue.
Force measurements were performed in fluid using DNP probes (0.06 N/m).
©2006 Veeco Instruments Inc.
Combining AFM and Optical Microscopy
Fully Compatible with high-end
inverted optical microscopes:
Minimal interference with normal
operation of optical microscope.
Facilitates use of standard off-the
shelf optical components:
•
•
•
•
•
≤ 0.55 NA condenser
Brightfield (Kohler Illumination)
Darkfield
DIC
Phase Contrast
©2006
Supports
high
mag. objectives (oil & water immersion)
Veeco Instruments
Inc.
Flexible SPM Platform:
Integration With Fluorescence Techniques
Integration with more advanced
optical techniques:
• Epifluorescence
• Confocal Laser Scanning
Microscopy (CLSM)
• Total Internal Reflectance
Fluorescence (TIRF)
• Fluorescence Recovery
After Photobleaching
(FRAP)
• Fluorescence Resonance
Energy Transfer (FRET)
Standard IR super luminescent
diode (SLD) for deflection
detection
• Eliminates interference with common
red emitting biological fluorophores.
©2006 Veeco Instruments Inc.
BioScope II Optical Access
Condenser
Tip Holder
Removable Sample Plate
and Sample
Objective
Cross-Section of the BioScope II from the side
©2006 Veeco Instruments Inc.
Combining AFM and Optical Microscopy
A1.273
©2006 Veeco Instruments Inc.
October 15 – 18, 2008
at the Hyatt Regency Monterey, California
www.afmbiomed.org
California Institute
for Quantitative Biosciences
Important Deadlines:
Abstract submissions deadline:
April 8, 2008
Pre-registration deadline for
discount rate: April 25, 2008
www.veeco.com/nanoconference
©2006 Veeco Instruments Inc.
Thanks for your attention!
Dr. Peter Schön (pschoen@veeco.de)
A1.273
©2006 Veeco Instruments Inc.
Bioscope-II ‘head’
Unique open “Hole In The Head” design
Ergonomic design for easy handling
Direct visibility and easy access to tip and/or sample
Allows introduction of materials into sample area
(chemicals, solutions, mechanical probes, etc.)
©2006 Veeco Instruments Inc.
Environmental Control:
Fluid Perfusion cell and heating stage
Confined Perfusion Chamber:
Controls and circulates fluid
Controls the gaseous environment of the
chamber
Fluid And Gaseous Control Enable:
Long term imaging of nutrient and
oxygen/pH sensitive samples such as live cells.
In situ imaging of self-assembly processes and
molecular interactions
The Integrated Heating Stage:
Controls the temperature of the sample stage
Directly monitors sample temperature using a
remote thermocouple
This Temperature Control Enables:
Live cell imaging over long periods.
The study of thermally activated
processes such as crystal formation and
dissolution.
Observation of lipid membrane phase transitions
©2006 Veeco Instruments Inc.
Compatibility with 4 Main Microscope
Vendors
4x
ZEISS
NIKON
OLYMPUS
LEICA
©2006 Veeco Instruments Inc.
1x
Force Volume Abbildung
links: Adhesionkraft
rechts: applizierte Kraft auf Zellinneres
©2006 Veeco Instruments Inc.