Optical Spectroscopy. Virginia Lorenz, Kai Wen Teng. PHYS 403 Spring 2017

Transcription

1 Optical Spectroscopy Virginia Lorenz, Kai Wen Teng PHYS 403 Spring 2017

2 Electromagnetic Spectrum of atoms and molecules From Diagram by Robert Clegg in Photosynth Res Aug-Sep;101(2-3):

3 Types of Fluorescent Molecules Synthetic Organic: Fluorescein Semiconductor Nanocrystal: Crystals: Naturally Occuring: Ruby and assorted minerals From mineralman.net Fluorescent Nanodiamonds Fluorescent Proteins: Green Fluorescent Protein Image from Zrazhevskiy et al Nano Lett., 2010, 10 (9), pp DOI: /nl

4 Perrin-Jablonski energy diagram illustrates the electronic states of a molecule and the transitions between them Alexander Jabłoński ground electronic singlet state Diagram by Ulai Noomnarm in Photosynth Res Aug-Sep;101(2-3):

5 O.D O.D Absorption (S 0 S 1 )? l Beer-Lambert s Law log(i 0 ) log(i) cl Extinction coefficient: Concentration Caffeine

6 Steady State Measurements: Absorbance One of the very first commercially available instrument that measures absorbance was the Beckman DU Machine nowadays that utilizes diffraction grating and diode array detector can acquire an absorbance spectra in less than 10 seconds.

7 Organic dye: Fluorescence (S 1 -S 0 ) Solvent Effect: Ruby:

8 Time-Dependent Fluorescence: Fluorescence Lifetime Fluorescence Lifetime: The average amount of time a molecule stays in excited state Probability of being in the excited state kf = rate constant for leaving excited state while emitting a photon ki = rate constant for leaving excited state through other means (ie. Dynamic quenching, Energy Transfer, etc) Excited State kf ki ki Diagram by Robert Clegg in Photosynth Res Aug-Sep;101(2-3): Fluorescence Lifetime: i 1 ki Lifetime is sensitive to other decaying pathway present!

9 The Bolognian Stone MarcAntonioCellio (1680) representing the light emission of heated barite

10 First mention of lifetimes? The Bologna stone, when placed in the sun attracts the rays, and retains them so long as to give light a considerable time after it is removed into the dark. Goethe The Sorrows of Werter

11 Lastusaari et al. 2011

12 Dr. Brand in attempted to distil human urine and in this way discovered phosphorus. Phosphorus (Greek phosphoros was the ancient name for the planet Venus) was discovered by German alchemist Hennig Brand in 1669 through a preparation from urine. Working in Hamburg, Brand attempted to distill salts by evaporating urine, and in the process produced a white material that glowed in the dark and burned brilliantly. Misnomer: Phosphorescence of phosphorous is due to slow oxidation Painting by Joseph Wright of Derby (18thcentury) representing the discovery of the phosphorescence of the phosphorus extracted from urine by Hennig Brand in 1669

13 Measuring the Depletion of the excited state * * kf kt t # x # xo e # F * x k Intensity that you = measure [x] * KF is rate constant of fluorescence Intensity measured is proportional to the # of molecules in the excited state!

14 Measuring Lifetime: Time Domain nsec What do you need? -Collect signal fast enough -Fitting

15 Measuring Lifetime: Frequency Domain E(t) = E o + Eωcos ωet + φe F(t) = F + F cos ω t + φ - φ o ω E E tan ω E F F 1 ω o M = = Eω Eo 1+ ωτ Mod 2 What do you need? -Intensity modulators -Synchronization

16 AOMs Intensity Modulator Annals of the New York Academy of Sciences Vol. 158 pp , modulation frequency limited by resonance frequency of the acoustooptic cell -variations in the intensity modulation caused by temperature

17 Pockels Cell Biophysical Journal Vol. 44 (1983) pp Hecht Optics

18 Directly Modulated Diode System in ESB Laser Diodes -> (405nm,436nm,473nm,635nm,690nm,780nm,830nm) LEDs -> (280nm,300nm,335nm,345nm,460nm,500nm,520nm)

19 Champaign, IL: domain of fluorescence lifetime imaging microscopy Robert Clegg UIUC Full-Field FLIM Enrico Gratton UIUC Scanning Confocal FLIM (FLIMBox) Beniamino Barbieri ISS Inc. Commercialization of FD FLIM

20 Applications of Fluorescence in Biology

21 Msinφ Msinφ Fluorescence Lifetime Imaging on Live Cells Top of cell: Intensity Top of cell: Lifetimes Optical Sections Rendered by Lifetime 0µm 0.5µm 1.0µm 1.5µm 2.0µm 2.5µm 3.0µm 3.5µm 4.0µm 4.5µm 5.0µm 5.5µm 6.0µm 6.5µm 7.0µm 7.5µm 8.0µm 8.5µm 9.0µm 9.5µm 10.0µm 10.5µm 4.5µm ns ns 1 4 ns 3 ns 4 ns 3 ns 2 #1 #2 0 Mcosφ Images courtesy of John Eichorst Mcosφ 1.0

22 Single Molecule Fluorescence Imaging Center of the distribution can be determined with ~1.5 nm accuracy if N is more than 10 4 Yildiz et al., Science 303, 676 (2004).

23 Super Resolution Fluorescence Imaging Huang et al., Science 319, 810 (2008).