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Cell parts can then be collected and purified. The disrupted rigid outer cell walls can be eliminated in another cycle of centrifugation and washing processes. After a series of purification steps a solution of crystalline cell parts containing only a minor fraction of dissolved biological material is obtained. Dependent on the various forms of treatment before the explosion, the results obtained can differ to a great extent. Variations of this procedure included tests on cells treated with detergent, non-treated live cells and fixated 2 cells. 5.2 Experimental Setups 5.2.1 Optical Microscopy and Fluorescence Microscopy In Pisa optical and fluorescence microscopy were carried out with a Zeiss Axioplan fluorescence microscope equipped with an epifluorescence system, various magnifications and a 100W mercury lamp. The following filter combination could be used: a UV-blue set (an 8 nm bandpass excitation filter, 365 nm; chromatic beam splitter, 395 nm; barrier filter, 397 nm; 800 µW/cm 2 ) and a blue-violet set (an 8 nm bandpass excitation filter, 436 nm; chromatic beam splitter, 460 nm; barrier filter, 470 nm; 1100 µW/cm 2 ). The chromatic beam splitters redirect light from the mercury lamp to be used as fluorescence excitation light (wavelengths smaller than 365 nm and 460 nm respectively). The bandpass filters only let bands around 365 nm and 436 nm wavelength pass. The barrier filters block reflected excitation light from the environment and transmit only the fluorescence light from the specimen.. Fluorescence spectra can characterize algal species (through their chloroplast absorption and emission spectra) and provide valuable information on the composition and photocycle of the Euglena gracilis photoreceptor. E.g. the embedded rhodopsin molecules can change their conformational state depending on the wavelength of absorbed light and thus fluoresce differently. Images during fluorescence microscopy were taken with a Canon digital camera. 5.2.2 Spectroscopy Some optical spectroscopy was carried out for educational purposes. Analysis of different parts of the cell was done, using a Jasco 7850 spectrometer and fluorescence spectra were recorded using a Perkin Elmer LS 50 B fluorimeter in Pisa. 2 Fixated cells are treated in such a way that enzymatic activity is shut down. This prevents tissue autodigestion (autolysis), i.e. that unregulated enzymes would otherwise start to decompose cell organelles and membranes. 68
Figure 5.3: This image shows cells that have been previously treated with detergent solution. Most of them are broken open and their chloroplasts float freely (red discs in the picture). The fluorescence seen is the emission during illumination by 436 nm light. The two visible green dots belong to two photoreceptors. Their rhodopsin molecules were previously excited by 365 nm light and change now back into the ground state by emitting characteristic and intense green light. Scale bar is 20 µm. Those spectra can be used to relate the type of alga to the type of pigments owned (chlorophyll in chloroplasts, carotenoids in eyespot droplets, rhodopsin in photoreceptor) and to position the algae within the phylogenetic tree. 5.2.3 AFM measurements AFM Setup Cells and cell organelles were investigated with a prototype MFP-3D atomic force microscope manufactured by Asylum Research (Santa Barbara, CA, USA). The AFM head was mounted on an inverted optical microscope (Axiovert MAT-100, Zeiss, Jena, GER) and equipped with top-view optics. This enabled precise optical positioning and investigation of transparent as well as opaque samples. Optical microscopy images were taken with a Canon digital camera. A closed-loop XY-stage allowed for AFM scanning across an area of up to 90 × 90 µm 2 as well as fine-grained sample positioning for local experiments. The stage used is compatible with different sample supports, including glass slides, coverslips (very thin glass slide usually placed on top of sample and supporting glass slide), or petri dishes from 35 mm to 85 mm in diameter. The setup included 69
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DIPLOMARBEIT Atomic Force Microscop
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»Later I have been asked many time
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Contents Abstract 4 1 Introduction
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5.2.1 Optical Microscopy and Fluore
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theory (see e.g. [1]). Now I call o
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understand phenomena at a larger sc
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of whole dried Euglena cells in air
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these assembly functions for us. 2
Page 17 and 18: Figure 2.2: Rise in public spending
Page 19 and 20: The presented three types of nanote
Page 21 and 22: 2.4.1 Self-Assembly and Emergent St
Page 23 and 24: After diligent investigation such s
Page 25 and 26: 1 µN) between a probe and the samp
Page 27 and 28: 3.2.1 Static Mode In Static Mode (a
Page 29 and 30: crystal and the cantilever tip (pha
Page 31 and 32: Figure 3.6: Highly ordered and fres
Page 33 and 34: Figure 3.8: Schematic drawing of th
Page 35 and 36: Figure 3.11: The cantilever deflect
Page 37 and 38: Figure 3.14: This force curve shows
Page 39 and 40: Figure 4.1: A group of Euglena orga
Page 41 and 42: several species with rigid pellicle
Page 43 and 44: Figure 4.2: The rightmost cell can
Page 45 and 46: cavity and is used for regulatory f
Page 47 and 48: as phobic reactions, because the ce
Page 49 and 50: Figure 4.7: Scheme of the photorece
Page 51 and 52: Figure 4.9: Measured monoclinic uni
Page 53 and 54: Figure 4.11: Twice the same Euglena
Page 55 and 56: 4.15), their ends are termed the (-
Page 57 and 58: Figure 4.15: Kinesin and dynein and
Page 59 and 60: Figure 4.17: (Image reproduced from
Page 61 and 62: into fully functional chloroplasts
Page 63 and 64: Figure 4.19: How a microtubule-kine
Page 65 and 66: Chapter 5 Materials and Methods The
Page 67: Cell Treatment - Demembranation In
Page 71 and 72: Figure 5.4: The AFM setup at the In
Page 73 and 74: Figure 5.6: If the user menu AFM_TU
Page 75 and 76: Figure 5.7: Single steps of the sam
Page 77 and 78: Chapter 6 Results High resolution t
Page 79 and 80: Figure 6.2: An embedded E. gracilis
Page 81 and 82: Figure 6.5: Scanning under liquid s
Page 83 and 84: Flagellum The flagellum of an E. gr
Page 85 and 86: Pellicle The pellicle of E. gracili
Page 87 and 88: Figure 6.11: The pellicle of an E.
Page 89 and 90: Crystalline Cell Parts Crystalline
Page 91 and 92: Figure 6.18: A layered structure th
Page 93 and 94: It might also be possible that comb
Page 95 and 96: Appendix A Acronyms SPM Scanning Pr
Page 97 and 98: [13] A. Junk, F. Riess: From an ide
Page 99 and 100: [45] J. Ruan, B. Bhushan: Atomic-sc
Page 101 and 102: [70] M. Rief, M. Gautel, F. Oesterh
Page 103 and 104: [97] P. Gualtieri, L. Barsanti: Alg
Page 105 and 106: Annex 1 This Igor script was used i
Page 107 and 108: w[i][j][laynr]=(w[i][j][3]-used_off
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