The Optical Microscope

The optical microscopes used in this work are based on the confocal principle [161], which is schematically shown in Fig. 3.4. In a confocal microscope the excitation light is focused onto an excitation pinhole, directed to an objective with a high numerical aperture, and focused to a diffraction limited spot in the focal plane of the objective. Reflected light or fluorescence is collected by the same objective and imaged onto a second pinhole, the detection pinhole, after which it can be detected. Scattered light (or fluorescence) from a region outside the focal volume of the objective can not pass the detection pinhole (dashed and dotted light path in Fig. 3.4). Therefore the confocal principle ensures that only photons from the focal spot of the objective receive the detector and out-of-focus light is efficiently blocked. This is the reason for the higher signal-to-background ratio that can be achieved with confocal microscopy as compared to conventional microscopic techniques [161, 162].

splitter

beam− detection pinhole

detector excitation pinhole

focal plane

objective

light source

Figure 3.4: Schematic illustration of the confocal principle. The light paths depicted as dotted and dashed lines represent light emitted or scattered from a region outside the focal volume of the objective. This light is effectively blocked by the detection pinhole, and only light emitted or scattered from the focal volume of the objective can be detected.

For the experiments described in this thesis two home-built microscopes were utilised:

a combined confocal and widefield microscope including a liquid-helium bath cryostat for measurements at 1.5 K (Fig. 3.5a), and additionally an inverted confocal microscope for room-temperature experiments (Fig. 3.5b). The low-temperature setup was realised during this work and is based on the room-temperature microscope, that was built during a Diploma thesis [163].

dichroic

Figure 3.5: Sketch of the confocal microscopes used in this work. a) Low-temperature micro-scope. b) Room-temperature micromicro-scope. For details see text.

Low-Temperature Setup

A schematic illustration of the low-temperature setup is shown in Fig. 3.5a. In confocal mode the excitation light passes a telescope consisting of two achromatic lenses with focal lengths of 40 mm and 160 mm and a pinhole with a diameter of 50µm to expand the beam diameter with concomitant beam shaping by the pinhole. The light is transmitted through a dichroic beamsplitter (DC670 or z440DCSP, AHF), directed into a home-built cryostat, and focused to a diffraction limited spot on the sample by an objective (NA = 0.85, Microthek). Both the objective and the sample are mounted in an insert that is immersed in suprafluid helium at 1.5 K in a home-built liquid-helium bath cryostat. The fluorescence light is collected with the same objective and reflected by the dichroic beam splitter. After passing suitable filters (BG 39 colour glass, Schott; HQ 500/100 bandpass filter, AHF; HQ467LP long pass filter, AHF), to suppress residual scattered laser light, the fluorescence is focused onto the detectors by achromatic lenses each with f = 100 mm.

The detection pinholes in this microscope are either the small active area of the APD or the entrance slits of the spectrographs in front of the CCD camera and the streak tube, respectively.

One limitation in confocal microscopy is that only light from a small diffraction lim-ited spot on the sample is detected and therefore only molecules in this volume can be investigated at any given time. To overcome this, either the laser spot has to move across

3.2 Experimental Setup

the sample (beam scanning) or the sample has to move across the focus of the objective (sample scanning). For the low-temperature microscope the former technique has been chosen and the laser spot is moved across the sample by tilting the scan mirror with a pair of computer-controlled, motorised micrometre screws (SM 440, Owis). To avoid vignetting the laser beam passes a pair of telecentric lenses (with f = 100 mm); in other words, the telecentric arrangement of lenses ensures that the parallel laser beam is always perfectly directed into the back aperture of the objective without being truncated [161]. Further-more, a widefield option is also available for this microscope. In this mode an additional lens (f = 200 mm) is positioned in the excitation path in front of the dichroic mirror, which permits to illuminate a large area of about 20 ×20µm² on the sample through the objective. The fluorescence light from this region is again collected by the same objective and imaged through the spectrographs (in imaging mode) onto the CCD camera.

The complete low-temperature setup, which was mainly used for the work described in this thesis, is displayed in Fig. 3.6 with the confocal and widefield option and the various

dichroic

Figure 3.6: Experimental setup for the low-temperature measurements with all excitation light sources and photodetectors.

excitation and detection schemes realised for this microscope.

Room-Temperature Setup

For room-temperature measurements a home-built inverted confocal microscope was also available (Fig. 3.5b). The main difference with respect to the low-temperature microscope, as described above, is the objective and the sample holder. Here, the excitation light was focused by an objective with an NA of 0.95 (PLAPO40X, Olympus) onto the sample that was mounted on a xyz piezo-stage (Tritor 102 SG, piezosystem jena). This stage allows to apply the sample scanning technique, where the sample can be positioned in three dimensions with a maximum displacement of 100µm and a resolution of about 2 nm for each axis. Therefore a telecentric system was not required for this microscope. A more detailed description of the room-temperature microscope can be found in Ref. [163].