Confocal optics

There are, however, many approaches to DNA sequencing, and even some recent instruments such as the sequencer made by LI-COR [28] do not use a CCD. The latter uses instead a very-low-noise silicon avalanche photodetector to excite near infrared-emitting dyes. The source is a laser diode emitting at 785 nm. It is a compact system that can be mounted on a focusing stage with confocal optics and it is meant for small laboratories that do not have HTS requirements. 

Various solutions have been proposed for the reduction or elimination of autofluorescence. One way is to chemically suppress the autofluorescence signal with some reagents such as sodium borohydride, glycine or toluidine blue. However, in many cases, these approaches are either infeasible or ineffective, and none of them fully eliminates the problem. The second way is to use spectral unmixing algorithms subtracting the background fluorescence. This is only possible if you have at your disposal complicated, expensive confocal optics with sophisticated automated software (http //www.cri-inc.com/applications/fluorescence-microscopy.asp). 

Bonilla, G., Tsapatsis, M., Vlachos, D.G., and Xomeritakis, G. (2001) Fluorescence confocal optical microscopy imaging of the grain bormdary structure of zeolite... 

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.

M.L. Taylor, A. Boyde, S.J. Jones, The effect of fluoride on the patterns of adherence of osteoclasts cultured on and resorbing dentine A 3-D assessment of vinculin-labelled cells using confocal optical microscopy, Anat. Embryol. (Berl.) 180 (1989) 427 35. 

Webb RH (1996) Confocal optical microscopy. Rep Prog Phys 59 427-471... 

Three major advancements in resolution have occurred since Hookes s discovery of the optical microscope in 1665 [46]. In 1873, Ernst Abbe established fundamental criteria for the resolution limit in optical microscopy [47], which did not exceed the range of a couple of 100 nanometers even after the introduction of the confocal optical microscope [43,48]. The invention of the transmission electron microscope by Ernst Ruska in 1933 extended the resolution of microscopes to the nanometer scale [49]. Finally, scanning tunnelling microscopy introduced, by Binnig and Rohrer in 1981, made a breakthrough when atomic... 

Fig. 5. Scanning confocal optical microscopy for single-molecule detection. Sample scanning configuration. The fiber exit and the active area of the SPAD serve as confocal pinholes. S sample, O high-NA microscope objective, DM dichroic mirror, L1,L2 lenses, F Filters.

R. Webb, Confocal optical microscopy, Rep. Prog. Phys. 59, 427 (1996). 

Figure 11.4a,b show the microcapsules as initially formed with Listeria monocytogenes and Staphylococcus aureus attached to the forming polyamide capsule wall. The bacteria, respectively rod and coccoid shaped, were clearly visible on the outside surface of the capsules when labelled with ethidium bromide, as evidenced by confocal optical sections. Variation of the initial bacterial concentration and... 

SERS Spectroscopy with Confocal Optical Microscopy... 

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. 

A scanning method for each observation may improve the spatial resolution. A LSM with confocal optics and a scanning near-field optical microscope (SNOM) can provide finer spatial resolution with a limit of a few nanometers. The highest... 

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