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A two-pulse experiment is described, using stimulated emission to reduce the fluorescence of 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium perchlorate (Pyridine 2) in a microscope of high numerical aperture. The experiment employed a 130 fs pulse at 375 nm for excitation and a 20 ± 5 ps pulse at 750 nm for stimulated emission. The pulses were provided by a mode-locked Ti:sapphire laser. The 375 nm excitation pulse was obtained by frequency-doubling and the 20 ± 5 ps infrared pulse by optical grating dispersion. The population of the excited state was monitored by varying the temporal delay between the excitation and stimulating pulse. This method enabled the measurement of the lifetime of the dye. For Pyridine 2 in glycerol, we determined a lifetime of 0.86 ± 0.2 ns. The decrease of fluorescence was due to stimulated emission, as was established by measurements of the temporal behaviour of the fluorescence signal upon amplitude modulation of the stimulating beam. We present a theoretical analysis of the temporal behaviour of depletion by stimulated emission in a two-pulse experiment.

We introduce a method employing ferroelectric monomolecular layers, by which it is possible to measure the light field polarization in the focus of a lens. This method allowed us to noninvasively establish the perpendicularly oriented focal field that is anticipated at high apertures. For a numerical aperture 1.4 oil immersion lens illuminated with linearly polarized plane waves, the integral of the modulus square of the perpendicular component amounts to (1.51±0.2) % of that of the initial polarization. It is proven that depolarization decreases with decreasing aperture angle. Whereas for regular imaging conditions depolarization is largely negligible, it plays a significant role in microscopy of highest resolution, microspectroscopy, and single molecule studies.

We studied the effect of electric field orientation on the point-spread function (PSF) of a 4Pi microscope. We show that in a standard 4Pi arrangement the orientation of the field can be used for changing between constructive- and destructive-mode 4Pi microscopy. The effect is counteracted by introduction of a phase shift of π into one of the half-arms. This compensation is compulsory during illumination with unpolarized or circularly polarized light. By performing our experiments with 1.2-N.A. water-immersion lenses, we demonstrate that water immersion is suitable for 4Pi confocal microscopy. At a two-photon excitation wavelength of 1064 nm, the water 4Pi confocal PSF features an axial lobe of 40% above and below the focal plane, which, by linear filtering, can be unambiguously removed. The measured axial full width at half-maximum of the PSF is 240 nm. This is 4.3 times narrower than its single-lens confocal counterpart. The 4Pi confocal microscope sets a new resolution benchmark in three-dimensional imaging of watery samples.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3–7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved threedimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190–280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy. # 2001 Elsevier Science B.V. All rights reserved.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100–140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane. The foci are scanned using a resonance scanner and imaged with a custom-made CCD camera. The MMM increases the imaging speed by parallelizing the illumination; the CCD camera can operate at a frame rate of 1428 Hz while maintaining a low read noise of 11 electrons per pixel by dividing its chip into 16 independent segments for parallelized readout. We image fluorescent specimens at a frame rate of 640 Hz. The calcium wave of fluo3 labeled cardiac myocytes is measured by imaging the spontaneous contraction of the cells in a 0.625 second sequence movie, consisting of 400 single images.

Summary We report the realization of stimulated emission on a microscopic scale. An experiment is presented describing significant depopulation of the excited state of the fluorophore Pyridine 2 with a mode-locked Ti: Sapphire laser. Stimulated emission is performed at 750 nm and excitation at the frequency doubled wavelength of 375 nm. The pulses are synchronized so that the stimulating pulse follows the excitation pulse after 5 ps. The set-up is a modified 4Pi-confocal microscope employing one of the objective lenses for excitation and the opposing one for stimulated emission.