Optical section

Figure Bl.18.11. Confocal scanning microscope in reflection the pinliole in front of the detector is in a conjugate position to the illumination pinliole. This arrangement allows the object to be optically sectioned. The lens is used to focus the light beam onto the sample and onto the pinliole. Thus, the resulting point spread fimctioii is sharpened and the resolution increased.

B1.18.5.2 OPTICAL SECTIONING, SMALLEST SLICE THICKNESS AND AXIAL RESOLUTION... 

Video-Enhanced Contrast. This technique is more expensive but much more effective than any other contrast-enhancing technique (15). Since the 1970s, the development of video processing of microscopical images has resulted in electronic control of contrast. As Shinya InouH, author of a classic text in the field, states "We can now see objects that are far too thin to be resolved, and extract clear images from scenes that appeared too fuzzy, too pale, or too dim, or that appeared to be nothing but noise" (16). The depth of the in-focus field can now be expanded or confined, very thin but very sharp optical sections can be produced, and a vertical succession of these images can be accumulated to reconstmct thicker stmctures in three dimensions (16). 

A final example of MDFI exploiting polarization resolution is given in Fig. 4.12. This shows the application of optically sectioned TR-FAIM to image ligand binding in a microfluidic reactor [67], Solutions of a small dye molecule (Hoechst 33258) and a (nonfluorescing) 5.8 kbp DNA plasmid were mixed in a 50-/mi wide... 

Neil, M. A. A., Squire, A., Juskaitis, R., Bastiaens, P. I. H. and Wilson, T. (2000). Wide-field optically sectioning fluorescence microscopy with laser illumination. J. Microsc. 197, 1-4. 

Grant, D. M., Elson, D. S., Schimpf, D., Dunsby, C., Requejo-Isidro, J., Auksorius, E., Munro, I., Neil, M. A. A., French, P. M. W. Nye, E., Stamp, G. and Courtney, P. (2005). Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source. Opt. Lett. 30, 3353-5. 

Cole, M. J., Siegel, J., Webb, S. E., Jones, R., Dowling, K., Dayel, M. J., Parsons-Karavassilis, D., French, P. M., Lever, M. J. Sucharov, L. O. et al. (2001). Time-domain whole-field fluorescence lifetime imaging with optical sectioning. J. Microsc. 203, 246-57. 

In this paper we start with a short introduction in Integrated Optics (section 2), continue with an introduction into 10 sensors (section 3), while main attention will be paid to treating (the design of) many types of refractive 10 sensors (section 4). Finally in section 5 an epilogue is presented. 

Observation. The time of observation is about 2-4 hocus for vital preparation and several days for fixed preparations. All the preparations may be analyzed by 3-channel simultaneous detection to receive common complicated interference image of the object and details of its structure (see experiment 1) by receiving 20 visual slices (optical sections) or the complete volume (the information must be also quantitatively extracted) (see experiment 2) by computer modelling the images as well as mathematical analysis (experiments). 

Prior DAM, Oparka KJ, Roberts IM. En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope. JMicrosc 1998 193 20-27. 

Confocal microscopy (CM) is another microscope technique for apparent optical sectioning, achieved by exclusion of out-of-focus emitted light with a set of image plane pinholes. CM has the clear advantage in versatility its method of optical sectioning works at any plane of the sample, not just at an interface between substances having dissimilar refractive indices. However, other differences exist which, in some special applications, can favor the use of TIRF ... 

Figure 27. Human osteoblast-like MG 63 cells in cultures on porous (A) or fibrous (B) poly(L-lactide-co-glycolide) scaffolds. A A summarizing picture of horizontal optical sections. The depth of cell ingrowth into the pores (average pore diameter of 400-600 mm) is indicated by spectral colors (blue 0-60 mm, green 80-160 mm, yellow 180-220 mm, orange 240-300 mm, red 320-400 mm, violet 420-480 mm). Day 14 after seeding, cells stained with propidium iodide. B cells grown for 4 days in static culture followed by 2 days in dynamic perfusion cell culture system. Cell membrane stained with Texas Red C2-maleimide and the nuclei counterstained with Hoechst 33342. Leica TCS SP2 confocal microscope, objective 5x (A) or lOx (B) [37].

Fig. 2. Hypostome (mouth) of a Hydra oligactis whole mount labeled with monoclonal antibody DB5. (A) Nonconfocal image of sensory neurons and processes (B) confocal optical section of the same field as (A) illustrating details of neuronal cell bodies and processes. Scale bar = 25 pm.

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