Confocal lens microscope

The spatial and temporal resolution of Raman scattering are determined by the spot size and pulse length, respectively, of the exciting laser. Femto-liter volumes (ca. 1 pm3) can be observed using a confocal lens microscope,... 

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.

Fluorescence Measurements, Fig. 3 The schematic concept of a confocal fluorescence microscope configuration. LS light source, OD optical detector, ExF excitation filter, EmF emission filter, DM dichroic mirror, L lens, EM position of fluorochrome molecules, LSP light-source pinhole, DP detector pinhole. Note that the emission light coming Irom out-of-focus fluorochrome molecules is blocked away by the detector pinhole... 

Plenary 8. J Grave et al, e-mail address J.Greve tn.utwente.nl (RS). Confocal direct unaging Raman microscope (CDIRM) for probing of the human eye lens. High spatial resolution of the distribution of water and cholesterol in lenses. 

There are two types of scanning acoustic microscopes. If the illumination and the reception of the acoustic waves are performed by two identical lenses arranged confocally, the SAM is called a transmission SAM. The lens geometry used for transmission imaging is shown schematically in Fig. 41 [93]. 

In a confocal microscope, invented in the mid-1950s, a focused spot of light scans the specimen. The fluorescence emitted by the specimen is separated from the incident beam by a dichroic mirror and is focused by the objective lens through a pinhole aperture to a photomultiplier. Fluorescence from out-of-focus planes above and below the specimen strikes the wall of the aperture and cannot pass through the pinhole (Figure 11.3). 

Fig. 1. Comparisons of the wide-field, flying spot, pinhole detector, and pinhole confocal microscopes. Components include an excitation light source (V), an excitation filter (E), a dichromatic mirror (DM), an emission barrier filter (B), an objective lens (n), a detector (D), and a pinhole (P).

Fig. 3. Comparisons of wide-field (A) and confocal fluorescence images (B, mesoglea level C, apical) of rhodamine phalloidin-stained F-actin in a whole-mount hydra tentacle. The hydra was fixed and stained as described in Chapter 18. The bar represents 25 pm. All images were collected with a Nikon (New York) Microphot FX microscope (x40 objective lens). Confocal images were collected with the microscope connected to a Bio-Rad (Hercules, CA) MRC600 laser-scanning confocal system.

Fig. 5. Optical sectioning of rhodamine phalloidin-stained F-actin in a neutrophil migrating through a 5-pm pore of a polycarbonate membrane. The neutrophil migration is stimulated in response to 10 M Af-formytmethionyl-leucy 1-phenylalanine. (A), (B), and (C) correspond to O.S-pm optical sections indicated as sections A, B, and C, respectively, in Fig. 4. The bar represents 10 pm. The images were collected with a Nikon Microphot FX microscope (x60 Plan-apochromat lens, numerical aperture, 1.6) connected to a Bio-Rad MRC600 laser-scanning confocal system.

The operation of a transmission scanning acoustic microscope requires the lenses to be set up so that they are accurately confocal. This requires holders that can be moved relative to each other along three axes, with rather fine adjustment, and that are rigid to better than a wavelength even when a specimen is vibrating between them. The separation must first be set. If ro is the radius of curvature of each lens, ciq the aperture radius, and n the refractive index, then the focal planes of the two lenses will coincide when the separation between their front surfaces is... 

Figure 1. UV field illumination of a Plan Apo 100x lens (1.4 NA) derived with a fluorescent plastic slide and the intensity measurement of 10-micron Spherotech beads (obtained from Spherotech, Libertyville, IL, USA). This illustrates the problem of using a lens with improper field illumination to make comparative measurements on a sample. The field illumination pattern shows a bull s eye intensity pattern slightly off-center and the five beads located in different parts of the field to illustrate the variation in intensity occurring by using a lens that has improper field illumination. The intensity of beads was derived by a small Region of Interest (ROI) inside the bead. The five beads show a decrease in intensity relative to the bead in the center of the illumination. Although this figure was obtained with UV optics, it represents the type of field illumination that can also occur with visible light excitation. This pattern is also unacceptable, if a confocal laser scanning microscope optical system is used for a FISH study, as the maximum intensity should be in the center of the objective and not in the corner.

Figure 16.19c shows the CTF of the reflection confocal microscope. As Wilson et al. pointed out, the spatial distribution shown in Figure 16.19a has no overlap with the CTF of a reflection confocal microscope unless we use an extremely high NA lens. 

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