Molecular Motors Research Group

  
 

MICROSCOPY TECHNIQUES

 
Single molecule fluorescence imaging is a highly powerful technique to understand how proteins physically perform their function in a time course. To be able to observe single proteins, we specifically label them with organic fluorophores and shine a laser light to observe the emitted fluorescent light under a microscope. The emitted light is collected with a highly sensitive CCD camera for quantitative analysis.

Our fluorescence microscope is equipped with 488 nm, 532 nm and 633 nm laser lines for the excitation of wide variety of fluorescent probes. All three lasers are combined to follow the same optical path and focused to the back focal plane of the microscope objective (Nikon 100X, 1.49 NA). The beam is then shifted laterally along the focal plane that creates tilted and parallel laser beam emitted from the microscope objective. When the tilt is larger than the critical angle, transmitted light will be total internally reflected back from the glass/water boundary. Total Internal Reflection (TIR) of light creates an evanescent field which penetrates only 100 nm into the flow chamber.


TIRF is ideal to study surface immobilized cells and in vitro systems since it results in minimal fluorescent background and sensitively detects fluorescent photons. Compared to the scanning confocal setup, TIRF is a wide field illumination technique that readily allows simultaneous observation of many single molecules on the surface. Fluorescent photons are filtered and collected by an EMCCD camera (Ixon EM+, Andor Technology) which can detect 92% of incoming photons with a minimal electronic noise.

 

To specifically label the motors, we removed all the solvent exposed cysteines and place a single cysteine to react with a single Cy3-maleimide molecule. Alternative labeling strategies are also possible. DHA tag was inserted into to C-terminus of dynein, which enables covalent linkage of TMR or biotin. Biotin can be linked with streptavidin coated quantum dots that are ~20 times brighter and more stable than Cy3 and TMR dyes. Labelled motors were introduced into the flow chamber after immobilizing microtubules on the glass surface. In the presence of limited ATP, individual steps of motors can be discerned.

 

FRET
We use our single molecule TIRF scope to detect fast conformational changes and particle tracking experiments. Specifically, by labeling a single protein with a pair of fluorescent dyes whose spectrum overlaps to allow Fluorescent Resonance Energy Transfer (FRET). Closely spaced fluorophores can transfer their energy via dipole-dipole interaction to each other. Excitation of the donor molecule whose emission is in resonance with the acceptor’s absorption yields the emission of an acceptor. The energy transfer depends on the distance (R) between the donor and the acceptor molecule with R6 that makes FRET highly sensitive to measure changes in the distance. Labeling of two sites of a protein with a FRET pair yields information on conformational dynamics for the distances between 2-10 nm. We are using FRET to study conformational changes in moving dynein molecules.

FIONA
To track the movement of motor proteins with high precision in vitro, we use Fluorescence Imaging with One Nanometer Accuracy (FIONA). The image of a point-like fluorescent object is as wide as 250 nm in the visible region of the light because of the diffraction limit. The position of an object, however, can be localized very precisely by determining the center of its emission pattern. The precision depends on maximizing the photon detection per image and minimizing the noise factors. Yildiz et al. 2003 showed that millions of photons can be collected from a single molecule before it photobleaches. Organic dyes (Cy3, TMR, Cy5) were localized within ~100 msec, which enables to measure the step size of the motor proteins kinesin and cytoplasmic dynein.

A. The Airy pattern of a diffraction-limited-spot in two dimensions. B. Fluorescence images of several single Cy3-DNA molecules immobilized on a glass surface. The data was taken with TIRF scope in 0.5 sec. C. Expanded view of one PSF with 2-D elliptical Gaussian curve fit (solid lines).  The center of this PSF can be located to within 1.5 nm (sm) (figure from Yildiz et al. Science 2003).

Polarization Microscopy
Single fluorescent particle tracking assumes that the dye acts as a point source and yields a symmetric PSF in far field. In reality, fluorophore excitation and emission is polarized. If the fluorophore dipole is fixed, its orientation can be tracked to study the rotational movement of single enzymes. To monitor the rotational dynamics of fluorescently labeled macromolecules, sample can be excited with a linearly polarized light. The changes in fluorescent intensity would represent the reorientation of an enzyme (different f), if the fluorophore does not undergo rapid rotational movement independent of the targeted molecule. To minimize the rotational mobility of a fluorophore independent of the head orientation, bifunctional probes can be attached to two closely spaced (~1 nm apart) binding sites. 3D orientation of the probes without angular degeneracy can readily be monitored by imaging the fluorescence intensity away from the focal plane.The position dependent intensity distribution of the fluorophore can be calculated based on its orientation (q, f) and its distance from the focal plane, z. Defocused imaging can also be combined with FIONA to track the translational and rotational movements simultaneously.

(Left) In plane (f) and out of plane (q) angles of a fluorophore dipole moment (green arrow) in xyz coordinates. (Right) Single molecule fluorescence polarization (SMFP) microscope. The sample is excited with two orthogonal laser beams (paths 1 and 2) each can switch between s and p polarized excitation. The fluorescence emission is separated onto two APDs by a polarizing beam splitter cube