TIRF

The substances methoprotryn (hRf 30-35), desmetryn (h/ f 40-45), ametryn (hRf 55-60), prometryn (h/Jf 65-70) and dipropretryn (h/ f 70-75) separated using mobile phase 1 and the components cyanazine (hRf 20-25), simazine (tiRf 30-35), atrazine (hRf 35-40), terbutylazine (h/Jj 45-50) and anilazine (h/ f 60-65) chromatographed with mobile phase 2 all yielded intense grey to brown-colored zones on a light brown background, that appear intense purple-red when viewed from the back of the plate (Wurster s red). 

Bisabolol oxide (h/ f 40-45) appeared as pink and bisabolol (tiRf 65-70) as mauve-colored chromatogram zones on a pale yellow background. The detection limits per chromatogram zone were 40 ng for bisabolol oxide and 250 ng for bisabolol. 

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

The major advantage of TIRF is that fluorophores outside the evanescent wave (typically more than 200 nm away from the surface) are not excited. Hence, TIRF has an intrinsic sectioning capability. Of interest is that the section capability (z-resolution) is far better than for confocal microscopy systems, which typically have a z-resolution of about 1 /mi. In addition and in contrast to confocal microscopy, TIRF does not cause out-of-focus bleaching because only the molecules at the surface will sense the evanescent wave. However, in comparison with confocal microscopy, a clear limitation of TIRF is that only one z-plane can be imaged the molecules immediately adjacent to the surface. As a consequence,... 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

Described below is how we upgraded our frequency-domain wide-held FLIM system in Amsterdam to incorporate TIRF. A TIRF upgrade to an existing wide-held FLIM setup is marginal both with respect to cost and time. Furthermore, after the upgrade, the system can still be used as a regular wide-held FLIM system. 

The upgrade of an existing TIRF setup to TIRF-FLIM is much more serious, both with respect to costs and with respect to investments in software and high-frequency or nanosecond gating detection systems and control software. 

To implement TIRF, we needed two modihcations hrstly we incorporated a Zeiss a-Plan Fluar 100 x NA 1.45 oil objective, and secondly, we modihed the conhguration of the output of the multi-mode hber to incorporate off axis coupling into the microscope. By using a micromanipulator moving the output of the hber off axis... 

Fig. 9.3. Optical alignment of the fiber-optic output with respect to the microscope axis (black line). A close up is shown of the side-port of the Axiovert 200 microscope and fiber-optic coupling of a modulated 514 nm laser source. Left the fiber output (coming from the right) is aligned onto the microscope axis enabling wide-held excitation. Right the fiber output is aligned slightly off axis, but sufficient to induce TIRF. The scale of the picture can be inferred from the optical table M6 screw mounts separated by 1 inch.

However, below I describe some simple troubleshooting experience of the TIRF-FLIM system in Amsterdam. 

To demonstrate an application of TIRF-FLIM, a FRET study of annexin A4 translocation and self-aggregation near the plasma membrane is shown in Fig. 9.4. This is a particularly useful application of TIRF-FLIM, since TIRF provides the spatial contrast of detecting only molecules immediately adjacent to the plasma membrane and the lifetime contrast reports on the aggregation state of annexin A4. Annexins are calcium-dependent lipid-binding domains with a different type of lipid binding domain compared to the common C2 domains (e.g., found in protein kinase C). Annex-ins consist of an N-terminal domain and a core domain binding calcium and phospholipids. The core domain is conserved in the... 

TIRF Total internal reflection fluorescence immunoassay WWTPs Wastewater treatment plants... 

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