Fluorescence Workshop UMN Physics June 8-10, Fluorescence Microscopy and Fluorescence Correlation Spectroscopy Joachim Mueller

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1 Fluorescence Workshop UMN Physics June 8-10, 2006 Fluorescence Microscopy and Fluorescence Correlation Spectroscopy Joachim Mueller

2 Fluorescence Microscopy Use a microscope as a fluorometer Advantages: superb optics very high collection efficiency imaging allows single cell measurements single molecule experiments

3 The microscope as a filter fluorometer with focusing optics

4 Basic design of a fluorescent microscope red fluorescence objective

5 Filters

6 Selecting Filters

8 Anatomy of a Fluorescence Microscope

9 Widefield-fluorescence microscope image Widefield fluorescence image of a 16 micron thick section of fluorescently-labeled mouse kidney. Copyright, J. Waters, 2004

10 Principle of Confocal Microscopy Pinhole only passes light that emanates from a thin section of the sample (indicated in red) Note confocal microscopy only observes a point (a tiny subvolume to be more precise) z

11 Laser Scanning Confocal Microscopy (LSCM): Observed volume Need to perform a raster scan to build up an image point by point Two electronically driven scan mirrors move the laser spot on the sample in a raster-like fashion.

12 Widefield Image Confocal Image Widefield fluorescence image of a 16 micron thick section of fluorescently-labeled mouse kidney. The same specimen show on the left, taken with a confocal microscope. Copyright, J. Waters, 2004

13 Photobleaching The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust can undergo thousands or millions of cycles before bleaching.

14 Photobleaching original image Photobleached image s

15 Two-Photon Microscopy: Principle Now consider two-photon absorption Energy Diagram: S 1 Internal Conversion ~10-12 s Absorption ~10-15 s Radiationless Decay <10-9 s Fluorescence ~10-9 s S 0 Two-photon absorption is an optical nonlinear process

16 Two-Photon Fluorescence Simultaneous absorption of two-photons is a rare process: Maximize two-photon effect by increasing the photon flux - spatially by focusing the light - temporally (ultrafast laser pulses) One photon beam two photon spot (volume: 1 fl) objective Inherent 3 - dimensional optical sectioning effect!

17 Two-photon spectroscopy One-photon two-photon Intensit y 1γ emission 2γ wavelength Two-photon absorption is spectrally well separated from the fluorescence! Note that Raman of the solvent will not occur within the fluorescence emission spectrum.

18 Two-photon Instrumentation Sample Objective Steering mirror Dichroic Mirror Beam Expander Short Pass Filter Ti-Sapphire laser APD Photon Counting Acquisition Card Data Analysis

19 Two-photon Imaging Purkinje neurone in a living brain slice filled with fluorescein dextran imaged with two-photon excitation laser scanning microscopy (Svoboda, Cold spring Harbor Laboratories) Two-photon image resolution is essentially the same as that of confocal microscopy. However, imaging in the presence of significant scatter (such as in thick tissue) requires two-photon excitation. Also note that photobleaching in twophoton microscopy is strictly restricted to the excitation volume! Denk, Nature Methods 2, (2005)

20 Fluorescence Fluctuation Spectroscopy Q: How many fluorescent molecules are (on average) in the two-photon (or confocal) volume of the microscope? A: That depends on the concentration. At a high concentration there are more molecules in the volume than at low concentrations. Q: Ok, many proteins in cells have nanomolar concentrations. How many proteins ( assuming c = 1 nm ) are now in the volume? A: Let me calculate (Volume is 1 femtoliter, Avogadro s number is 6x10 23, c = 1 nm). The number I get is a single molecule per observation volume. Well that s ok, fluorescence is very sensitive and can detect single molecules. Q: Proteins in a solution (and in a cell) are typically mobile. They diffuse around. What will happen if the single molecule moves around? Also a single molecule is in the volume on average. Is there a chance that sometimes there will be two or no molecules in the volume? A: Yes, the number of molecules will fluctuate as they diffuse in and out of the observation volume. Because two molecules produce more fluorescence than a single molecule there will be fluctuations in the fluorescence intensity.

21 Fluorescence Fluctuation Spectroscopy Fluorescence intensity: F fluctuation time Raw data: Photon Counts Coverslip objective

22 50 Statistical Analysis of the Fluctuations required Photon counts 0.04 Fluorescence Correlation Spectroscopy (FCS) Counts Auto Correlation Parameters: G(0) & k kinetics Fit Data Autocorrelation Time Time (ms) Autocorrelation Function: Number of Occurances Photon Counting Histogram (PCH) PCH Parameters: <N> & ε G( τ ) = df() t df( t + τ ) F Counts per Bin

23 F(t) in photon counts Calculating the Autocorrelation Function Fluorescence Fluctuation df() t = F() t F time τ Average Fluorescence F t t + τ G( τ ) = df() t df( t + τ ) F 2

24 The Autocorrelation Function t 2 t 3 t 4 t 1 t 5 Detected Intensity (kcps) Time (s) G(0) 1/N as time t approaches 0 G(τ) Diffusion G( τ ) = df() t df( t + τ ) F Time(s)

25 The Effects of Particle Concentration on the Autocorrelation Curve <N> = G(t) <N> = Time (s)

26 What about the excitation (or observation) volume shape?

27 Correlation function of diffusing molecules For a 3-dimensional Gaussian excitation volume: Dτ 8Dτ G( τ ) = N w0 z0 N: average number of particles inside volume D: Diffusion coefficient w o : radial beam waist of two-photon laser spot z o : axial beam waist of two-photon laser spot photon equation contains a 4, instead of 8

28 The Effects of Particle Size on the Autocorrelation Curve Diffusion Constants um 2 /s 90 um 2 /s 71 um 2 /s Stokes-Einstein Equation: D = and k T 6 π η r G(t) Slow Diffusion Fast Diffusion Time (s) MW Volume 3 r Monomer --> Dimer Only a change in D by a factor of 2 1/3, or 1.26

29 FCS inside living cells Correlation Analysis D solution D nucleus = 3.3 inside nucleus g(τ) 0.4 in solution E-5 1E-4 1E τ (sec) Measure the diffusion coefficient of Green Fluorescent Protein (GFP) in aqueous solution in inside the nucleus of a cell.

30 Statistical Analysis: Brightness Brightness ε is the average fluorescence intensity of a single particle Illustration: EGFP Protein Sample Fluorescence F Brightness ε time

31 Brightness Encodes Stoichiometry Sample Fluorescence Brightness EGFP Protein F time ε N 1 F 2ε time N 2 + F 2ε ε time N 1 N 2

32 Photon Counting Histogram (PCH) Aim: To resolve species from differences in their molecular brightness Molecular brightness ε : The average photon count rate of a single fluorophore PCH: probability distribution function p(k) where p(k) is the probability of observing k photon counts ε = 16000cpsm N = 0.3 Single Species: p(k) = PCH(ε, N ) Note: PCH is Non-Poissonian! Sources of Non-Poissonian Noise Detector Noise Diffusing Particles in an Inhomogeneous Excitation Beam* Particle Number Fluctuations* Multiple Species*

33 PCH in cells: Brightness of EGFP Chen Y, Mueller JD, Ruan Q, Gratton E (2002) Biophysical Journal, 82, 133. Molecular Brightness (cpsm) 3x10 4 2x10 4 1x autofluorescence cytoplasm Excitation=895nm autofluorescence nucleus EGFP cytoplasm EGFP solution EGFP nucleus The molecular brightness of EGFP is a factor ten higher than that of the autofluorescence in HeLa cells

34 Brightness and Stoichiometry Intensity (cps) x EGFP Brightness ε app (cpsm) EGFP 2 EGFP EGFP Brightness Concentration [nm] EGFP EGFP 2 Brightness of EGFP 2 is twice the brightness of EGFP Chen Y, Wei LN, Mueller JD, PNAS (2003) 100,

35 The End

Two-photon FCS Tutorial. Berland Lab Department of Physics Emory University

Two-photon FCS Tutorial. Berland Lab Department of Physics Emory University Two-photon FCS Tutorial Berland Lab Department of Physics Emory University What is FCS? FCS : Fluorescence Correlation Spectroscopy FCS is a technique for acquiring dynamical information from spontaneous