Biophotonics II general remarks

Transcription

1 general remarks BIOPHOTONICS I (WS 2017/18) I. Imaging Systems human vision microscopy II. Light Scattering Mie scattering light propagation in tissue BIOPHOTONICS II (SS 2018) III. Biospectroscopy Fluorescence spectroscopy Phosphorescence, bio- and chemiluminescence Vibrational spectroscopy IV. Lasers in medicine Laser interaction with tissue Applications Literature: Bergmann-Schäfer, Optik (Walter de Gruyter) E. Hecht, Optik (Addison-Wesley) J. Bille, W. Schlegel, Medizinische Physik 3 (Springer) P.N. Prasad, Biophotonics (Wiley) T. Vo-Dinh, Biomedical Photonics Handbook (CRC Press) V.V Tuchin, Handbook of Optical Biomedical Diagnostics (SPIE Press) G.G. Hammes, Spectroscopy for the biological sciences (Wiley) J.R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer) It is NOT required to have attended the Biophotonics I lecture prior to visiting Biophotonics II. Lecture Biophotonics II will be credited with 2 CP subject to successfully passing the written exam. If you intend to obtain credit points, i.e. to participate in the exam, you will have to register at: 1

2 lecture #5 (May 21 st, 2018): summary III.3.4. fluorophores (continued) Quantum dots III.3.5. special methods and applications A) flow cytometry and fluorescence-assisted cell sorting (FACS) B) Fluorescence in-situ hybridisation (FISH) C) microarrays broad absorption spectrum narrow emission spectrum photostable D) Förster resonance energy transfer (FRET) FRET Efficiency: source: classes.midlandstech.edu Förster radius: 2 4 R0 ~ D n J( ) 2

3 fluorescence lifetime of the NADH coenzyme GlucDH-NADH complex: t = ns from: Alexa v. Ketteler, PhD thesis, University of Heidelberg (2012) Prof. Dr. Petrich Biophotonics II (SS 2018) 3

4 Based on previous published work [18], we attribute this short fluorescence lifetime to a high concentration of melanin (with a dominant short 200 ps decay component) relative to NAD(P)H (mean fluorescence lifetime ~0.4 2 ns depending on protein binding state), and this immediately enables the determination of the melanocytic nature of the lesion Seidenari S, Arginelli F, Dunsby C, French PMW, et al. (2013) Multiphoton Laser Tomography and Fluorescence Lifetime Imaging of Melanoma: Morphologic Features and Quantitative Data for Sensitive and Specific Non-Invasive Diagnostics. PLoS ONE 8(7): e doi: /journal.pone Figure 3. Fluorescence lifetime imaging of melanoma at different depth. 4

5 Fluorescence lifetime imaging (FLIM) Multi-photon lifetime imaging of fluorescent proteins Multi-photon excited fluorescence lifetime images of cultured cells transfected with a GFP-tagged protein (a), YFP-tagged protein (b) and both GFP- and YFP- tagged proteins (c), (d). The colour bars show the calibration of fluorescence lifetime from approx 2.1 ns (red) to 3.0 ns (dark blue). Although their fluorescence emissions overlap significantly in the green portion of the spectrum, the GFP tags show a significantly shorter lifetime than the YFP tags in the single labelled cells and can be distinguished in the co-transfected cells. Separation of these probes can be further improved by correcting for the instrument response of around Nick White and Rachel Errington, Oxford University, UK source: 5

6 Biophotonics II Fluorescence correlation spectroscopy (FCS) Source: Stowers Institute for Medical Research ) ( 1 1 ) ( ' 2 3 G R N G D D D 6

7 III.3.5 special methods and applications (continued) G) fluorescence anistropy 7

8 III.3.5 special methods and applications (continued) H) fluorescence recovery after photobleaching (FRAP) 8

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