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Confocal Microscopy Techniques

Confocal Raman and Luminescence Microscopy at Room Temperature

Abbildung: Aufbau eines konfokalen Raman-Mikroskopes
Foto: Facettenauge einer Hummel

In a confocal microscope, a laser beam is focused to a diffraction-limited spot on the sample with a microscope objective of high Numerical Aperture. Light scattered from this spot in backward direction is collected and collimated by the same objective and focused through a tiny pinhole onto a photodetector. Only light from the focus can penetrate through the pinhole; light from other depths in the sample is efficiently blocked.

Foto: EPROM-Chip

As a consequence, out-of-focus regions do not blur the signal, but are dark. This provides the possibility to investigate different depth layers separately and to obtain three-dimensional microscope images. In contrast to a regular microscope, the sample must be raster-scanned with respect to the laser focus (or vice versa), however, since only one point is illuminated at a time. Raster-scanning can be accomplished with a three-piezo stage.

Foto: Anordnung von Salzkörnchen

With a confocal optical set-up it is not only possible to detect elastically scattered light, but also other types of secondary radiation emerging from the laser focus. Examples are fluorescence, different types of photoluminescence,  Raman scattering, or - in the case of nonlinear-optical materials - higher harmonics.

Abbildung: holographische Gitter in modernen Funktionspolymeren

In this way the instrument operates as a confocal luminescence or Raman microscope. The different frequency components of the collected light are dispersed in a monochromator, whose entrance slit is replaced with the pinhole. A CCD line detector is placed in its exit focal plane so the whole spectrum is recorded in parallel.

Foto: Spinnenfäden

Measuring the integrated intensity of characteristic Raman or luminescence lines as a function of the focal position yields a three-dimensional image of the chemical composition of the sample by purely optical means in a non-destructive fashion.

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM)

Abbildung: Aufbau der FLIM-Apparatur
Foto: Fluoreszenzlebensdauer

Regular fluorescence microscopy can be extended to measure the fluorescence lifetime(s) at each spatial position. This is known as fluorescence lifetime imaging microscopy (FLIM). Our set-up works in the frequency domain. The cw laser which is used for excitation is amplitude-modulated in the range of several ten MHz with an acousto-optic modulator (AOM), and the modulated fluorescence is detected phase-sensitively. The non-zero fluorescence lifetime leads to a phase shift and a decreased modulation amplitude of the fluorescence signal with respect to the excitation. The lifetime can be calculated from both effects.

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