Fluorescence excitation-detection geometries

The preferred excitation-detection geometry for the study of macroscopic samples is the right angle geometry [24]. In order to maximize the polarization of the emitted fluorescence, excitation is made with linearly polarized radiation. Tire direction of polarization is usually the vertical direction (see Fig. 7.11). 

Figure 11.7 Excitation-detection geometries for the determination of the transition strength for (a) Linearly polarized and (b) circularly polarized two-photon excitation. Fluorescence intensity measurements are made at magic angle polarization settings with respect to the quantization (symmetry) axis for the two polarizations. Alternatively, the fluorescence intensity can be constructed from J /V(t) + 2Jh (t) and 2J /(t) + Jn(t) measurements for linear and circular polarizations respectively, (c) Excitation-detection geometry for fluorescence anisotropy measurements following linearly polarized two-photon excitation...

Sensors based on fluorescence are quite robust because the wavelength and the orthogonal detection geometry of the incident and emitted radiation results in a high signal-to-noise ratio. The sensors described here utilize quenching of fluorescence. Thus, the analyte is the quencher Q and the indicator is a fluorescing dye F, which when excited to F, emits fluorescence with a characteristic decay time. 

Figure 6.19 Stark quantum beats in BaO A1 +(u = 2, J = 1). The J = 1 level is excited via the R(0) line by radiation from an N2-laser-pumped dye laser. The pump radiation is linearly polarized at 45° to the 5-field direction in order to produce a coherent superposition of At = 0 with M = 1 components. The top trace shows the signal resulting when the polarization of the detected fluorescence is selected to be at 45° to and at 90° to the excitation polarization. The middle trace is for parallel excitation and detection polarizations. The bottom trace is the difference between the two detection geometries. [From Schweda, et ai.(1985).J...

Excitation and detection geometry. The sample volume from which the fluorescence is detected can differ considerably. In two-photon imaging the excited volume is of the order of 0.1 pm. Confocal imaging with a wide pinhole detects from a considerably larger sample volume. Consequently, the fluorescence comes from a larger number of molecules, and a correspondingly higher intensity is available. The majority of FLIM experiments are performed in two-photon systems with a small focal volume and low intensity. 

Figure 2. Experimental geometry of a 90 fluorescence anisotropy experiment. Excitation is along the X-axis, fluorescence is detected along the Y-axis.

One of the main tasks in PCS is to obtain the maximum amount of (useful) information from the fluorescence light with reasonable effort and costs. Therefore, as many photons as possible should be detected including their parameters such as polarization, color, detection time, etc. [67]. Improvements have, however, not only been restricted to an improved detection of photons and their parameters, but also implemented new excitation and detection geometries. 

Fig. 18.9 Single molecule fluorescence detection in LC ARROW chip, (a) Top view of experi mental beam geometry of dye molecule in sub picoliter excitation volume (dotted ellipse) (2exc excitation beam, dF fluorescence signal) (b) fluorescence signal as function of molecules in excitation volume symbols, different experimental runs, dashed line linear fit...

The optically active 1,2-dioxetane of 2,4-adamantanedione (89) was synthesized. Thermal activation of 89 yielded chemiluminescence (Xmax = 420 nm characteristic of ketone fluorescence), pointing to intermediate 90 which is chiral only in its excited state due to the out-of-plane geometry of one of the two carbonyl groups. However, circular polarization of chemiluminescence measurement of 90 has not detected optical activity at the moment of emission. The authors have concluded that fast, relative to the lifetime of ketone singlet excited state, intramolecular n, it energy transfer caused racemization of 90196. 

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