Nondescanned detection

Figure 5.71, left, shows the principle of a laser scanning microscope with one-photon excitation. The laser is fed into the optical path via a dichroic mirror. It passes the optical scanner, and is focused into the sample by the microscope objective. The focused laser excites fluorescence inside a double cone throughout the complete depth of the sample. The fluorescence light is collected by the objective lens. Detection of the fluorescence light can be accomplished by either descanned or nondescanned detection. 

Nondescanned (or direct ) detection solves a problem endemic to fluorescence scattering in deep sample layers. Fluorescence photons have a shorter wavelength than the excitation photons and experience stronger scattering. Photons from deep sample layers therefore emerge from a relatively large area of the sample surface. To make matters worse, the surface is out of the focus of the objective tens. Therefore the fluorescence cannot be focused into a pinhole. 

Nondescanned detection splits off the fluorescence tight directly behind the microscope lens and directs it to a large-area detector. Consequently, acceptable light collection efficiency is obtained even for deep layers of highly scattering samples. Two-photon imaging with nondescanned detection can be used to image tissue layers several 100 pm (in extreme cases 1 mm) deep [85, 278, 344, 462, 534]. 

The absence of a pinhole in a two-photon microscope with nondescanned detection makes the optical path relatively easy to align. Two-photon microscopes can be built by upgrading a one-photon system with a Ti Sapphire laser or by attaching the laser and an optical scanner to a conventional microscope [136, 137]. 

Commercial laser scanning microscopes use the same microscope body and the same scan optics for one-photon and two-photon excitation. Most two-photon microscopes have lasers for one-photon excitation as well. They can switch between both modes, and between descanned and nondescanned detection. Moreover, in both the descanned and the nondescanned detection path, the light is split spectrally by additional dichroic mirrors or dispersion prisms and several detectors are used to record images in selectable wavelength ranges. The dichroic mirrors and filters are assembled on motor-driven wheels and are changed on command. The laser power... 

Applications of a system consisting of a Zeiss LSM 510 with fibre output and a Becker Hickl SPC-730 TCSPC module to FRET and autofluorescence are described in [32, 33, 36]. Unfortunately, fibre outputs are often relatively inefficient compared to the internal detection light paths of the scanning head or to nondescanned detection. Therefore, the sensitivity of fibre-coupled systems is often not satisfactory [147]. 

Fig. 5.76 TCSPC laser scanning microscope with nondescanned (direct) detection...

A nondescanned ( direct ) FLIM detection module with two wavelength channels was developed for the Radiance 2000 microscope from Biorad. The detector module contains computer-controlled dichroic beamsplitters and filters, preamplifiers, and overload shutdown of the detectors. The preamplifiers simultaneously deliver photon pulses to a Becker Hickl SPC-830 TCSPC and intensity signals to the standard steady-state recording electronics. Unfortunately all Radiance scanning microscopes were discontinued in 2004. 

A typical image obtained by nondescanned detection and two-photon excitation is shown in Fig. 5.78. The autofluorescence of aortic tissue was excited at 800 nm. The figure shows the intensity image, an image of the average lifetime, and the lifetime distribution over the pixels. The fluorescence decay displayed for a selected pixel is multiexponential, as is typical for autofluorescence. 

Fig. 5.79 One-photon excitation with nondescanned detection. Elodea spec., excitation at 405 nm, acquisition time 16 seconds. Bine to red corresponds to a lifetime range of 0.5 to 1.5 ns...

In practice, the only feasible solution is often to transfer the light to the poly-chromator slit plane by an optical fibre. The slit is removed, and the numerical aperture at the input of the fibre is reduced to match the numerical aperture of the polyehromator. Because only moderate wavelength resolution is required, a relatively thick fibre (up to 1 mm) can be used. Therefore a reasonably high coupling efficiency with a single fibre can be obtained, even for nondescanned detection systems. The fibre should be not longer than 50 cm to avoid broadening of the IRF by pulse dispersion. 

Another possibility is to use a fibre bundle. The input side of the bundle is made circular, the out side is flattened to match the polyehromator slit. The large area of the fibre bundle makes it relatively simple to collect the light from the nondescanned detection path of a scanning microscope. However, the aperture of the microscope objective lens must be correctly imaged onto the input of the bundle. Otherwise the illuminated spot scans over the bundle and causes the fibre structure to appear in the image. 

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

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