Laser scanning microscopy

Confocal laser scanning microscopy (CLSM)

Confocal laser scanning techniques have caused a revolution in fluorescence microscopy. The optical sectioning capability of confocal microscopes has resulted in a new quality of biological imaging.
Only light emitted from objects inside the focal plane can reach the detector, and light coming from objects outside the focal plane is obstructed by the pinhole. Since objects in the focal plane are not blurred by out-of focus fluorescence, the contrast is enhanced. This does not only yield clean optical sections. By acquiring a stack of images, whose focal plane is shifted in small steps, three-dimensional views of the sample can be obtained.

Currently, the Biomolecular Photonics Group is equipped with a confocal laser scanning microscope from Zeiss (LSM 510) (see Fig. 1). A new confocal laser scanning microscope (Leica TCS SP8) will be installed in October 2015.

Multiphoton fluorescence microscopy

The third dimension can also be added to fluorescence microscopy by exciting the sample via two-photon absorption. By virtue of the non-linear absorption character, two-photon excitation is limited to the high-intensity region at the focal point of the strongly focused excitation laser. Out-of focus fluorescence is not generated and does not need to be cancelled out.