Two-photon microscope

Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. and Barry, N. (2003). Fluorescence lifetime imaging for the two-photon microscope time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381-90. 

Another difference in these FRET methods is the cost of the microscopes. The two-photon microscope and its mode-locked laser used for sRET and FLIM-FRET cost approximately an order of magnitude more than the E-FRET system. Clearly, if cost is a limiting factor then the E-FRET approach is superior. 

Helmchen, F., M.S. Fee, D.W. Tank, and W. Denk. 2001. A miniature head-mounted two-photon microscope High-resolution brain imaging in freely moving animals. Neuron 31 903-912. 

Neil MAA, Juskaitis R, Booth MJ, Wilson T, Tanaka T, Kawata S (2000) Adaptive aberration correction in a two-photon microscope. J Microsc 200 105—108... 

One of the developments in the area of confocal microscopy is the use of fluorescence to highlight or visualise certain features that either fluoresce naturally or are derivatised with probes prior to microscopic examination. Another new development is two-photon microscopes, which allow operation in the UV spectrum and, hence, deeper penetration of structures such as living cells, skin and other biological samples. 

The recently developed fluorescence correlation spectroscopy permits studies of molecular associafion in one femtoliter of solufion using a confocal or two-photon microscope. Two lasers are used to excite two fluorophores of different colors, each one on a different type of molecule. Fluorescence of single molecules can be defected, and molecular associations can be detected by changes in the distribution of fhe flucfua-tions in fluorescence intensity caused by Brownian rnohon. A different type of advance is development of compufer programs that analyze chromosomes stained with a mixture of dyes with overlapping spectra and display the result as if each chromosome were painted with a specific color. 

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... 

Both the frequency-domain and the time-domain techniques can be classified into camera, or direct imaging techniques, and scanning, or point-detector techniques. There are considerable differences between the ways the two detection principles interact with laser scanning. Figure 5.72 shows the excitation of a sample in the focus of a one-photon microscope (left) and a two-photon microscope (right). 

Fig. 5.72 Intensity distribution around the laser focus, seen from the surface of a thick sample. Left One-photon excitation. Fluorescence comes from the complete excitation light cone. The confocal microscope obtains a sharp image by detecting through a pinhole. Right Two-photon excitation. Fluorescence is excited only in the focal plane. Nevertheless, scattering in a thick sample blurs the image seen from the sample surface. The two photon microscope obtains a sharp image by assigning all photons to the pixel in the current scan position...

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. 

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... 

The logical way to record an IRF in a two-photon microscope is to use second-harmonic generation (SHG). SHG in a crystal is not very useful because the SHG is emitted in the direction of the laser radiation. Returning it to the microscope lens with the right NA is difficult. The best way to record an IRF in a microscope is SHG by hyper Rayleigh scattering in a suspension of gold nanoparticles [206, 375]. 

A crucial point of detector selection is whether or not an accurate IRF can be recorded in the given optical system. IRF recording is often a problem in micro-seopes or other systems that use the same beam path for excitation and detection. Reflection and scattering makes it difficult to record an accurate IRF in these systems. In two-photon microscopes the detector may not even be sensitive at the laser wavelength, or the laser wavelength may be blocked by filters. If an accurate IRF is not available, lifetimes much shorter than the detector IRF cannot be reliably deconvoluted. The rule of thumb is to use a detector with an IRF width shorter than the shortest lifetime to be measured. 

A. Diaspro, Building a two-photon microscope using a laser scanning confo-cal architecture, in A. Periasamy (ed.), Methods in Cellular Imaging, Oxford University Press, 162-179 (2001)... 

Q-T. Nguyen, N. Callamaras, C. Hsieh, I. Parker, Construction of a two-photon microscope for video-rate Ca imaging. Cell Calcium 30(6), 383-393(2001)... 

Levi, V, Ruan, QQ, and Gratton, E, 3-D particle tracking in a two-photon microscope Application to the study of molecular dynamics in cells. Biophysical Journal 88 (2005) 2919-2928. 

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