Objective numerical aperture

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. 

Table 7.4. Examples of Working Distance Resolution and Depth of Field as a Function of Objective Numerical Aperture...

Using a lOx objective (numerical aperture = 0.45), 9 fields/well are acquired to maximize imaging of the whole cell surface see Note 36). 

The magnification is rather chosen to be about 500 to 1000 where is the numerical aperture of the objective (see the next section) The eyepiece is then necessary to magnify the real image so that it can conveniently be inspected. 

Laser trapping is a technique to manipulate small sized materials, which was developed by Ashkin in 1970 [20, 21]. In this experiment, a laser beam is tightly focused by an objective lens with high numerical aperture (NA), and a dielectric... 

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 constants Aohi and Boh characterize the collection efficiency of the objective and originate from the integration over Q. They are related to the numerical aperture NA of the objective so that... 

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. 

The angle between the most divergent rays that can pass through an objective is termed the angular aperture (AA) of the objective. It is customary to express the aperture of an objective in terms of the sine of this angle, and to define the numerical aperture (NA) ... 

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... 

Because the excitation intensity varies as the square of the distance from the focal plane, the probability of two-photon absorption outside the focal region falls off with the fourth power of the distance along the z optical axis. Excitation of fluorophores can occur only at the point of focus. Using an objective with a numerical aperture of 1.25 and an excitation beam at 780 nm, over 80% of total fluorescence intensity is confined to within 1 pm of the focal plane. The excitation volume is of the order of 0.1-1 femtoliter. Compared to conventional fluorometers, this represents a reduction by a factor of 1010 of the excitation volume. 

Figure 7.S. Collection efficiencies QllL versus z, for an objective of numerical aperture 1.4 centered on the normal to the interface. Results are given both for the objective positioned beneath the interface to collect light emitted through the glass and above the interface to collect light emitted in the water, (a) Bare glass (b) aluminum film on glass. All optical parameters are the same as in Figure 7.3.

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