Multiphoton microscopy offers several advantages over confocal laser scanning microscopy (CLSM), such as the use of less damaging longer wavelengths, reduced scattering, deeper imaging, elimination of pinholes, and higher light-collection efficiency. Perhaps more important, excitation and photobleaching are limited to the focal region.
The beamsplitter module allows the microscope to produce high-data-rate images such as this two-photon excited fluorescence of Rhodamine 6G (inset).
Multiphoton microscopy shares one disadvantage with CLSM, thoughlow image acquisition rate. Images must be reconstructed pixel by pixel, and with a 1- to 2-s image acquisition time, the technique is not suitable for investigating fast biological processes. To speed up image acquisition, researchers have used line scans, polygon mirrors, resonant scanners and microlens arrays.1 The problems with the microlens array design are low light throughput, non-uniform intensity foci, and lens aberrations. In our approach, a special beamsplitter divides the incoming laser beam into N beamlets that are coupled to the microscope and scanned simultaneously in the object plane, speeding image acquisition by a factor of N; depending on laser power, N can be as high as 256.2
The beamsplitter module consists of a set of wide-band reflecting and semireflecting mirrors that generate a linear array of beamlets (see figure). Compared to a microlens array-based design, the beamsplitter provides higher light throughput (above 90%), uniform beamlet intensity, and convenient control on the polarization, number and inter-foci distance of beamlets, and field of view. In addition, the slight difference in optical paths introduces intrinsic time-multiplexing capabilities, and the use of flat optical elements minimizes aberrations. We couple the outgoing beamlets into the microscope via an XY scanner and intermediate optics; a microscope objective focuses the beamlets into multiple foci on the object plane.
The optical system yields a linear array of foci with an inter-foci separation in the 0.4- to 2-µm range. The XY scanner allows the positioning of the beamlet-array anywhere within the field of view of the microscope objective. The scanner can be operated either in the resonant or non-resonant mode. Scanning in the XY plane generates a plane of two-photon excited fluorescence; in combination with the Z-drive, the scanner allows the system to acquire a 3-D image. Although the two-axis galvanometer scanner can achieve frame rates of up to 3500 Hz, which can accommodate real-time observation and acquisition of 3-D stacks, the final frame rate of the imaging system is limited by the readout speed of the CCD camera and the intensity of the fluorescence signal. The scanner configuration also allows free selection of the position and the size of the field of view.
The beamsplitter can be configured to generate either a linear or area array of beamlets. We generate area array by coupling the output of the first stage beamsplitter via a periscope to the input of the second stage beamsplitter. The linear array offers several advantages. In addition to the standard XY scanning mode, it also allows the acquisition of XZ-planes and convenient coupling to the input slit of an imaging spectrograph that enables high-speed spectral imaging and sectioning.
The number of beamlets in the linear array can easily be switched from 64 to 32, 16, 4, 2, or a single-beam by moving the semi-reflecting mirror in and out of the optical path. This option is quite important when doing imaging deeper into the sample where higher laser intensity is required. By changing the intermediate optics we can shrink the distance between neighboring foci to the extent that individual foci start overlapping. This overlap illuminates the sample homogeneously along a shortened line, enabling line imaging without the need for scanning.
With a few modifications, this design can also provide a powerful tool for whole field 3-D fluorescence lifetime imaging microscopy (FLIM).3 By coupling a picosecond-gated, high-repetition-rate image intensifier to the CCD camera, we can acquire a series of time-gated images at various delay values with respect to the laser pulse and generate a FLIM image. oe
1. J. Bewersdorf, R. Pick, et al., Opt. Lett. 23, 655-657, (1998).
2. T. Nielsen, M. Fricke, et al., J. Microscopy 201, 368-376, (2002).
3. M. Straub, S. W. Hell, App. Phys. Lett. 73, 1769-71, (1998)
Ramesh Ahuja is president of TauTec LLC, Columbia, MD.