Numerical aperture, microscope objective

The optical layout for the measurement of biological samples (cells) is shown in Figure 29.3b. The sample was irradiated with co-linear IR and visible light beams. The transient fluorescence from the sample was collected from the opposite side by an objective lens. In this optical layout, the spatial resolution was determined by the objective numerical aperture (NA) and the visible fluorescence wavelength IR superresolution smaller than the diffraction limit of IR light was achieved. Here, Arabidopsis thaliana roots stained with Rhodamine-6G were used as a sample. We applied this super-resolution infrared microscope to the Arabidopsis thaliana root cells, and also report the results of time-resolved measurements. 

The advantage of Raman spectromicroscopy is that very small specimens can be studied while still allowing the determination of the second and fourth moments of the ODF. However, the expressions for the Raman intensities are more complex since the optical effects induced by the microscope objective have to be considered. Although the corrections may be small, they are not necessarily negligible [59]. This problem was first treated by Turrell [59-61] and later by Sourisseau and coworkers [5]. Turrell has mathematically quantified the depolarization of the incident electric field in the focal plane of the objective and the collection efficiency of the scattered light by high numerical aperture objectives. For brevity, only the main results of the calculations will be presented. Readers interested in more details are referred to book chapters and reviews of Turrell or Sourisseau [5,59,61]. The intensity in Raman spectromicroscopy is given by [59-61]... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

For very high quality microscopes (NA = 1.4), R is on the order of 0.2 /urn that is, particles separated by less than this distance cannot be distinguished from each other. Magnification is the product of the eyepiece and objective numbers maximum magnification is 1000 times the numerical aperture. Depth of field is also related to the aperture, decreasing when the latter increases. A large depth of field is useful when evaluating particles of many different... 

The first fluorescence correlation spectroscopy experiments were carried out several decades ago,62 64 but the general use of the technique was made possible with the introduction of lasers with high beam quality and long-term temporal stability, low noise detectors, and high-quality microscope objectives with high numeric apertures.58,63 The most common set-up is using a confocal inverted epi-fluorescence... 

Somljo It is not a question of diffusion, it is because the confocal microscope does not have a 1 A confocal plane. At best, given a x 40 objective with a numerical aperture of 1.3, it will have a -axis resolution of about 0.7 [im. 

Dark-field illumination is classified into three types. The first one is for a microscope equipped with low numerical aperture (NA) objective lenses (see Fig. 1). To cast a shadow at the objective lens, a ring-slit as shown in Fig. IB is inserted into the light path. The second is for highNA (>0.5) objective lenses. Special, ready-made dark-field condensers or lenses are used for dark-field illumination. The third is independent... 

As Raman scattering is a weak process, photons are precious and high collection efficiency is desired for a higher signal-to-noise ratio. Specialized optics such as Cassegrain microscope objectives and nonimaging paraboloidal mirrors have been employed to increase both the collection spot size and the effective numerical aperture of the optical system.10... 

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