The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.
Broadband, sub-10-fs pulses, can be propagated through polarization maintaining
silica core single mode fibers for use in NLOM. In a manner similar to all fiber chirped
pulse amplifiers, we stretch the pulse sufficiently, ~-4000 fs2 post fiber residual chirp,
such that nonlinearity is minimized owing to the significant impact of dispersion on pulse
width. The optics of the imaging system provide the remaining positive dispersion
delivering a near transform limited, 12.7 fs pulse, to the specimen plane. We are able to
achieve average powers up to 75 mW from the fiber with minimal changes in spectra at a
fiber length of 400 mm. Image intensity analysis of identical images taken with and
without the fiber indicates that the fiber based system is capable of generating signals that
are within a factor of two of our traditional NLOM. Autocorrelations and pulse spectra
are also presented following propagation through the fiber and imaging system.
The intrinsic optical sectioning, reduced light-scattering, and reduced photodamage of multiphoton laser-scanning microscopy (MPLSM) has generated great interest for this technique in experimental Neuroscience, as it enables to study both structure and function of fine neuronal processes within living brain tissue. At present, virtually all MPLSM systems employ galvanometric beam positioning. Due to this inertia-limited approach, single-dimension line scans are employed to achieve frame rates sufficient for functional imaging. Although such line scans allow adequate sampling rates (≤1kHz), two significant drawbacks remain. First, the majority of scan time is wasted by illuminating regions of no interest, while sacrificing signal integration time at sites-of-interest. Second, the sites from which signals can be recorded are limited to those along a single line. Alternatively, acousto-optic (AO) beam positioning with high-resolution TeO2 deflectors allows inertia-free skipping between arbitrary sites within the field-of-view in <15μs. This achieves high sampling rate recording at multiple, non-adjacent sites quasi-simultaneously (1-5kHz frame rate, 12-60 sites). Such a multi-site optical recording system would greatly advance studying complex neuronal function, by enabling membrane potential or calcium transients to be observed throughout the complex geometry of neuronal dendrites. This paper presents images and functional recordings from living neurons within brain slices, acquired with AO-MPLSM. Our novel imaging system allows a user to collect structural images first and subsequently select sites of interest for fast functional imaging. To demonstrate the system’s power, we present high-speed recordings (1kHz) from >10 sites within the dendrites of pyramidal neurons in acute brain slices, at signal-to-noise ratios comparable to line-scan systems.
The femtoliter excitation volume of multiphoton microscopy renders all emitted photons useful in detecting fluorescence signals. We have previously demonstrated the viability of transmitting the emitted fluorescence via multimode optical fibers onto a photomultiplier tube (PMT) in a full-field arrangement. A custom MPLSM, based on a commercially available confocal microscope was developed to readily switch between the regular descanned path for confocal microscopy and our non-descanned pathways (direct detection) supporting fiber-coupled detection. We now wish to demonstrate the efficacy of fiber-based detection using a side-by-side comparison of our fiber-coupled paradigm to the traditional method of directly focusing the fluorescence, through air, onto a PMT. To effectively compare the two methods, we have incorporated a second direct detection epi-fluorescence pathway for air-coupling onto a PMT that does not affect the performance of the fiber-based MPLSM. We found that fiber-based detection compares favorably against traditional direct detection. We demonstrate the viability of fiber-based detection for high-resolution neuronal brain slice imaging.
The extremely small (femtoliter) excitation volume of multiphoton (MP) microscopy renders all emitted photons useful in detecting fluorescence signals. Hence, multiphoton laser scanning microscopy (MPLSM) systems can collect fluorescence through the objective (epi-fluorescence), as well as the condenser (trans-fluorescence). For maximal collection efficiency, both optical paths can be used concurrently (4π detection). Most MPLSM systems incorporate photodetectors directly in or adjacent to the epi- and trans-fluorescence optical paths of the microscope, generally photomultiplier tubes with associated optics. These arrangements are optically straightforward, but are often bulky and difficult to reconfigure. Here, we demonstrate that all fluorescence from the specimen can be efficiently coupled into two multimode optical fibers -- one each for the epi- and trans-fluorescence pathways. Fiber-coupled detection enables a modular detection paradigm where light can be routed to easily reconfigurable and interchangeable detection module(s). A novel MPLSM system was constructed, which is readily switched between the original de-scanned detection path for confocal microscopy, and the newly added pathways supporting fiber-coupled non-descanned 4π detection for MP microscopy. Sample MP images of fluorescent beads and fluorescent-labeled hippocampal neurons are presented, demonstrating the viability of fiber-coupled detection.
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