TIRF, STORM, FRAP, FRET, FCS, Laser Ablation

Location: Thomas Building, DE-512
Contact phone: (206) 667-4205
Contact e-mail: imaging@fredhutch.org

The scientific imaging shared resource provides instrumentation and support with a number of advanced microscopy techniques. Some examples of available techniques are listed below. We are always interested in trying out new things. If you are interested in an application that is not listed here, please contact the resource.

TIRF:

Total internal reflection fluorescence microscopy uses a special illumination
Cell with immunofluorescent staining for vinculin, a focal adhesion marker, imaged in conventional widefield fluorescence mode (left) and TIRF (right). Image courtesy of Anjali Teckchandani (Jon Cooper lab).


Power of TIRF
Cell with immunofluorescent staining for vinculin imaged in conventional widefield fluorescence mode (left) and TIRF (right).

technique to achieve selective fluorescence excitation of a very thin plane of approximately 100 nm, resulting in outstanding optical sectioning. This method is particularly powerful for the visualization of dim signals that are obscured by string fluorescence from the background. Typical applications include the imaging of molecules at the cell surface, such as coated pits, that would be obscured by strong cytoplasmic signal, or the imaging of molecules in vitro, such as reassembled microtubules.

STORM:

Stochastic optical reconstruction microscopy is a recent super-resolution microscopy technique based on the principle of localization microscopy, that can provide images of cellular or in vitro components with resolutions down to 20 nm, or ten times better than conventional light microscopy. Several localization techniques have been implemented, but their basic principle is the same: a very small subset (less than 0.1 %) of the molecules in the field of view are imaged at any given time. This allows precise localization of each molecule by curve-fitting. The imaged molecules are bleached, and the cycle is repeated hundreds or thousans of times until most of the molecules have been localized. At our resource, we use Nikon's implementation (N-Storm). More details can be found at the vendor's web site.

FRAP:

Fluorescence recovery after photobleaching (and the related technique of fluorescence loss in photobleaching FLIP) are live-cell techniques for studying the motion of fluorescent molecules, such as protein localization or turn-over. In FRAP, fluorescent molecules are photobleached in a specified region with a pulse of intense light, and the recovery of fluorescence in the bleached region is measured over time with time lapse microscopy. The increase in fluorescence intensity over the bleached region indicates the relocalization to that region of fluorescent molecules from the unbleached portion of the cell. In FLIP, bleaching of a specified region will lead to a decrease in fluorescence in the non-bleached areas, due to re-localization of unbleached molecules. Both techniques can provide information about molecular motility (such as diffusion vs active transport), indicate whether molecular associations are stable or labile, and provide information about rates of protein synthesis and degradation. Our resource has equipment for performing FRAP and FLIP experiments, including the Ultraview spinning disk confocal system and the Zeiss LSM 780 confocal system.

FRET:

Fluorescence (or Forster) resonance energy transfer is a microscopy technique for the study of molecular interactions. In FRET, if two fluorescent molecules are chosen so that the emission peak of the first molecule (donor) overlaps with the excitation peak of the second molecule (acceptor), energy transfer can happen when the two molecules are in close contact, so that when the donor molecule is excited, it transfers its energy to the donor which then fluoresces. Such effect can be obtained, for example, between a Cyan (CFP) and A Green (GFP) fluorescent protein in close contact. The technique can be used to determine whether two molecules tagged with the appropriate fluorescent labels are in close enough proximity to suggest direct molecular contact. Several implementations of FRET exist, including sensitized emission, whereby the acceptor molecule fluoresces upon excitatin of the donor molecule, and acceptor photobleaching whereby the signal from the donor molecule increases upon photobleaching of the acceptor. Our resource has equipment for the implementation of both methods.

FCS:

Fluorescence correlation spectroscopy is a microscopy technique to measure rates of molecular motion. Several implementations of the technique exist. In the imaging resource, Fluorescence intensity fluctuations in a small volume can be acquired at very fast frame rates with the Zeiss LSM 780, and the data can be exported for analysis with third party software. For more details about FCS, please consult the resource.

Laser ablation:

Laser ablation is a technique that allows researchers to destroy cells or other structures under the microscope to gain insights into their role. In our resource, the Ultraview spinning disk confocal microscope is equipped with a high power nitrogen laser from Photonic Instruments (now Andor) that can be precisely targeted to a defined spot or region in the field of view. The operation is extremely simple and fully controlled by the software for precise control of the laser power and region to be targeted.

Scheduling time for instruments, training, and support:

To schedule time on any of our microscopes, or to schedule staff assistance or training, please use iLab. The resource is open to all from 9:00 AM - 5:00 PM, Monday through Friday. After hours access can be granted to experienced users and requires training by the resource's staff. Please contact Scientific Imaging for further details.

How to access your data:

Data acquired on Scientific Imaging instruments is transferred to the si folder in the user's fred account. If you do not have a fred account, please submit the computing account application form. Once you have obtained a fred account, please contact Scientific Imaging so that we can create an si link for you. External users without access to fred can obtain their data via ftp, or can download to their own data storage device from one of our dedicated computers. Please note that no portable drives are allowed on instrument computers.