Fluorescence Microscopy for an NMR- Biosensor Project

Transcription

1 Fluorescence Microscopy for an NMR- Biosensor Project Ole Hirsch Physikalisch-Technische Bundesanstalt Medical Optics Abbestr. -1, Berlin, Germany Overview NMR Sensor Project Dimensions in biological systems Widefield Fluorescence Microscopy Confocal Microscopy -Photon Microscopy Comparison of Methods Contributions to Sensor Project Examples Auswärtsseminar der AG Optische Technologien WSH Zwickau

2 19 Xe-NMR-Biosensor Magnetic Resonance Population difference Chemical shift n 1 ΔE=ħ γ B 0 I. J. Dmochovski n 0 MRI with hyperpolarized gases Cryptophane Molecular cage S. Patz et al., Eur. J. Radiol. 64(3), 007. E. Dubost et al, Org. Lett O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

3 NMR spectroscopy Optical Microscopy MRMI Typical dimensions 1 mm 100 µm 10 µm Cell diameter Cell organelles 1 µm Wavelength of green light 100 nm 10 nm Thickness of cell membrane 1 nm Typical molecule 100 pm = 1Å C-C bond H-Atom O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

4 Widefield Fluorescence Microscopy Rhodamine B Cell culture, transmission image and WF fluorescence Inverse Microscope (Zeiss) O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

5 Principle of Confocal Microscopy Patent Minsky 1957, commercially available since the 1980s in-plane light off-plane light (background) Pollens Zeiss online campus O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

6 Principle of -Photon Microscopy -Photon Microscopy - almost simultaneous absorption of photons - light source with high peak power required - excitation at longer wavelength deeper penetration and less sample bleaching - excitation of fluorescence only in a small focal volume - main application: deep imaging in scattering media, e.g. tissue - detection without pinhole - light detection close to objective O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

7 Comparison of methods Widefield fluorescence Confocal Microscopy PH 1AU Confocal Microscopy PH < 0.5 AU -Photon Microscopy Lateral Resolution 0.51 em 0.51 em 0.37 Conf. x Axial Resolution n em n 0.88 n exc n 0.64 n Conf. x FWHM of PSF (z-direction) 1.77 n exc 1.77 n exc 1.8 n Conf. x Thickness of optical slice n n em n PH n 0.64 n (S. Wilhelm, Confocal Laser Scanning Microscopy, CZ Jena) AU: Airy unit PH: pinhole diameter

8 How can optical Microscopy contribute to the Biosensor-Project? Test application for NMR-Biosensor: Observation of the uptake of certain peptides by cells model for controlled delivery of pharmaceuticals Contributions of Fluorescence Microscopy: - Visualisation of peptides uptaken by the cells - Quantification of the amount of peptides uptaken by the cells - Localisation of tagged peptides on cell level - Distinction between peptides in and at cells - Estimating the velocity of peptide uptake, allows conclusions on the transport mechanism O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

9 Experiments and Microscopy on cells Cell grow at MDC Image processing and analysis Transport to PTB laboratory Microscopy Mixing of nutrition solution with labeled peptide solution Removal of liquid container, Application of cover slip Incubation of cells for different times Rinse with buffer solution, Removal of unbound compounds O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau

10 Fluorescence Microscopy First Results - Estimation of practical substance concentrations and incubation times - Verification of different penetration behaviour of cationic and anionic peptides - Control samples treated with poor dye-solution Cat. Peptiode+Fluoresceine an. Peptide+Fluoresceine Autofluorescence of a sample Transmitted light (top) and Widefield fluorescence (bottom) O. Hirsch Auswärtsseminar der AG Optische Technologien WSH Zwickau