NIR laser microscope

From these vievqjoints, we have developed a femtosecond NIR laser microscope with a home-built cavity dumped chromium forsterite (Cr F) laser as an excitation light source whose output wavelength is centered at 1260 run. In the following the set-up of the NIR laser microscope and its application to multiphoton imaging are presented. 

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. 

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

Two-photon excitation by femtosecond NIR laser pulses can be used to obtain clear images of tissue layers as deep as 1 mm [132, 278, 279, 344, 462, 495, 534]. The efficiency of two-photon excitation depends on the square of the power density. It therefore works with noticeable efficiency only in the focus of the laser beam. With a microscope lens of high numerical aperture a lateral resolution around 300 nm and a longitudinal resolution of about 1 pm is obtained. Two-photon laser scanning microscopy has therefore become a standard technique of tissue microscopy. Two-photon laser scanning can be combined with... 

A Raman microscope consists of five basic components excitation source (laser), focusing component (microscope), signal analyser (spectrometer or interferometer), photon detector (either monochannel or 2D array) and mapping unit such as a computer-controlled micromanipulator. Raman microscopes are usually equipped with low-power UVA IS or NIR lasers, with laser spot sizes (focused laser beam) below 10 pm. Raman microscopy... 

Table 5.50 lists the main features of Raman microspectroscopy. Virtually any object which can be observed under a microscope can be analysed with Raman microscopy. Here, the usual constraints inherent in electron beam methods (vacuum, metallisation, etc.) are totally absent. Although micro-Raman spectrometers mainly use visible excitation, the confocal configuration almost eliminates fluorescence which falls outside of the focal volume. The focus area for visible lasers is <1 /xm, whereas the focus diameter for NIR lasers is 20 fim. 

Bruker has introduced an FT-Raman microscope which is an accessory to an FT-IR spectrometer (42). The coupling between the microscope and the Raman module is made by NIR-fiber optics. In the wavelength range of the Raman experiment excited by a Nd YAG laser, the fiber optics transmission is at a maximum, thus allowing the experiment to be successful (43). Spatial resolution down to 5//m can be achieved. The technique appears to be a capable adjunct to FT-IR microscopy. 

If the spatial resolving power has to be high, then the Raman radiation must be observed through microscope objectives (Fig. 3.5-10 b). Unfortunately, these objectives have a somewhat lower optical conductance than the regular sample arrangement (Schrader, 1990). As a result, the observed Raman spectrum is also considerably weaker. A microscope may be connected to the spectrometer by a mirror system or by optical fibers, as shown in Fig. 3.5-10 b. Optical fibers are e.specially useful for NIR FT Raman spectroscopy, because the transmission of the fibers may be at its maximum exactly in the range of a Raman spectrum excited by a Nd YAG laser (Fig. 3.3-5). 

FT-Raman spectroscopy is relatively insensitive compared to dispersive Raman spectroscopy, due to the longer wavelength of excitahon and the poor noise performance of detectors in the NIR. This means that FT-Raman microscopes lack the sensihvity to analyze small samples unless high laser powers are employed, which leads to the problems noted above. 

For a two-photon microscope the situation is even more complicated. Even if the NIR blocking filter is removable, a detector with a biaUcali or GaAsP cathode is insensitive at the laser wavelength. Of course, by increasing the laser power something is detected in any PMT. However, in the NIR a photocathode for the... 

It is well known that the dependence of the SERS bands on the excitation energy can affect the relative intensity ratios, especially in cases where the first-layer effect plays an important role [12]. Consequently, it is crucial to corroborate the indepence of the surfactant SERS spectra from the excitation energy. Unfortunately, it is not possible to examine the CH bands around 3000 1/cm from the C PyBr by means of NIR FT-SERS spectroscopy [33]. As an alternative to overcome this drawback silver colloids were used. The laser beam was focused onto the sample cell and collected in backscatter-ing configuration, using conventional optics instead of a microscope to improve the signal to noise ratio. 

It is difficult to say with certainty how Raman instrumentation will evolve over the next 10 years. However, there are several instrumentation developments that are beginning to appear as commercial products. These include more systems engineered for dedicated process and/or QA/QC applications, NIR multichannel detectors for use on a dispersive Raman instrument with long-wavelength lasers, UV microprobes, and near-field Raman microprobes that can complement atomic force microscopes (AFMs) or scanning tunneling microscopes (STMs), or their variants. It is an exciting time to be a Raman researcher. 

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