We demonstrate a widefield multiphoton microscope and a temporally decorrelated, multifocal, multiphoton microscope that is based on a high-efficiency array of cascaded beamsplitters. Because these microscopes use ultrashort pulse excitation over large areas of the sample, they allow efficient use of the high-average power available from modern ultrashort pulse lasers.
©2000 Optical Society of America
1. 1. Introduction
State-of-the-art ultrashort-pulse lasers used for multiphoton microscopy[1,2] have more pulse energy than is required for many biological-imaging applications. Thus, to avoid undesirable effects such as saturation or physical damage to the specimens, the input beam is attenuated from the hundreds of milliwatt or even the watt level to the milliwatt level. This is not an efficient use of expensive photons, especially when we consider that the nonlinear intensity dependence of the absorption makes multiphoton microscopy inherently confocal, which aids in efficient use of the excitation and emitted light by eliminating the need for confocal pinholes.
The low average powers needed to reach high intensities with ultrashort laser pulses (<50 fs), however, open the door to methods for efficiently exciting multiphoton fluorescence over large areas. Simple imaging schemes can be developed for multiphoton microscopy that make more efficient use of the high average power available from the modern ultrashort pulse lasers. In this article, we present two new techniques that can use high-average power laser sources more efficiently than existing multiphoton microscopes. The first method, widefield multiphoton microscopy relies on directly exciting an entire plane in the sample, while the second method, temporally decorrelated multifocal microscopy, excites the sample with multiple foci. By temporally decorrelating the foci, this microscope avoids the interference effects that prevent multifocal microscopes based on spinning or rastered lenslet arrays from achieving the highest possible resolution and contrast.
2. Wide-field multiphoton microscopy
2.1 Experimental Setup
In a standard multiphoton microscope, the sample is excited using a single diffraction limited focal spot at the focus of an objective. An image is then built up by either rastering the sample or scanning the focus within the sample. While this method allows high-resolution imaging, the high peak power and intensity of the tight focus does limit the average power that can be used. Rather than simply attenuating the power in the beam and throwing away expensive photons, a more efficient method of imaging is to use widefield illumination. In widefield illumination, the idea is to excite an entire plane in the focal plane of the objective, instead of exciting a single point, and then to detect the emitted multiphoton fluorescence with high resolution. Thus the entire image plane is excited simultaneously with a much higher average beam power than can be used for a point focus with a high numerical aperture (NA) objective. This method has been used for imaging single molecules in supported phospholipid membranes but not in real-time video rate imaging of motile, living cells.  Fig. 1 shows a schematic of the widefield multiphoton microscope. The input excitation is the ~20-fs, 800-nm pulses from a Ti:sapphire oscillator running at 76 MHz. A prism compressor was used to compensate for the dispersion of the optics in the microscope and maintain a short pulse at the focus. A lens focused the light through a dichroic mirror and on to the objective lens, greatly underfilling it and thereby reducing the effective NA of the objective for the excitation. This widefield illumination of the sample in the focal plane of the objective excites multiphoton fluorescence in the sample, which is then collected with the full NA of the objective. It is possible to estimate the field of view from the effective NA using Gaussian optics. For a Gaussian beam the intensity in the focus is
Here, P is total power in the beam, and is the spot size for a beam with confocal parameter, zR≈1.169(nλ/NA2), and wavelength, λ, in a medium with refractive index, n. The emission intensity of the two-photon fluorescence then varies as I 2P(r, z)∝I 2 (r, z). In practice, however, it is simpler to measure the field of view by illuminating a cell containing 1- µm fluorescent microspheres and fitting the fluorescence distribution to a Gaussian.
In this work, we imaged the two-photon autofluorescence centered at 640nm from the chlorophyll in the motile microorganism Euglena, so the dichroic used transmitted 800-nm wavelengths and reflected ~600-nm wavelengths. The autofluorescence was collected and collimated in the backward direction by the objective, and then separated from the 800-nm light by the dichroic mirror before being focused on a Hamamatsu C5985 CCD camera to produce the image. An interference filter, which transmitted 670nm - 20nm, was used to block any additional unwanted light.
We used three objectives for imaging the Euglena: 1) a Zeiss 100×/1.25-NA Achroplan oil objective, 2) a Zeiss 40×/0.65-NA A-plan air objective and 3) a Zeiss 20×/0.45-NA A-plan air objective. All the objectives are infinity corrected. Using 50 mW of average power measured in front of the objectives, we were able to make video frame rate (30 Hz) movies of the live Euglena with all three objectives. As examples, Fig. 2 is a video frame rate movie of a Euglena with a 100× objective, and Fig. 3 is a video frame rate movie of a Euglena with a 40× objective. Measuring the full-width at half-maximum of the fluorescence from a sample of 1-µm polystyrene microspheres (Molecular Probes, 365/415 blue FluoSpheres), the illuminated field of view was approximately 5 µm for the 100× objective and 15 µm for the 40× objective. Both movies show the two-photon autofluorescence of the chlorophyll in the Euglena. In Fig. 2, the Euglena is contracted from its normal length of ~18 µm and tumbling in place as it seeks a new direction of travel, while in Fig. 3 the Euglena begins at its normal length and then contracts and tumbles. To our knowledge these are the first real-time video movies of living cells using widefield two-photon microscopy. We note that we have observed the tumbling behavior of the Euglena using brightfield illumination without the laser on, so it is not necessarily caused by the laser illumination. When not tumbling, the Euglena are capable of swimming rapidly as is seen in the movies, and the difficulty of tracking them made filming other behaviors difficult.
While this widefield microscope is much simpler and less expensive to build than a typical resonantly scanned multiphoton microscope and easily provides video frame rate imaging, there are limitations. A widefield-multiphoton microscope does not have the advantage of being inherently confocal that a standard multiphoton microscope has because of the strong intensity dependence of the two-photon absorption in the typical high-NA focus. While the resolution of the detection system may still be diffraction limited, ~1.5 µm axial resolution for a 1.25NA objective at 800 nm for example, the excitation now includes a much longer region in the axial dimension due to the reduced NA of the illumination. Since there are no confocal pinholes, the CCD camera detects the out-of-plane light that reaches it, which reduces the resolution and contrast relative to a confocal system. These effects are apparent in Figure 4, which shows the measured axial point spread functions (PSFs) for the widefield microscope using 1.25-NA objective. Two measurements are shown, the peak of the signal and the integrated signal as a coumarin dye cell is scanned into the widefield focus. While the peak signal, which has essentially confocal detection, still shows a very sharp rise as the interface between the coverslip and the dye enters the peak focal intensity, the integrated signal shows a very slow rise due to the long depth of focus of the illumination. Moreover, there is a large background in the peak measurement, even when the interface is ~50-µm from the peak of the focus. Thus, while the widefield multiphoton microscopy is simple and allows video-frame-rate imaging with 2-photon excitation, it does not allow clean sectioning for performing 3-D microscopy. For efficient 3-D multiphoton imaging with sectioning, a different method is required.
3. Temporally decorrelated multifocal microscopy
3.1 Scaling with number of foci & laser power
One means of increasing the efficiency (and potentially increasing the image acquisition speed) in multiphoton microscopy is to distribute the power over many foci and scan all the foci instead of only one single focus. This way more of the available laser power may be used while keeping the power in each beam below the damage threshold. The cell viability data of K nig et al.  indicated a loss of cell viability of Chinese hamster ovary cells under exposure to 70-pJ, 150-fs, 800-nm pulses at 80 MHz repetition rate. Now consider the extended-cavity Ti:sapphire oscillator with a 21.6 MHz repetition rate that we have in our lab. This oscillator is capable of producing 20-nJ, 20-fs pulses. Dividing this energy among multiple foci and scaling for pulsewidth and other parameters, there is a potential improvement in the efficiency of excitation of more than two orders of magnitude over a single focus. While different damage mechanisms may introduce other limitations, the potential increase in efficiency makes using multiple foci attractive.
3.2 Temporally decorrelated multifocal arrays
The initial use of multiple foci in multiphoton microscopes has relied on placing a microlens array in the beam to produce a series of beamlets [6–8]. These beamlets were then appropriately relayed to the entrance pupil of the objective, and scanning was achieved with galvanometric scanners  or by rotating the lenslet array. The multiphoton signal was then imaged to a CCD array. A limitation of this method is that, as the foci are placed closer together, interference between the overlapping, out-of-focus, tails of adjacent beamlets can generate significant amounts of multiphoton signal. For wavelengths near 800 nm and numerical apertures (NA) near 1.3, this effect greatly degrades the contrast for separations between the foci that are less that ~7–10 µm.
Recently, we demonstrated a temporally decorrelated, multifocal multiphoton microscope that does not have these limitations. Egner et al. have also shown the theoretical advantage of decorrelating the pulses using diffraction theory. In a temporally decorrelated multifocal microscope, which was first suggested by Buist et al., the light pulses are successively delayed in time such that the foci do not interfere. In our original work on temporal decorrelation of the foci, we used an etalon to produce an array of beamlets, which were then imaged to produce a series of collimated beams with slightly differing incidence angles at the entrance aperture of the objective as shown in Fig. 5. This produced a line of equal intensity, evenly spaced foci, which were also temporally decorrelated. We note that the intensity of the focused beamlets on the first mirror M1 is below the damage threshold of the mirror. This method allows multifocal multiphoton imaging with the resolution of single point illumination. The etalon that produces the temporal decorrelation, however, has an optical efficiency of only a few percent because of the high reflectivity mirrors in it, which were used to ensure equal powers in the beamlets. To image or machine a large area with a more efficient use of the source, it is desirable to produce a temporally decorrelated array that uses all of the incident laser light.
3.4 High-efficiency multifocal arrays
Fig. 6 shows a new beamlet array design based on cascaded 50/50 beamsplitters, which we have used to make a multifocal microscope. By first dividing the power into two equal beams, then into four beams, eight beams, and so on, we can produce rows of 2N beamlets, where N is the number of cascaded beamsplitters. The lens, L1, indicates where the corresponding lens L1 from Fig. 4 would be. The rest of the microscope would be identical. We can achieve a two-dimensional temporally decorrelated array of size 2N×2M, simply by placing M beamsplitters and a mirror to operate on the entire row of 2N beamlets in the plane orthogonal to the plane of the initial row of beamlets. Fig. 7 shows a two-photon absorption fluorescence image of the 8×2 array of temporally decorrelated foci produced by cascaded beamsplitters. The difference in intensity between the two rows is due to a beamsplitter that is not exactly 50/50. With a 50/50 beamsplitter all the foci can have equal intensity. The measured axial PSF for the 16 foci array is ~1.5 µm. Using this 8×2 array, we can image by asynchronously rastering the scanning mirrors to fill in the gaps between the foci. As an example of the sectioning capability of the multifocal microscope, we took a stack of 2-photon absorption autofluorescence images of a section through a grain of pollen from Clivia Mineata., which is shown in a brightfield image in Figure 8. We took forty-six sections with an exposure time of two seconds for each image. The axial spacing between the sections was 0.5 µm and the region covered in the stack is shown on Figure 8. The objective used was the Zeiss 100×/1.25-NA Achroplan oil objective. The total average power in all 16 beamlets was 200 mW, measured in front of the objective, from an extended-cavity Ti:sapphire oscillator with a 21.6-MHz repetition rate. Per beam this is comparable to the average powers that are typically used in two-photon imaging. Figure 9 shows a movie of the 3-D rendering of the image stack. The autofluorescence is confined largely to two sac-like internal membranes, one of which rests on top of the other and is most likely autofluorescence in the green from flavins, which are known to be concentrated in internal membranes of pollen . The clear separation of the membranes, which are only ~1.5 µm thick in the image, shows the high-resolution section ability of the multifocal microscope. We note that other than thresholding for the 3-D rendering in Fig. 9, Fig. 9 and all the images in this paper are raw data without image processing or sampling.
In this article, we have presented two new techniques for high-efficiency multiphoton microscopy in living systems that can use the high-average power of modern ultrashort pulse lasers. The first method, widefield multiphoton microscopy relies on directly exciting an entire plane in the sample, while the second method, temporally decorrelated multifocal microscopy, excites the sample with multiple foci, which are temporally decorrelated to avoid interference effects.
The widefield multiphoton microscope is simple to build and allows video frame rate imaging of biological samples such as motile Euglena without scanning. Because this microscope has a Gaussian excitation profile in the lateral plane and lacks the inherent sectioning of a standard multiphoton microscope, the technique does not allow threedimensional rendering of sectioned data. We note, however, that deconvolution techniques, which are used for widefield microscopy , might also work for widefield multiphoton microscopy.
For the multifocal, multiphoton microscope, we have demonstrated a cascaded beamsplitter design, which allows efficient use of all the available light with the resolution of a single-focus multiphoton microscope. By increasing the number of foci, it will be possible to use high average power lasers to illuminate large areas of samples simultaneously instead of attenuating the beam and throwing away the majority of the available photons, as is currently done in most multiphoton microscopes.
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