Water immersion objectives

The other important technique for the study of films at the air/water interface which has recently been introduced is fluorescent microscopy. This technique was introduced by von Tscharner and McConnell [90] and Mohwald [91, 92]. It depends on the fact that certain amphiphilic fluorescent dyes become incorporated into islands of the surface active material under study. Furthermore, where two phases of the surface active material coexist, the dye can often be chosen so that it segregates preferentially into one phase. A shallow Teflon trough is employed with a water immersion objective incorporated into the bottom. The depth of water is adjusted so that the objective focuses on the water surface. The layer of material at the air/water interface is illuminated by a xenon lamp. The fluorescent light so generated passes via the objective and suitable filters to an image-intensified video camera and the image is displayed on a television screen. In some versions of this technique the fluoresence is viewed from above. Most of the pioneering work in this field was devoted to the study of phospholipids, a topic to which we will return. Recently this technique has been applied to the study of pen-tadecanoic acid and this work will be considered here as it relates directly to other papers discussed in this section. 

Fig. 4.8. Schematic of SERS experiment on pollen cellular fraction. Freeze-dried pollen was incubated with water, the supernatant was probed by SERS by adding a small amount to a solution of gold nanoparticles. The Raman experiments were carried out using a water immersion objective...

In the Raman experiments, an excitation wavelength of 785 nm (intensity 1.8 105 W/cm2) was used. The sample, i.e. a drop of Au nanoparticle suspension with soluble pollen content was placed under a (60x) water immersion objective. Raman spectra were recorded with 1 s acquisition time. The control preparations (pollen supernatant with water) did not yield any spectral features. A spectrum of rye pollen supernatant with Au nanoparticles is shown in Fig. 4.9, together with a normal Raman spectrum of a rye pollen grain. The difference in spectral information that can be obtained by both approaches is evident from a comparison of these two spectra. Although an estimate of an enhancement factor is not possible from this experiment, it is clear that... 

Figure 4.11 A three-dimensional reconstruction of multiphoton microscope acquired image slices of a series of small vessels in the dorsal skinfold window chamber. Taken with a 40x water immersion objective and digitally zoomed to approximately 60x.

An In Via Confocal Raman Micrscope AFM system (Renishaw, UK) fitted with a 50x Leica water immersion objective (NA of 0.8) and a 785 run diode laser delivering up to 7 mW of laser power was used. 

A practical example is shown in Fig. 5.103. An aqueous Rhodamine-110 solution was excited at a wavelength of 496 nm. The pulse period was 13.6 ns, the pulse width 180 ps. The CW-equivalent power density in the focus was approximately 24 kW/cm. The photons were acquired for 40 seconds. The optical setup was based on an Olympus IX 70 microscope with a 60x water immersion objective lens of NA = 1.2. A pinhole of 100 pm diameter was used in front of the detectors. The effective sample volume was about 2 fl [442]. The photons were detected by two... 

Transmitted and fluorescent images of cells in buffer are collected using the appropriate laser line and a 60x planapochromat water immersion objective (N.A. 1.2). 

FIGURE 38.19 Two 100 aM sample electropherograms of ITP/CE separation of Alexa Fluor 488 (the peaks near 73.5 s) and Bodipy (peaks near 76.5 s). A glass microchip (microchannel cross-sectional dimensions are 50 xm wide and 20 p.m deep) and 60x water immersion objective (N.A. = 0.9) were used. The detector was located 30 mm downstream of the injection region. 

To monitor mercury deposition in situ, a microscope reaction cell can be used. The working Pt UME and counter electrode are inserted through a hole at the base of the cell while the reference electrode is positioned in a side compartment as shown in Figure 6.3.8.1. Once mounted on an optical miCToscope equipped with a water immersion objective (Olympus FLxw40), a camera and personal computer can then be used to record images of the mercury deposition. 

Figure 4.13 Photomicrography showing a single hve cancer cell growing on a quartz window and the cellular compartments such as the nucleus and cytoplasm. Micro-Raman spectra corresponding to these compartments measured with an xlOO water immersion objective, 50 mW of a 785 nm laser, and a collection time of 20 s. (Courtesy of F. Draux.)...

Figure 3 compares the power collection efficiency of two optical collection schemes, system I, a trans-illumination scheme and system II, an epi-illumination scheme. The power collection efficiency is computed with respect to system 0 gathering the power Pq from a randomly oriented fluorophore located at a distance z > 0 from a glass-water interface using a 1.20NA water immersion objective positioned at z<0. The dotted line describes the fluorescence power Pi collected by system I, consisting of a 1.20NA water... 

For physiological experiments, the preparation is placed in a small recording chamber (<0.5 ml) and best viewed in transmitted light on an upright compound microscope fitted with differential interference contrast (Nomarski) optics and a 40-1 OOx (typically 40x) water-immersion objective (Broadie and Bate 1993a). 

Under normal conditions, the segmental nerves of all preparations are severed with dissecting scissors near the VNC to eliminate CNS-mediated spontaneous transmission. For whole-cell recording, the preparation can be adequately viewed on a standard dissecting microscope (40x magnification). For detailed observation, the preparation is best visualized with a 40x water-immersion objective on an upright compound microscope using Nomarski optics. 

---