Observing a nanomachine at work: Single-molecule imaging or spectroscopy (SMI or SMS)

Transcription

1 Observing a nanomachine at work: Single-molecule imaging or spectroscopy (SMI or SMS) Principle: SMS allows one to observe the function and the motion of nano-objects in realtime in living systems. Usually, fluorescence measurements are performed on an ensemble of molecules (several thousands). In SMS, the fluorescence of a single molecule is detected, and then statistics is performed over the results obtained on several molecules. The fact that SMS is based on fluorescence makes it non-invasive and highly specific. In order to observe the behaviour of a non-fluorescent biomolecule of interest, this molecule is specifically labelled using a fluorophore.

2 Measurement principle: Fluorescence microscopy Laser In order to discriminate molecules one from each others, it is necessary to dilute the concentration of fluorophores to such an extent that the average distance between two fluorophores is larger than the resolution of the microscope (confocal or wide-field). Depending on the experiment, the dilution will occur: in a cell membrane in an artificial membrane on a biomodified surface in a thin polymer sheet spin coated on a glass surface CCD

3 Typical images of single molecules MHC I molecules labelled with a fluorophore on the surface of living cells

4 Benefits of single-molecule experiments Avoid ensemble averaging (for instance in the case of receptors existing in different conformations, membrane domains in a cell, biomotors that can be in an active or in an inactive state, ). Avoid temporal averaging (no need to synchronize). Give access to novel experimental parameters (in particular to a higher position accuracy).

5 Single-molecule photophysics: Jablonski's diagram s Non-radiative decay very variable Fluorescence lifetime: 1/ k f =τ f =1/(k em +Σk i ), radiative lifetime τ r =1/k em Fluorescence quantum yield q f = τ f /τ r = # emitted photons/# absorbed photons

6 Signal emitted by a single molecule RI ( ) = R max ex = R max I ex / ex I sat 1 + I / I kq f f (1 + k / k ) ISC T sat I sat = ( kf ) σ (1 + k / k ) 2 σ = 3σav cos θ ISC R = fluorescence emission rate, I ex = laser excitation intensity, I sat = saturation intensity, k T = triplet decay rate, k ISC = inter-system crossing rate, σ = absorption cross-section T For TMR oriented // to the laser beam: σ av = 1.9*10-16 cm 2 q f = 0.28 τ f = 2.1 ns τ T = 1/k T = 2 µs k ISC = 0.03/ τ f I sat = 11 kw/cm 2 ; R max = 4500 photons/ms

7 Fluorescence timetrace Antibunching Bunching Time Blinking can have several origins: 60 0 Triplet and other dark states. Modifications in the environment: Changes of the spectrum or of the fluorescence quantum yield. Chemical reactions (e.g. protonation) or complex formations (with O 2 ). photo-ionisation. etc Counts Time

8 Single nanoparticles in a transparent polymer Blinking is very pronounced due to photo-ionisation

9 Photobleaching T + O S + O A considerable amount of energy is flowing through single molecules. After a limited amount of time, they will undergo photodestruction. Most common mechanism: The molecule goes into the triplet state (S1->T1) and then decays producing higly reactive singlet oxygen that will oxidize the fluorophore. Photobleaching quantum yields are typically in the order of Photobleaching drastically reduces the measurement time.

10 Typical fluorophores

11 Set-up: Wide-field microscopy Illumination of a wide region using a laser (clean gaussian illumination). The polarization, the excitation intensity and the excitation wavelength are controlled. Detection using highly-sensitive CCD cameras. Sample Microscope objective Laser illumination: Truncated gaussian profile Laser + Beam expander Aperture f Condenser Dichroic mirror Filter objective illumination = r = 0.61 aperture fcondenser Camera Resolution: λ NA NA = n sinϑ Resolution = capability to discriminate the light emitted by two different sources

12 Set-up: Confocal microscopy Confocal illumination: Only a small volume (the confocal volume) is efficiently detected and illuminated. An image is obtained by scanning the sample and recording the fluorescence intensity as a function of the position. Laser Sample Micr oscope objective Exci tation pinhole Dic hroic mirr or Measurements are very slow, but the contrast is better than in wide-field illumination. Detection using highly-sensitive singlephoton avalanche photo-diodes (SPAD). Using a cube polarizer or a dichroic, the fluorescence can be split into components. Elements to control intensity and polarization El ement to se parate polarization or colour Filter Detection pinhole SPADs

13 Set-up: Total-Internal Reflection Fluorescence (TIRF) Microscopy The set-up is similar to a wide-field microscope except the fact that the objective back-aperture is only illuminated at its edge. As a result, the excitation beam reaches the sample with such a large angle that it is totally reflected at the glass-sample interface. The sample is only illuminated by a so-called evanescent wave that rapidly decays in z with characteristic lengths in the order of 100 nm. Therefore, only a small slice of the sample is illuminated, drastically decreasing the background. Glass Coverslip Microscope object ive z I Other less used set-ups: Near-field optical microscopy Two-photon excitation microscopy

14 Detection efficiency Objective Dichroic mirror Filter Optics Detector η 2 obj =0.5 [1-1-( NA / n ) ] For NA=1.2 and n=1.33, η obj =28% But it depends on the orientation of the fluorophore Filters and dichroics: η fil =40% Optics: η opt =90% Detector: η det =50% In total: η tot =5%

15 Signal/noise ratio Detected signa l Signal from the molecule Background SNR = η η det RT int RT + C PT + N T det int b int d int T int =integration time C b P=background Las er intensity N d =noise from the detector The noise consists of the statistical noise of the signal, of the noise induced by the background and of the noise from the detector. The background has multiple origins: stray photons; autofluorescence from the filters and optics; impurities in the sample (cell autofluorescence). The minimal background is due to the Raman signal from the solvent. Practically: It is best to work slightly below I sat. Reduce as much as possible the autofluorescence. Often detected counts from the molecule are sufficient to get an SNR of This typically represents 5-10 ms measurement time. The effective number of images that can be recorded depends on the photo-bleaching.

16 How to be sure that these are single molecules? 1: Well characterize the system without single molecules Cell autofluorescence Many biomolecules are fluorescent: flavines, NADH, FAD, chlorophyll. Cell autofluorescence is localized. Sometimes there is autofluorescence in the membrane. Most of all, the Golgi apparatus is usually very brillant showing small vesicles. In general stressed cells are more autofluorescent. Only a detailed analysis of the autofluorescence allows unambiguous statements about single-molecule measurements.

17 2: Careful control of the concentration Concentration Molecules of a dye (Rhodamine 6G) diluted in a polymer (PVA). It is important to have full control on the concentration of the fluophores.

18 3: Detected molecules must exhibit several characteristic features of single molecules E: The spots have a limited size due to diffraction.

19 Determination of the position of a single molecule Intensity Position The image of a single molecule on the camera can be very well approximated by a gaussian. The width of the gaussian is given by the resolution (~0.5*λ): This is the minimal distance needed to discriminate two single molecules. The maximum indicate the effective position of the single molecule: Theoretically it could be determined to an infinite precision! Pratically, due to noise, the precision is in the order of 30 nm. This is 10 times better than conventional techniques. A series of images allows one to track a single molecule with a precision of 30 nm.

20 Example: The motion of the molecular motor myosin V The precision is in the order of 1 nm! A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, P.R. Selvin, Science 300, 2061 (2003)

21 Single-molecule tracking The position of the single molecule is monitored, allowing its diffusion coefficient to be measured. First, suitable single molecules are selected that fulfill several criteria: The size of the spot should be diffraction-limited, single-step photobleaching should be observed, the molecule should be present in several consecutive frames. Example: single molecules of the acetylcholine receptors in a living cell.

22 Single-molecule tracking 2 In a second step, single-molecule images are fitted using a two-dimensional gaussian.

23 Single-molecule tracking 3 The mean square displacement (MSD) is calculated r 2 [µm 2 ] D = µm 2 /s = cm 2 /s time [s] MSD( τ ) = 4Dτ With MSD: mean square displacement, D diffusion coefficient, τ=nδt time, N total # of measurements

24 Single-molecule tracking 4 Due to noise, even an immobilized molecule will show an apparent diffusion delta x = ±36 nm

25 Single-molecule tracking 5 How to proceed when the sequence of images is too short (a couple of images)? Series of traces are measured (at least 100) with varying delay times between images t lag. For all series, P(r 2, t lag ), the probability to find a molecule located at the origin at time 0 in a circle of radius r at time t lag, is calculated, yielding the MSD (r 02 ). Example: Single-molecule diffusion of the membrane anchor of H-Ras: Two types of diffusion are observed: Brownian diffusion and confined diffusion. P. Lommerse, Th. Schmidt, et al. Biophys. J. 86, 609 (2004)

26 Single-molecule tracking 6 In case of non-brownian diffusion, MSD deviates from a linear relationship.