Combining High Resolution Optical and Scanning Probe Microscopy

Transcription

1 Combining High Resolution Optical and Scanning Probe Microscopy Fernando Vargas WITec, Ulm, Germany

2 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

3 Company Background

4 Company Background Headquarters Ulm, Germany US office Urbana, IL

5 Products Scanning Probe Microscopes and Accessories High Resolution Optical Microscopes

6 Atomic Force Microscopy (AFM) position sensitive photo diode laser diode piezoscanner Binnig, Quate, Gerber IBM Lab Rüschlikon 1986 AFM tip cantilever probe sample

7 end-cap AFM Probe AFM Objective

8 Mercury 100 AFM

9 HeLa Cells human epithelial carcinoma cells fluoroalcane macromolecules 40x40 microns 400x400 nm

10 MFM - Magnetic Force Microscopy MFM 20 microns scan range Topography MFM 5 microns scan range

11 Pulsed Force Mode free cantilever oscillation

12 Pulsed Force Mode AFM SBS-PMMA polymer blend Topography Stiffness Adhesion 10x10x0.08 µm 3

13 Confocal Raman Microscopy Confocal Microscopy Raman Spectroscopy Confocal Raman Microscopy Examples

14 confocal microscopy detector pinhole classical Laser beamsplitter confocal sample objective focal plane - much smaller background - 3-D information - slightly higher resolution possible

15 Raman Spectroscopy

16 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.

17 Raman spectrum of Aspirin (acetylsalicylacid) => chemical, but NO spacial resolution

18 Confocal Microscope + Raman Spectrometer => Imaging with chemical sensitivity

19 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 => s = 4,5 h!!! => short integration times needed (ð 100ms / pixel!!!) => system must have highest efficiency

20 CCD APD multi mode fiber video camera spectrometer single mode fiber Super Notch filter holographic beam splitter laser coupler objective XYZ stage

21 Application examples

22 biological samples (living) epithelium cells of rat in physiological buffer (RR1022 virus transfected) samples prepared by A. Rück, ILM, Ulm

23 epithelium cells of rat in physiological buffer 100 x 100 pixel = 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 / water

24 epithelium cells of rat in physiological buffer Video Raman (CH 2) Intensity in the CH 2 streching band ( / cm) Peak position of the CH 2 streching band ( / cm) 100 x 100 pixel = spectra objective: Nikon 60x, NA = 1.0 water 532 nm excitation, 10 mw 100 ms / spectrum => 17 min.

25 epithelium cells of rat in physiological buffer - determination of basic spectra - fit of measured spectra with linear combination of basic spectra

26 epithelium cells of rat in physiological buffer

27 epithelium cells of rat in physiological buffer nucleus endoplasmatic reticulum nucleoli cytoplasm nuclear membrane mitochondriae

28 inner coating, Tropicana orange juice container, polymer, different layers 50µm x 100µm scan, 200 x 120= spectra, 50ms. 100 x objective (NA= 0.9), 532nm.

29 Vickers indent (Si) Topography shift of Si Raman line

30 Carbon nanotubes AFM Raman