We have developed a random access multiphoton microscope that allows for fast 3D imaging without physically moving scan mirrors or the objective lens. This is achieved by a unique arrangement of four AODs for laser beam steering. We have used this microscope for structural and functional imaging of pyramidal cells. We have recorded calcium transients from different lateral and axial positions on a neuron with acquisition rates that are in the kilohertz range by utilizing the random access capabilities of this inertia-free 3D laser scanner.
One current limitation of our approach is the restriction to a 50μm axial scan range when using a high magnification objective lens. While this exact number is flexible and the axial range can be stretched further, we have noticed a substantial drop in excitation power at further axial distances. This is primarily due to the birefringent diffraction principle of the utilized slow shear TeO2
AODs. A hallmark of this diffractive effect is a significantly narrow incident angular aperture. This constraint reduces the diffraction efficiency of uncollimated incident laser beams, such as the beams at the third and fourth AODs of our scanner when the axial position moves away from the inherent objective focus. By the acoustic bandwidth considerations alone, an axial scan range of approximately 200μm and an lateral scan range of approximately 350μm should be feasable32
. One possibility to avoid this effect would be to use deflectors whose diffraction is not birefringent-based, unfortunately, with larger angular apertures, such AODs have significantly lower overall diffraction efficiencies. One intriguing possibility would be to utilize specialized wide-angular-aperture deflectors. These deflectors have been shown to provide high diffraction efficiencies while maintaining larger angular apertures40,41
, thus making them optimal for AOD-based focusing applications. We are currently working on an improved scanning scheme with extended axial scan range, utilizing custom-made wide-angular-aperture AODs.
In general, larger axial ranges would also reduce the effective spatial resolution. Indeed, in addition to minor reductions in effective objective numerical aperature32
, there is also an anticipated increase in spherical aberration as the excitation beam at the BFA of an infinity-corrected objective lens deviates from the perfectly collimated condition42
. The amount of spherical aberration introduced in this work did not extensively effect the imaging resolution over the used axial range, as shown in supplementary Figure 1
, with the largest measured change in lateral resolution being a factor of 1.76 and the largest change in axial resolution a factor of 1.52. While such resolution changes had a small but noticeable effect on structural imaging, as can be seen in and , the effect on functional imaging was negligible, since the tiniest functional units we are interested in, i.e., spines, are not significantly smaller than our largest spot size. Other groups have shown minimal reductions in spherical aberration from collimation changes in water immersion lenses over a similar axial range43
. With larger axial ranges, however, it is generally anticipated that spherical aberration will become more important. In such cases, it might prove useful to utilize a reverse Gaussian aperture44
to preserve the resolution over the entire axial range at the expense of a mildly reduced resolution at the inherent focal plane.
One issue we did not address in this context is temporal pulse broadening. While the scanning mechanism we employed inherently compensates for spatial dispersion32
, it does not compensate for temporal dispersion. Consequentially, the pulsewidth is broadened from 200fs when exiting the Ti:S laser to approximately 1.8ps at the BFA. This required an increase in laser power for two-photon fluorescence excitation, and average powers of approximately 40-100mW at the BFA were used for these studies. This power increase also compensated for the decrease in intensity at positions not at the focal point of the objective due to increased spot sizes (see supplementary Figure 1
), which would otherwise also reduce excitation efficiency. Notably, this amount of power was approximately 10%-25% of the amount available at the BFA, thus we were not power limited by the broadened pulsewidth. In addition, while it might seem that the required higher power levels would lead to increased photodamage, previous studies have shown equivalent levels of photodamage in multiphoton microscopy when using pulsewidths ranging from less than hundred femtoseconds to several picoseconds45
. It has even been suggested that potential photodamage can be reduced with longer pulsewidths46
. For these reasons, combined with the physical impracticality of a prism-based prechirper large enough to compensate for the ~80,000fs2
group velocity dispersion (GVD) introduced by the four AODs, an external temporal compensation device was not utilized. On the other hand, it has been also been suggested that using narrow pulses in multiphoton microscopy will lead to increased penetration depth47
, since higher peak powers can be applied to deeper tissue. Thus, with increasing axial ranges, or with “in vivo” imaging where more tissue is transversed48
, external temporal dispersion compensation might become increasing valuable, in which case, compensation scheme utilizing diffraction gratings49
or photonic crystal fibers50
offer possible methods for generating the large amount of compensatory negative GVD values. Another possibility is to use moderately longer pulselengths than what we used in this experiment to start with, which minimizes both the temporal dispersion and spatial dispersion induced by the system. However, these optimal mid-range femtosecond pulse lengths are not widely commercially available on most laser systems and typically require either off-label manipulations to the laser cavity15
or custom designed laser systems35
Two recent manuscripts have reported the development of other methods for 3D functional neuronal recordings, both of which are significantly different from the method proposed here. The first is from Gobel et al., who combine a scanning system consisting of galvanometer driven pivoting mirrors for lateral imaging and a piezoelectric actuator for axial imaging with a novel scanning strategy31
. In essence, this technique represents the furthest extent to which inertia-based scanning techniques can be taken. Indeed, when used for scanning neuronal populations, it has been shown that up to 90% of user-selected cells can be imaged at acquisition rates on the order of 10Hz. The remaining fundamental limitation is the dependence upon actuator-and galvanometer-based scanning technologies. In fact, even the fastest piezoelectric actuators are only capable of speeds around 100Hz, if not slowed down by the inertia of a microscope objective. However, at the higher speeds, the vibration from physical movement of the objective lens can easily disturb image quality and disrupt biological samples. In contrast, the AOD-based scanning approach presented here is easily capable of acquisition rates that are in the kilohertz range, without imparting any physical vibrations to the sample or the image. While the present 50μm range of our scanner is smaller than the axial range of many piezoactuators, which can be 100s of microns, as mentioned above, this axial range can be extended to similar values by using either, a lower magnification objective lens, wide-angular-aperture deflectors, or a combination of both.
The second manuscript which demonstrates a method only for fast 3D scanning is by Vucinic et al, in which two AODs are used to quickly scan a volume35
. This approach can be seen as a simplified version of the scanning scheme presented here, wherein the acoustic frequencies applied to the first X and Y deflectors are kept constant, or the first two deflectors are bypassed altogether, but chirped frequencies are applied to the latter X and Y deflectors. Since these acoustic frequencies are chirped, the laser beam after the AODs is no longer collimated, as shown in . In addition, similar to the method presented here, the degree of collimation, and hence the axial position can be changed by changing the degree of chirp. However, unlike the method presented here, there is no secondary set of deflectors in the path. As a result, there is a continuous lateral movement of the laser beam that is not compensated and forces the scanner into a continuous “raster” mode. While this approach does allow for 3D structural imaging, it has two consequences for functional imaging. First, it limits the effective speed of the system since time is wasted at sites that are not of interest as this is true with all raster scanning systems. Second, this scheme drastically reduces the dwell time per point of interest. This severely limits the number of detected photons, and reduces the achievable signal-to-noise ratio, a fact which the authors acknowledge. These two consequences make this approach non-optimal for functional 3D imaging. Indeed, while Vucinic et al have shown functional imaging signals, they have been able to do this only when not scanning in three dimensions. Thus, when acquiring functional data, their imaging scheme resembles previously published 2D schemes15
. In this regard, while both approaches allow for fast 3D structural imaging, unlike our approach, their approach does not permit fast 3D functional imaging, where high scanning speeds are arguably more important.
In summary, we have developed a multiphoton microscope that represents the first device we know of capable of recording from multiple sites in a tissue volume at acquisition rates that are in the tens of kilohertz range. We have utilized this instrument to monitor calcium dynamics in a single neuron. Such experiments have previously be limited to a single focal plane, which often restricts researchers to very few sites as dendritic processes can rapidly pass through this plane. Our approach can also be combined with similarly designed 3D random-access photolysis of caged compounds, which opens the door for countless experiments examining the spatio-temporal dynamics of neural computation. Additionally, this technique can be immediately applied to a variety of other neurophysiological situations, most notably in the study of cells populations. In this case lower magnification would be employed, and indeed, we have previously shown an axial range of approximately 1mm when the AOD-based system is combined with a 10X objective, giving a large axial range over which cells could be imaged. Given its applicability to a wide range of neurophysiological experiments, this novel imaging tool represents an important technological advancement in the current experimental arsenal of neurophysiologists.