Diffraction-limited excitation spot

Once a diffraction-limited excitation spot is achieved, the same microscope objective that is used for illumination can also be used to collect light from the sample similar to the case of SNOM. If the incoming beam of light is collimated, the beam of collected light is also collimated for a chromatically corrected microscope objective. The use of collimated beams... 

Fig. 9 Schematic illustration of STED. (a) Jablonski diagram indicating the So and Si state of a molecule (left). A femtosecond pulse excites the fluorophore from So to a high vibrational level in Si. The following slow STED pulse induces the stimulated emission from the lowest Si level to a high vibrational level in So. The STED pulse depletes the Si state and quenches the fluorescence. The corresponding wavelength diagram is shown on the right-hand side the dotted and solid lines are the absorption and emission spectra, respectively. The spontaneous emission at A-oet is detected as the signal in the microscopy measurement, (b) The combination of the diffraction-limited excitation spot and the engineered STED pulse yields a narrowed point spread function...

In principle, Raman microspectroscopy is attractive because the practical diffraction limit is on the order of the excitation wavelength, which is about 10-fold smaller for Raman spectroscopy with a visible laser than for mid-lR spectroscopy. It is therefore possible to focus visible or NIR laser light to much smaller spot... 

A molecule can also be excited by absorbing two photons simultaneously [189]. The sum of the energy of the photons must be larger than the energy gap between SI and SO. Because two photons are required to excite one molecule, the excitation efficiency increases with the square of the photon flux. Efficient two-photon excitation requires a high photon flux, which is achieved in practice only by a pulsed laser and by focusing into a diffraction-limited spot. Due to the nonlinearity of two-photon absorption, the excitation is almost entirely confined to the central part of the diffraction pattern. 

Beam shaping of the excitation beam. The beam from a laser diode has a no-tieeable astigmatism and is not focused into a diffraction limited spot. Improv-... 

Diode lasers have an extremely small cavity. Most lasers in the power range below 200 mW (CW) are single-mode lasers, i.e. the height and width are so small (a few pm) that only one transversal mode is excited. This implies that the radiation ean, in prineiple, be focused into a diffraction-limited spot. However, beeause the eavity is only a few pm long, the light is emitted over a wide angle. The general beam profile of a laser diode is shown in Fig. 7.1. 

The experimental techniques for single molecule spectroscopy described in the previous chapters differ mainly in the method employed to reduce the excitation volume of the sample (combined with different fluorescence collection methods). This was achieved in four different ways (i) the laser was focused to a tiny spot on the sample by a lens immersed in liquid helium, (ii) the excitation light was coupled into an optical fiber carrying the sample at its end, (iii) the sample was mounted behind a small aperture (pinhole with typically 5 pm diameter). All these methods reduce the excitation area to a few pm. The near-field technique (iv) allows investigations beyond the classical diffraction limit the tapered tip used had a typical diameter in the order of 50-100 mn. 

Since spatial resolution is diffraction limited, short wavelength lasers are optimal for analyzing small sample features. In order to achieve micron-level spatial resolution, the alignment of the Raman microscope is critical. The visual light path, the excitation laser beam path, and the Raman scatter beam path from the sample to the detector must aU be targeted precisely on the same spot. 

A beam of a TEMqo laser (Sect. 5.3) with a Gaussian intensity profile is focused to a diffraction-limited spot with the diameter d — 2kf/D by an adapted lens system with the focal length / and the limiting aperture D. For example, with flD= I at = 500 nm, a focal diameter of J 1.0 xm can be achieved with a corrected microscope lens system. This allows the spatial resolution of single cells and their selective excitation by the laser. 

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