Effect of a finite-size pinhole on noise performance in single-, two-, and three-photon confocal fluorescence microscopy.

It is known that signal level in single-, two- and three-photon confocal fluorescence microscopy increases with the size of the detector. Here we evaluate the signal-to-noise and the signal-to-background criteria for these microscopes. We investigate the effect of pinhole size on their ability to detect a weakly fluorescent point object in the presence of a uniformly fluorescence background.

Three-dimensional second-harmonic generation imaging with femtosecond laser pulses.

A three-dimensional reflectance scanning optical microscope based on the nonlinear optical phenomenon of second-harmonic generation is presented. A mode-locked Ti:sapphire laser producing <90-fs pulses at approximately 790 nm was used, and the images were constructed by scanning of an object, which possessed local second-order nonlinearity, relative to a focused spot from the laser. The second-harmonic light at approximately 395 nm generated by the specimen was separated from the fundamental beam by use of dichroic and interference filters and was detected by a photodiode.

Optimization of second-harmonic generation microscopy.

We describe the principles and characteristics of second-harmonic generation imaging (SHGI) and explore various methods for optimization of the technique. Second-harmonic imaging is optimized for ultrashort laser pulses, high numerical aperture microscope objectives, a highly sensitive non-descanned large area detector, pseudo-phase-matching, and specimens with large second-order non linearity or which exhibit surface plasmon enhanced phenomena. We also compare and contrast the techniques of SHGI and two-photon excited fluorescence imaging.

Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy.

Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, in reflection and transmission respectively, and the simultaneous nature of the acquisition of the two images allows pure TPF and SHG images in precise registration to be obtained.

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level.