Confocal microscopic optical systems

Confocal microscopic optical systems form the foundation of the read-out technologies employed in the EVOscreen system. Using confocal optics, sub-microliter miniaturiza-... 

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.

Toriumi, A., and Kawata, S. Reflection confocal microscope readout system for three-dimensional photochromic optical data storage. Opt. Lett. 23,1924, 1998. 

The optical focus system or optical profilometer system is based on the dynamic focusing principle of a confocal microscope. These systems... 

Cork T and Kino G S 1996 Confocal Scanning Optical Microscopy and Related Imaging Systems (New York Academic) Gu Min 1996 Principles of Three Dimensional Imaging In Confocal Microscopes (Singapore World Scientific)... 

The experimental set-up for the FCS measurement is illustrated schematically in Figure 8.6. A CW Ar laser (LGK7872M, LASOS lasertechnik GmbH) at 488 nm was coupled to a single mode optical fiber to isolate the laser device from an experimental table on which the confocal microscope system was constructed. This excitation laser light transmitted through the optical fiber was collimated with a pair of lenses, and then was guided into a microscope objective (lOOX, NA 1.35, Olympus). 

With the aim of elucidating molecular dynamics in a small domain, we have constmcted several microspectroscopic systems, that is, (i) the confocal microscope with the excitation light source being a femtosecond NIR laser emitting a 35 fs pulse, and (ii) the fluorescence correlation spectroscopic system with optical tweezers. 

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].

Figure 14.4 gives the optical system particulars of the CSBLM (confocal scanning-beam laser microscope) used. The reader should keep in mind that a detailed understanding of this system requires a good knowledge of optics. Some readers may choose to skip the next section on first reading. 

Figure 16.15 shows the readout result of the four-layered optical memory. The readout system was a reflection-type confocal microscope. For reading, a He-Ne laser was used as a light source, because the urethane-urea copolymer has little absorption for red light. The scattered light at the recorded bit data... 

The reflection confocal microscope (RCM) is an attractive configuration as a readout system of multilayered optical memories because it has extremely high axial resolution and its configuration is substantially easier than that of transmission confocal microscopes, A typical RCM system is shown in Figure... 

The readout result with RCM optics is shown in Figure 16.15. The four recording layers are clearly detected. As a result, it has been determined that the reflection type confocal microscope configuration can be used as a readout system of multilayered optical memory by using the recording medium in which the photosensitive thin films and transparent films are piled up alternately. 

One of the important functions of this infrared microscope is the measurement of the IR spectrum from a spatial region smaller than the diffraction limit. This possibility is already illustrated in Figure 29.4e. The TFD-IR spectrum, that corresponds to the IR absorption spectrum, was measured from a fluorescence region smaller than the IR diffraction limit. Infrared spectroscopy in a sub-micron region will be possible by using a high NA objective lens with the confocal optical system. 

We have performed super-resolution infrared microscopy by combining a laser fluorescence microscope with picosecond time-resolved TFD-IR spectroscopy. In this chapter, we have demonstrated that the spatial resolution of the infrared microscope improved to more than twice the diffraction limit of IR light. It should he relatively straightforward to improve the spatial resolution to less than 1 pm by building a confocal optical system. Thus, in the near future, the spatial resolution of our infrared microscope will be improved to a sub-micron scale. 

---