Infrastructure

– Biophysics and medical technology

Confocal microscopes (CLSM)

  • Leica TCS SP8 SMD MP (CLSM)
  • Zeiss LSM510 META MP (CLSM)
  • Leica TCS SP5 (CLSM)

Atomic force microscopes (AFM)

  • BioScope Catalyst (AFM)
  • MultiMode 8 (AFM)

Other equipment

  • Total internal reflection microscope Zeiss Laser TIRF I
  • Flow cytometer Gallios
  • Computer for offline data analysis

Methods

We provide infrastructure for the different techniques and methods listed below

Confocal laser scanning microscopy (CLSM)

Confocal laser scanning microscopy has been established as a valuable tool for obtaining high resolution images and three-dimensional reconstructions of a variety of biological specimens.

Confocal laser scanning microscopy (CLSM)

Confocal laser scanning microscopy has been established as a valuable tool for obtaining high resolution images and three-dimensional reconstructions of a variety of biological specimens.

The microscope works as follows

A beam of laser light is focused by an objective lens onto a fluorescent specimen. The fluorescent energy from the sample is then collected through the same objective and recorded by a photo detector. The optical system is designed so that the laser's focal point in the sample is imaged exactly on the face of the photo detector. This means that fluorescence coming from the point of laser focus will be focused on the photo detector, and fluorescence coming from any other points will be out of focus on the photo detector. By inserting a small aperture (pinhole) in front of the photo detector, the gathered fluorescence can be limited to a region very close to the focal point. In CLSM, the laser is moved across the sample in a raster (x-y) pattern, and by moving the focus vertically (z), multiple slices can be used to build up a full three-dimensional image.

Confocal laser scanning microscopy (CLSM)


Multi-photon microscopy

In traditional fluorescence spectroscopy, one photon is used to excite a molecule from its ground state to a higher energy state. Afterwards, the molecule decays to an intermediate energy state, emitting a photon of light (fluorescence) with energy equal to the difference of those two levels. In multi-photon excitation, however, two or more photons with lower energy (longer wavelength) excite the molecule together.

Multi-photon microscopy

– Coherent Mira Model 900-F Laser

In traditional fluorescence spectroscopy, one photon is used to excite a molecule from its ground state to a higher energy state. Afterwards, the molecule decays to an intermediate energy state, emitting a photon of light (fluorescence) with energy equal to the difference of those two levels. In multi-photon excitation, however, two or more photons with lower energy (longer wavelength) excite the molecule together.

One advantage of multi-photon excitation is that the induced fluorescence only occurs at, or near, the focal point of the beam. This improves the signal-to-noise ratio by eliminating fluorescence from outside the focal plane. The pinhole is therefore unnecessary, because all of the fluorescent light originates from the laser focus spot. Furthermore, longer wavelengths penetrate deeper in biological materials and are not scattered as much as shorter wavelengths.

Multi-photon microscopy

Fluorescence recovery after photo bleaching (FRAP)

Fluorescence recovery after photo bleaching (FRAP) is sequentially performed in three steps.

Fluorescence recovery after photo bleaching (FRAP)

Fluorescence recovery after photo bleaching (FRAP) is sequentially performed in three steps.

The prebleach equilibrium fluorescence signal is measured in a region of interest (ROI) using low laser intensity for a certain period of time.

Sufficiently low laser intensity is preferable to avoid bleaching (1). The ROI is then rapidly bleached with a pulsed IR laser (Ti:Sapphire) with high intensity (2). Finally, the recovery of postbleach fluorescence intensity is detected as a function of time using low laser intensity (3).

Figure 1

The recovery curve can have different shapes, depending on the dynamic of the tracer molecules and the surrounding environment (figure 2). The diffusion coefficient can be found by fitting the recovery curve to an appropriate diffusion model.

Figure 2

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) or fluorescence cross correlation spectroscopy (FCCS).

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) or fluorescence cross correlation spectroscopy (FCCS).

In this method the fluctuation in fluorescence in a small focal volume is monitored (approx. 10-15 l and concentration in the order of nM).

The random fluctuations are due to diffusion of tracer molecules in and out of the focal volume (figure 1).

Figure 1

The probability that a fluorescent particle inside the volume at time t is still there at time t+¿, gives the autocorrelation function (figure 2).

Figure 2

Advantages with TPFCM compared to FRAP are

  1. single molecules can be measured
  2. low laser intensity is used so the photodamage is limited
Wed, 02 Sep 2015 14:26:23 +0200