Fluorescence recovery after photobleaching (FRAP)

FRAP is a method to measure the mobility of molecules in living specimen; in addition FRAP can be used to measure the connectivity of compartments and binding properties. In a nutshell the equilibrium of fluorescent molecules is perturbed by bleaching a region of interest with a strong pulse of light. Subsequently the recovery of fluorescence in the bleached region is monitored over time. FRAP is part of our regular internal course series about basics in microscopy (link). FRAP can be performed at any current commercially available point-scanning confocal microscope; wide-field or confocal spinning disk systems can be upgraded with special in-coupling of a bleaching laser for example with scanner to perform FRAP experiments (like on our PerkinElmer Ultraview VoX).

  • Availability in the ALMF: all confocal microscopes and the PerkinElmer Ultraview Vox with Pk unit
  • External information: WikipediaLiterature list

Fluorescence (Förster) resonance energy transfer (FRET)

FRET is a phenomenon of non-radiative energy transfer, which can occur between two fluorophores (donor and acceptor) in very close proximity, usually less than 10 nm. As this distance is in the scale of biological molecules it is often exploited to probe for molecular interactions or in optical sensors (with donor and acceptor on one molecule) measuring physiological values in the cell.

Preconditions for FRET:

  1. Proximity:
    Distance between donor and acceptor molecules has to be in the range of the Förster radius (the distance of 50% FRET efficiency) of the used FRET pair. This is usually below 10 nm. The FRET efficiency is inversely proportional to the sixth power of the distance.
  2. Orientation:
    The non-radiative energy transfer via dipole-dipole interaction depend strongly on the orientation of the fluorophore dipoles. In most cases molecules can freely rotate, enabling FRET measurements with an average FRET efficiency readout.
  3. Spectral overlap:
    Donor emission and acceptor absorption spectra need to overlap significantly (even without emitted photons the transferred energy needs to be in a range the acceptor can absorb).

FRET measurement methods:

Acceptor photobleaching: A set of images of donor and acceptor channels is acquired before and after bleaching a region of interest in the acceptor channel. Donor molecules which interacted with acceptor molecules before bleaching, cannot transfer energy after acceptor photobleaching and emit more fluorescence instead leading to a brighter donor image in the region of bleached acceptor. Usually used with fixed samples, since differences between pre- and post-bleach images due to movement lead to artifacts. 

Donor photobleaching: Donor molecules, which can return to ground state by transferring energy onto the acceptor, bleach slower (less chances of excited states to chemically react). This can be exploited to qualitatively detect FRET. Unfortunately measurements are often influenced by acceptor bleaching, this is why we do not recommend this method. 

Sensitized emission: The emission of acceptor molecules excited by energy transfer from donor molecules is called sensitized emission. For the majority of FRET pairs this sensitized emission signal is overlaid by cross-talk of donor emission and cross-excitation of acceptor molecules. These contributions can be determined by donor-only and acceptor-only samples using the same imaging conditions. Three channel datasets are needed for the measurement of sensitized emission: donor excitation + emission ('donor',ch1), donor excitation + acceptor emission ('FRET',ch2) and acceptor excitation + emission ('Acceptor', ch3). 

Ratiometric imaging: FRET readout of optical probes containing both donor and acceptor on a single molecule (e.g. cameleons) can be measured by ratiometric imaging. With a fixed ratio between donor and acceptor the fluorescence emission, the FRET changes can be measured by the ratio between donor channel and 'FRET' channel (donor excitation + acceptor emission). 

Fluorescence Lifetime Imaging Microscopy (FLIM): The time of donor molecules in the excited state is reduced, if engaged in FRET, since energy transfer is an additional pathway for the molecules to return to ground state. As a consequence the average time until fluorescence is emitted (fluorescence lifetime) is decreased. There are two methods to measure the fluorescent lifetime (link to FLIM below).

  • Availability in the ALMF:
    Acceptor photobleaching: all confocal microscopes and the PerkinElmer Ultraview Vox with Pk unit
    Sensitized emission + Ratiometric imaging: Available on most laser scanning and spinning disk confocals, Leica AF7000 (epifluorescence/TIRF) + Olympus xCellence (epifluorescence/TIRF)
    FLIM: Lambert LIFA FLIM (frequency domain) 
  • External information:
    FRET basics website
    FRET basics presentation

Fluorescence lifetime imaging microscopy (FLIM)

FLIM is a technique for producing an image based on the time that fluorophores stay in their excited state (i.e. the fluorescence lifetime). There are two ways of measuring the lifetime of molecules:

  • Time domain: Direct method measuring the time between excitation pulse and fluorescence emission by photon counting (time-correlated single photon counting (TCSPC)). It needs special equipment which is normally implemented on confocal microscopes.
  • Frequency domain: Indirect method, normally used on widefield and spinning disc microscopes).

FLIM is thus useful to measure anything that significantly changes this lifetime. For instance for some fluorophores the lifetime depends on the PH of their surrounding. A very popular application is to use FLIM for measuring the occurrence of FRET. In FRET the lifetime of the donor fluorophore is shortened due to the additional decay channel, namely the energy transfer.


Fluorescence correlation spectroscopy (FCS)

FCS is a method to measure diffusion dynamics and interaction of fluorescent particles in liquid environment. It is based on the measurement of autocorrelation of the fluorescence signal from molecules moving through the focal volume of confocal microscope. The autocorrelation curve calculated by the software gives information on apparent diffusion and concentration of molecules. If the size of the focal volume is calibrated, the diffusion coefficient of the investigated molecules can be accurately calculated. If two types of particles are marked with different fluorophores, one can measure also cross-correlated between the signals (fluorescence cross-correlation spectroscopy, FCCS). If particles move independently there will be no cross-correlation provided that there is no bleed through between fluorescence channels. If particles interact, which implies that they move together, it will create a significant cross-correlation between the signals. Thus the degree of cross-correlation can be an indication for both interaction and the percentage of interacting species. Method can be used for in vitro and in vivo measurements.


Laser nano-surgery

Focused intense laser light can be used to selectively alter or destroy volumes in biological specimen down to the size of the diffraction limited focus spot. Pulsed lasers are needed to achieve sufficient energy levels to form plasma localized to the focus volume. Regions of interest can be treated by moving the laser spot by a scanner, enabling different types of experiment from line cuts (e.g. cutting actin stress fibres in cells or even cutting a cell into two pieces) to ablating clusters of cells by scaning a larger region.

  • Availability in the ALMF: Olympus FV1200 (355 nm and 532 nm laser with ps pulses via second scanner) and Zeiss LSM 780 NLO (two-photon laser)
  • Selected literature: 
    • At the cutting edge: applications and perspectives of laser nanosurgery in cell biology. Ronchi, P., Terjung, S. & Pepperkok, R. Biol Chem. 2012 Apr;393(4):235-48. doi: 10.1515/hsz-2011-0237. Europe PMC
    • Golgi depletion from living cells with laser nanosurgery. Ronchi, P. & Pepperkok, R. Methods Cell Biol. 2013;118:311-24. doi: 10.1016/B978-0-12-417164-0.00019-7. Europe PMC

    • Long-range Ca2+ waves transmit brain-damage signals to microglia. Sieger, D., Moritz, C., Ziegenhals, T., Prykhozhij, S. & Peri, F. Dev Cell. 2012 Jun 12;22(6):1138-48. doi: 10.1016/j.devcel.2012.04.012. Epub 2012May 24. Europe PMC

    • Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization. Colombelli, J., Besser, A., Kress, H., Reynaud, E.G., Girard, P., Caussinus, E., Haselmann, U., Small, J.V., Schwarz, U.S., Stelzer, E.H. Journal of Cell Science 2009; 122: 1665-1679 . DOI: 10.1242/jcs.042986. Europe PMC

Photo-activation, photo-conversion and photo-uncaging

A number of fluorescent proteins and dyes are available, which can be activated (switched from a dark state to a fluorescent state; e.g. paGFP) or converted (e.g. from green to red fluorescence like Kaede or EOS2) by irradiation with low doses of UV/blue light. Using a scanner, it is possible to activate/ convert selected regions of interest. This can be used to mark cells or to measure molecule mobility similar to FRAP experiments.

Similar equipment can be used for photo-uncaging experiments: UV-light can cleave off so called cages from specifically synthesized molecules. The cage can either mask a bio-active side-chain of the molecule, rendering it biologically inactive, or quench fluorescence until uncaging. Alternative names of this method are photo-activation or photo-stimulation.

  • Availability in the ALMF:
    photo-activation and conversion: all confocal microscopes and the PerkinElmer Ultraview Vox with Pk unit
    photo-uncaging: Olympus FV1200 (375 nm laser via second scanner) / Zeiss LSM 780 NLO (if cage can be removed by 2-photon excitation)

Super-resolution: Localization microscopy (dSTORM, GSDIM)

Localization Microscopy is a super-resolution technique based on single molecule detection. The resolution can be improved approx. 10fold compared to diffraction limited widefield microscopy resulting in a resolution of ~20nm laterally and ~50 nm axially. Localization Microscopy can be performed in TIRF- and  Epifluorescence and is therefore well suited for a wide range of (chemically fixed) samples. Photoactiveted Localization microscopy, direct Stochastical Optical Reconstruction Microscopy (dSTORM) and GSDIM (ground state depletion microscopy followed by individual molecular return) are all techniques based on the principle of single molecule detection. Whereas PALM is typically performed with photoactivatable or photo-convertible fluorescent proteins, dSTORM / GSDIM is using organic dyes, which are temporally switched off to achieve single molecule detection. GSDIM / dSTORM is often used in combination with immunofluorescence (IF) staining. Although many standard organic dyes (e.g. Alexa, Atto and Cy-Dyes) can be used, special buffers / embedding media are required for embedding / mounting.

  • Availability in the ALMF: Leica SR GSD 3D

Super-resolution: Stimulated emission depletion microscopy with gated detection (gSTED)

STED is a scanning confocal microscopy based super-resolution technique. The resolution is improved more than 5 fold (<50 nm in x, and y) by shrinking the confocal volume using stimulated emission depletion of excited fluorophores in the outer part of the confocal spot.  A wide range of organic dyes emitting from green to red (including common dyes like Alexa 488 and Alexa 568) provides multi-colour capability for super-resolution co-localization studies using fixed specimen. The compatibility with fluorescent protein like eYFP, eGFP or mStrawberry additionally allows the imaging of living cells. The system is equipped gated detection which provides enhanced resolution and better suitability for life cell imaging compared to standard CW-STED. In embedding media (nearly) matching the refractive index of immersion oil, STED-3D is available as an option reducing the resolution in x,y and z below 130 nm.

  • Availability in the ALMF: Leica SP8 STED 3X

Total internal reflection microscopy (TIRF)

TIRF microscopy allows the selective illumination of a very thin optical slice (~70nm to ~250nm thick) at the cover-slip surface. This is achieved by shining the excitation laser at a high angle onto the coverslip-sample interface. This leads to the total reflection of the excitation laser and only a thin layer directly at the coverslip surface (the so-called "evanescence wave") contains light to excite fluorophores. The advantage of this method is that no fluorophores in the bulk solution will be excited leading to a very high signal to background ratio. Typical applications of TIRF microscopy include: single molecule detection in in vitro experiments; studies related to the basal plasma membrane, such as endo- or exocytosis, focal adhesions as well as membrane translocation of signal molecules.  

  • Availability in the ALMF: Leica AF7000 TIRF MC, Olympus xCellence TIRF
  • External information: WikipediaLiterature list