techniques

Techniques

In this section a brief overview is given of the main techniques being used in LCAM-FNWI:

     Controled Light Exposure Microscopy (CLEM)
     Fluorescence Fluctuation Spectroscopy (FFS)
     Fluorescence Lifetime Imaging Microscopy (FLIM)
     Fluorescence Recovery After Photobleaching (FRAP)
     Förster Resonance Energy Transfer (FRET)
     Photoactivation Localization Microscopy (PALM)
     Spinning disk microscopy
     Total Internal Reflection Microscopy (TIRF)
     Multi-Photon Excitation (MPE)

For more info about microscopy one can visit the following websites:
    Nikon microscopy education website
    Zeiss microscopy education website
    Olympus microscopy education website

also handy:
Invitrogen spectra viewer (spectra of fluorescent dyes)
SIP chart analysis (calibration of your sectioning fluorescence microscope)

CLEM: Controled Light Exposure Microscopy

Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Controlled light-exposure microscopy (CLEM),a simple imaging approach that reduces photobleaching and phototoxicity two- to tenfold, depends on the fluorophore distribution in the object. By spatially controlling the lightexposure time, CLEM reduces the excitation-light dose without compromising image quality.

recommended literature:
Hoebe RA, Van Oven CH, Gadella Jr TWJ, Dhonukshe PB, Van Noorden CJF, Manders EMM. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging, Nat. Biotech. 23, 249 (2005).

Available CAM microscope(s): Nikon C1 and Nikon A1

FFS: Fluorescence Fluctuation Spectroscopy

Fluorescence fluctuation techniques like fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) monitor concentrations and mobility-, binding- and conformational state dynamics of fluorescent molecules and their complexes in situ. Since FCS and PCH are single-molecule techniques, molecules f.e. fluorescently labelled proteins can be studied at the nanomolar level. For many proteins (especially those involved in signal transduction) this is the physiological relevant concentration in a living cell, thus no over-expression of the protein is required. For FCS and PCH the fluorescence intensity is monitored in the small observation volume of a confocal microscope (green), which is continuously illuminated (blue). A particle (red) with a given molecular brightness produces an intensity fluctuation as it passes the observation volume. Particles with a higher molecular brightness will result in stronger intensity fluctuations. Since small particles will diffuse more rapidly through the observationvolume than large molecules, the duration of the fluorescence bursts contains information on the diffusion speed of the particles.
Both PCH and FCS analysis use the same experimental data, but each technique focuses on a different property of the signal. While FCS is a measure of the time-dependent decay of the fluorescence fluctuations yielding parameters like particle number, diffusion time and dark-state kinetics, PCH calculates the amplitude distribution of these fluctuations yielding the distribution of molecular brightness per particle (Chen et al., 1999). When no fluorescence quenching occurs this distribution provides a direct readout of the oligomerization state of the particle.

recommended literature:
Schwille & Haustein. Fluorescence Correlation Spectroscopy: An introduction to its concepts and applications, www.biophysics.org/education/schwille.pdf
Chen Y, Müller JD, So PT, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J. 77, 553 (1999).

Available CAM microscope(s): Olympus/Picoquant FLCCS microscope

FLIM: Fluorescence Lifetime Imaging Microscopy

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful tool for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique. The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample. FLIM is very useful for biomedical tissue imaging, allowing to probe greater tissue depths than conventional fluorescence microscopy.

recommended literature:
T.W.J. Gadella (Ed.), FRET and FLIM Techniques. Laboratory techniques in biochemistry and molecular biology 33, Elsevier Science, Amsterdam (2009)

Available CAM microscope(s): FLIM (widefield-frequency domain) and Olympus/Picoquant FLCCS microscope (time domain)

FRAP: Fluorescence Recovery After Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) is a technique to monitor and quantify the immobility and binding and/or diffusion rate of fluorescent particles. FRAP is not an equilibrium technique since a selected region will be photobleached by an intense laser pulse. Hereafter mobile molecules will enter the photobleached region from the surrounding area and bleached molecules will move out of the region. The recovery of the fluorescence intensity contains information about the bindingkinetcs and/or mobility of the molecules. A related technique is Fluorescence Loss In Photobleaching (FLIP): After the bleaching pulse fluorescence intensity is monitored in a region other than the photobleached region. Due to the altered concentration gradient between bleached area and surroundings, a loss in intensity may be seen when the two regions are connected.

recommended literature:
Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., and Tilley, L. Fluorescence photobleaching analysis for the study of cellular dynamics. Eur. Biophys. J. 31, 36 (2002).
Sprague, B. L. and McNally, J. G. FRAP analysis of binding: Proper and fitting. Trends Cell Biol. 15: 84 (2005).

Available CAM microscope(s): all microscopes

FRET: Forster Resonance Energy Transfer

Fluorescence Resonance Energy Transfer (FRET) involves the energy transfer through dipole-dipole coupling of an donor and acceptor chromophore. The resonance conditions necessary for this process dictate that the fluorescence emission spectra of the donor overlaps with the absorption spectra of the acceptor molecule. The degree of overlap is used to calculate the spatial separation, R, for which energy transfer efficiency, E, is 50% (called the the Förster radius R₀), which typically ranges from 2-7 nm. This range makes FRET an ideal mechanism for the study of protein-protein interactions and can be determined by the measurement of fluorescence lifetime, or intensity of donor or acceptor.

recommended literature:
Jares-Erijman, E.A. and Jovin, T.M. FRET imaging Nat. Biotech. 21, 1387 (2003)
T.W.J. Gadella (Ed.), FRET and FLIM Techniques. Laboratory techniques in biochemistry and molecular biology 33, Elsevier Science, Amsterdam (2009)

Available CAM microscope(s): all microscopes

MPE: Multi-Photon Excitation

In the early 1930s two-photon excitation of molecules was predicted by Maria Göppert-Mayer in the case that high photon densities are present so that two or more low energy photons can be simultaneously absorbed in a single quantum event. Since the energy of a photon is inversely proportional to its wavelength, the two photons should be twice the wavelength necessary for single-photon excitation. It was until the beginning of the 1990s when Denk et al. introduced the two-photon laser scanning microscope resulting in the first biological applications of TPE. Because excitation in multiphoton microscopy occurs only at the focal point of a diffraction-limited spot, it is possible to create thin optical sections of thick biological specimens in order to obtain three-dimensional resolution.

Two-photon microscopy has some major advances over SPE-confocal microscopy for 3D imaging. First of all is the penetration of near-infrared light used for TPE much deeper than that of visible light used in conventional (SPE) confocal microscopy. TPE of thick biological samples allows imaging to a depth of more than 200 mm whereas SPE confocal microscopy is limited to depths of approximately 50 mm. In SPE confocal microscopy fluorescent light is generated throughout the sample along the optical axis but only the signal from a thin focal plane is detected by placing an aperture (the so-called pinhole) at the image plane. However, by using TPE molecules are excited at the focal plane only and therefore no pinhole is required. In addition, TPE minimizes photobleaching and photodamage in out-of-focus regions that are usually limiting factors in conventional live cell imaging.

recommended literature:
Diaspro, A., Chirico, G., and Collini, M. Two-photon fluorescence excitation and related techniques in biological microscopy. Quart. Rev. Biophys. 38, 97 (2005).

Available CAM microscope(s): Zeiss LSM510

PALM: Photo-Activation Localization Microscopy

It is well known that there is a spatial limit to which light can focus: approximately half of the wavelength of the light you are using. But this is not a true barrier, because this diffraction limit is only true in the far-field and localization precision can be increased with many photons and careful analysis. The image of a point source on a microscope detector is called the point-spread function (PSF), which is limited by diffraction to be approximately half the wavelength of the light. But it is possible to simply fit that PSF with a Gaussian to locate the center of the PSF, and thus the location of the fluorophore with a much higher accuracy (compare the 'standard' LSM image <left> with the PALM image <right>).

Betzig et al. (see image from Science) developed photo-activated localization microscopy (PALM) while Zhuang and co-workers used a similar technique called stochastic optical reconstruction microscopy (STORM). In both techniques samples filled with many dark fluorophores are imaged. The dyes can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, well separated molecules "turn on". Then Gaussians are fit to their PSFs in order to localize the centre of the particle. After the few bright molecules photobleach (sometimes actively by using another differently colored excitation source), the next flash of the photoactivating light activates random fluorophores again and the PSFs are fit of these different molecules. This process is repeated many times, building up an image. Because the molecules were switched on-and-off (and thus localized) at different times, the 'resolution' of the final image can be much higher than that limited by diffraction. The current limitation of these techniques is that it can take on the order of hours to collect enough photons per molecule.

recommended literature:
Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642 (2006).
Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793 (2006).

Available CAM microscope(s): Andor Spinning disk microscope

Spinning disk microscopy

Confocal Dual Spinning Disk

Confocal Dual Spinning Disk

The speed limitations imposed by standard confocals can be overcome by parallelism. An array of beams can be used in parallel either by using either a line of pinholes or an array of pinholes. The principal of generating images using an array of pinholes was first proposed in 1883 by German Physicist Paul Nipkow. The array of scanning pinholes is called a Nipkow disc after him and they formed the basis of the first television cameras. The diagram above shows the heart of a practical Nipkow spinning disc system. A laser is used to illuminate the sample and this is focused through a pinhole as well to ensure the laser only illuminates the region and plane of the sample that it is to be imaged. The fluorescence light from the sample is separated from the illumination by filters. The light from the sample is re-imaged through the pinhole to reject out of plane light which will obscure the image and directed towards an imaging system such as an CCD camera.

recommended literature:
Gräf, R., Rietdorf, J., and Zimmermann, T. Live Cell Spinning Disk Microscopy. Advances in Biochemical Engineering/Biotechnology 95, 57 (2005).

Available CAM microscope(s): Andor Spinning disk microscope and FLIM

TIRF: Total Internal Reflection Fluorescence

Total Internal Reflection Fluorescence (TIRF) can be implemented in two ways. Here ‘’objective-based’’ TIRF is implemented. The excitation light that enters the objective under an angle will hit the interface between the sample glass and the cell/medium. When this angle is larger than the critical angle, which is defined by the difference in refractive index between glass and the sample, the excitation light will be reflected completely. As a consequence a small evanescent wave is generated in the sample that will excite only those fluorophores that are in close proximity (~100 nm) of the glass substrate. TIRF is therefore ideally suited to study membrane processes without having too much ‘background’-signal from fluorophores located in the cytoplasm.

recommended literature:
Schneckenburger, H. Total internal reflection fluorescence microscopy: technical innovations and novel applications. Current Opinion in Biotechnology 16, 13 (2005).

Available CAM microscope(s):