Fluorescence image from

Figure 7.1 (a,b] Fluorescence images from combinatorial pattern with excitation wavelength of 514 nm and 633 nm, respectively, (c,d) plots of fluorescence enhancement versus width of square section nanopillars for varying spacing for the same patterns as in ( a) and (b], respectively. Center-to-center spacings in nm indicated Nanopillar heights 75 nm [36],... 

For this purpose, the combination of an inverted optical microscope with an atomic force microscope on top has proved very useful (Fig. 9.2), especially in the biological sciences, where AFMs are nowadays a very important experimental tool (Morris et al. 1999). Note that the most versatile solution is an AFM which allows optical imaging also from the top via, e.g., phase contrast microscopy. This setup gives access both to transparent objects (fluorescence imaging from below) as well as nontransparent materials and is therefore also very well suited for simultaneous morphological and optical imaging of submicron scaled circuits and other elements of new solid state electronics. 

Wang G, Smith SJ (2012) Sub-diflfaction limit localization of proteins in volumetric space using Bayesian restoration of fluorescence images from ultrathin specimens. PLoS Comput Biol 8 el002671... 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

Figure Cl.5.3. Near-field fluorescence image 4.5 p.m square) of single oxazine 720 molecules dispersed on die surface of a PMMA film. Each peak (fwhm 100 nm) is due to a single molecule. The different intensities are due to different molecular orientations and spectra. Reprinted widi pennission from Xie 11221. Copyright 1996 American Chemical Society.

Figure Cl.5.4. Comparison of near-field and far-field fluorescence images, spectra and lifetimes for the same set of isolated single molecules of a carbocyanine dye at a PMMA-air interface. Note the much higher resolution of the near-field image. The spectmm and lifetime of the molecule indicated with the arrow were recorded with near-field excitation and with far-field excitation at two different excitation powers. Reproduced with pennission from Trautman and Macklin [125].

Figure Cl.5.14. Fluorescence images of tliree different single molecules observed under the imaging conditions of figure Cl.5.13. The observed dipole emission patterns (left column) are indicative of the 3D orientation of each molecule. The right-hand column shows the calculated fit to each observed intensity pattern. Molecules 1, 2 and 3 are found to have polar angles of (0,( ))=(4.5°,-24.6°), (-5.3°,51.6°) and (85.4°,-3.9°), respectively. Reprinted with pennission from Bartko and Dickson [148]. Copyright 1999 American Chemical Society.

Fig. 5 Polypeptide vesicles demonstrate the ability to utilize the EPR effect, (a) Chemical structure of the amphiphilic block polypeptide PSar-b-PMLG. (b) Fluorescence image using fluorescently labeled PEG. Fluorescence is not observed in the cancer site although accumulation is observed in the bladder, (c) Fluorescence image using ICG-labeled vesicles, showing evidence of vesicle accumulation due to the EPR effect. Adapted from [41] with permission. Copyright 2008 American Chemical Society...

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

While fluorescent imaging techniques offer very high sensitivity, there remains the problem of background noise arising from fluorescence from the sample itself (autofluorescence). There are two strategies to overcome this (i) two-photon excitation,32 and (ii) the use of phosphorescent... 

Fig. 4.4. RLD FLIM of (A) unstained freshly resected human pancreas imaged though a macroscope at 7.7 frames per second and (B) unstained sheep s kidney imaged through a rigid endoscope at 7 frames per second. Both samples illuminated at excitation wavelength of 355 nm and fluorescence imaged through a 375 nm long pass filter. Adapted from Fig. 3 of Ref. [18].

Fig. 4.9. Schematic of time-resolved fluorescence anisotropy sample is excited with linearly polarized light and time-resolved fluorescence images are acquired with polarization analyzed parallel and perpendicular to excitation polarization. Assuming a spherical fluorophore, the temporal decay of the fluorescence anisotropy, r(t), can be fitted to an exponential decay model from which the rotational correlation time, 6, can be calculated.

The acquired images are composite images that consist of fluorescence stemming from different molecular species donors, acceptors, or FRET pairs (Figs. 7.1 and 7.2). These fluorescent... 

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