Combining High Resolution Optical and Scanning Probe Microscopy Fernando Vargas WITec, Ulm, Germany www.witec.de
Company Background Foundation 1997 by O. Hollricher, J. Koenen, K. Weishaupt WITec = Wissenschaftliche Instrumente und Technologie = Scientific Instruments and Technology WITec today established company with world-wide distribution US branch office since 2002: WITec Instruments 3D confocal image of a phone card chip
Company Background
Company Background Headquarters Ulm, Germany US office Urbana, IL
Products Scanning Probe Microscopes and Accessories High Resolution Optical Microscopes
Atomic Force Microscopy (AFM) position sensitive photo diode laser diode piezoscanner Binnig, Quate, Gerber IBM Lab Rüschlikon 1986 AFM tip cantilever probe sample
end-cap AFM Probe AFM Objective
Mercury 100 AFM
HeLa Cells human epithelial carcinoma cells fluoroalcane macromolecules 40x40 microns 400x400 nm
MFM - Magnetic Force Microscopy MFM 20 microns scan range Topography MFM 5 microns scan range
Pulsed Force Mode free cantilever oscillation
Pulsed Force Mode AFM SBS-PMMA polymer blend Topography Stiffness Adhesion 10x10x0.08 µm 3
Confocal Raman Microscopy Confocal Microscopy Raman Spectroscopy Confocal Raman Microscopy Examples
confocal microscopy detector pinhole classical Laser beamsplitter confocal sample objective focal plane - much smaller background - 3-D information - slightly higher resolution possible
Raman Spectroscopy
Raman effect: - Some of the incidents photons are able to excite (or anhilate) vibrational quantum states in molecules of the sample. - Each molecule has characteristics vibrational quantums states which are possible to be stimulated.
Raman spectrum of Aspirin (acetylsalicylacid) => chemical, but NO spacial resolution
Confocal Microscope + Raman Spectrometer => Imaging with chemical sensitivity
main problem in Raman microscopy: signal intensity max. intensity: 1-10 mw at the sample 5 2 1mW, 0,5µm spot => 4x10 W/cm time: 128x128 pixel image, 1s/pixel integration time => 16384 s = 4,5 h!!! => short integration times needed (ð 100ms / pixel!!!) => system must have highest efficiency
CCD APD multi mode fiber video camera spectrometer single mode fiber Super Notch filter holographic beam splitter laser coupler objective XYZ stage
Application examples
biological samples (living) epithelium cells of rat in physiological buffer (RR1022 virus transfected) samples prepared by A. Rück, ILM, Ulm
epithelium cells of rat in physiological buffer 100 x 100 pixel = 10000 spectra objective: Nikon 60x, NA = 1.0 water 532 nm excitation, 10 mw 100 ms / spectrum => 17 min. single spectrum, 0.1 s integration time CH 2 Proteins: 1450/cm 1660/cm Lipids: 1070/cm 1300/cm 1440/cm 1070 1265/ 1310 1440 1660 water
epithelium cells of rat in physiological buffer Video Raman (CH 2) Intensity in the CH 2 streching band (2800-3000 / cm) Peak position of the CH 2 streching band (2885-2935 / cm) 100 x 100 pixel = 10000 spectra objective: Nikon 60x, NA = 1.0 water 532 nm excitation, 10 mw 100 ms / spectrum => 17 min.
epithelium cells of rat in physiological buffer - determination of basic spectra - fit of measured spectra with linear combination of basic spectra
epithelium cells of rat in physiological buffer
epithelium cells of rat in physiological buffer nucleus endoplasmatic reticulum nucleoli cytoplasm nuclear membrane mitochondriae
inner coating, Tropicana orange juice container, polymer, different layers 50µm x 100µm scan, 200 x 120= 24000 spectra, 50ms. 100 x objective (NA= 0.9), 532nm.
Vickers indent (Si) Topography shift of Si Raman line
Carbon nanotubes AFM Raman