For my PhD work I made extensive use of 2-photon imaging of layer 6 cell bodies at depths of up to ~850μm using GCaMP. This is somewhat deeper than we (and others) have been able to image comfortably using other mouse lines. While we didn’t empirically test all the edge cases of our protocol to validate which parameters were actually needed to achieve this imaging depth, here is a very rough overview of the likely reasons we were able to acquire reasonable images in L6. In brief: there’s no very interesting tricks involved, other than somewhat sparse expression and clean window surgeries.
This is all work that was done in collaboration with Chris Deister in Chris Moore’s lab.
L6 cell bodies, montage from multiple frames where each cell was active. Individual frames usually only show very few active cells.
We used the NTSR1 line to restrict GCaMP6s expression to L6 CT cells. We used AAV2/1-hSyn-Flex-GCaMP6s (HHMI/Janelia Farm, GENIE Project; produced by the U. Penn Vector Core), with a titer of ~2*10^12/ml with an injection of ~0.3μl, through a burr hole, >2 weeks prior to window implant surgery. This gives us a relatively localized expression in L6 (approximate diameter of region with cell bodies ~300 μm), and results in relatively little fluophore above the imaged cells.
Compounding this effect, the L6 CT processes above L4/L5a are relatively sparse. Together, this means that we were able to image at large depths without risking significant excitation of fluophores above the focal point. See also Durr et al. 2011 for a nice quantification of superficial/out of focus fluorescence.
“The maximum imaging depth was limited by out-of-focus background fluorescence and not by the available laser power. For specimens with sparser staining patterns or staining limited to deeper layers, larger imaging depths seem entirely possible.” from: Theer, P., Hasan, M.T., and Denk, W. (2003). Two-photon imaging to a depth of 1000 mu m in living brains by use of a Ti: l2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024.
On top of the local expression pattern achieved through the AAV injection, the highly sparse spiking activity in L6 CT cells are very friendly to GCaMP imaging. Because neighboring cells rarely were co-active, the identification of cells and segmentation of fluorescence traces was relatively easy, even with the significantly degraded z-resolution. Edge case: We imaged a few animals where GCaMP expression was much more spread out, likely due to variation in the AAV spread, and some reporter line crosses that expressed YFP in all L6 CT cells, in addition to AAV-mediated GCaMP. Imaging at depths past L4/5 was harder in these animals with laser powers that would safely avoid any tissue heating or bleaching, suggesting that local expression/sparsity of superficial fluorescence was a requirement for imaging. Part of this was that increased background fluorescence from the dense L4/5 innervation by the L6 CT made it harder to distinguish cell bodies, but it seems likely that the overall increased out-of-focus fluorescence starts being an issue in some cases.
At depths below L2/3, the window diameter can start to affect imaging quality. With large NA objectives (we almost exclusively used a 16x 0.8NA here), deeper imaging planes, and imaging locations away from the center of the window, progressively more excitation light can get cut off by the edge of the window, resulting in power and effective NA loss.
Here is a plot of the available 2-photon excitation power for a completely uniformly filled 0.8NA objective through a 1mm window, ignoring tissue scattering. Realistic beam profiles that deliver more power at lower angles will be affected less in terms of power, but will still lead to effective NA loss, so this plot only works as an upper bound on how bad things could get. The plot shows the squared fraction of photons that make it to the focal spot, for imaging in the center of the (1mm) window (red), or 200μm of center (black).
While a 2mm window should be big enough from this point of view when imaging in the window center, we used a 3mm imaging windows, giving us plenty of room to search for sensory driven barrels to image in without risking any light cut off. Also, the edges of windows are rarely as clear as the center, so the extra safety margin is good to have. This can mean not having to wait for an extra week for the window to clear sufficiently, which is a big help. Past 3mm, window size seems to offer little further advantages, at least for S1 imaging, and bigger windows are much harder to position flat on the cortex.
Large windows could also make it somewhat easier to collect the emitted (scattered) visible light. The rule of thumb for the surface area from which scattered photons are emitted is ~1.5*imaging depth (Beaurepaire&Mertz 2002), so a window that doesn’t cut off excitation light should be near optimal for collection as well.
‘Stacked’/’Plug’ Imaging window
We used the window design described in Andermann et al. 2011 and Goldey et al. 2014, made from 3 and 5mm cover slips (Warner CS-3R and CS-5R, ~100-120μm thickness), directly on the dura without any agar (or any topical pharmaceuticals). This, together with somewhat thinning the skull under the 5mm portion of the glass (especially rostral&caudal of the window for S1 implants, these are the ‘high spots’ that would make the window rock in the medial/latral direction otherwise) to ensure flat position of the glass on the brain, positions the bottom of the window at, or slightly below the level of the inner surface of the skull, which pushes back any swelling that will have occurred during the craniotomy, and compensates the distance between the glass and the brain surface cause by the curvature of the skull.
imaging window ‘plug’ design.
When setting the window into place, it is important to carefully inspect blood flow and to avoid applying too much pressure on the brain and chronically affecting blood flow, especially at the borders of the window. If flow is reduced immediately after window insertion but recovers within a few minutes we usually had no issues.
The main effect of the window design is that the edge formed by the 3mm cover slips seems to keep dura/bone regrowth out of the imaging area – we’re usually able to image for as long as we want to (>2-3months) – usually AAV over-expression rather than window clarity limits the imaging schedule.
Flat 5mm windows without the stacked 3mm cover slips seem to give approximately the same initial imaging quality, but quickly degrade due to tissue regrowth, suggesting that the flat positioning of the window is not always a limiting factor for good optical access.
We made sure to minimize any damage to the dura during the craniotomy and window implant. If bleeding occurred post-operatively, or if there was any amount of subdural blood, L6 imaging was impossible. Due to the window design, superficial blood usually cleared up within 1-2 weeks. In some cases, window clarity still improved after ~4 weeks. The main reason we saw bleeding was when we had performed viral injections ~2 weeks before the window implant, and the burr hole left a small spot of dura adhesion that ripped out when removing the bone – it seems possible that performing injections at the time of window implant could be preferable in some cases.
Occasionally windows deteriorated after >2 months – the first sign of this is the appearance of freely moving csf(?) under the window, and/or increased dura autofluorescence elicited by blue light. In any of these cases, L6 imaging became almost impossible immediately, even though axons/dendrites down to L4 could still be imaged without problems.
Edge case: We had 2 cases of animals with very mild cases of superficial blood in the tissue in which L6 imaging was possible with laser powers of ~70mW total that were barely ok to use in other cases (that is we didn’t observe beaching or any evidence of tissue damage), but that caused superficial tissue damage in the mice with mild residual blood. We don’t know whether this is due to a higher IR absorption and subsequent damage by superficial layers/dura in these mice, or whether the blood increased the likelihood of a immune reaction, or whether the problem was purely coincidental. The take away is that it’s better to wait a few days for windows to clear up rather than pushing to potentially dangerous laser powers.
We’re using a microscope with a 2″ collection path and a Nikon 16x/.8NA objective. This objective seems to represent a nice sweet spot of good enough NA and great collection efficiency (see also Labrigger). We’re slightly under-filling the back aperture, which sacrifices z-resolution but somewhat increases the proportion of photons that make it to the focal spot because lights coming in at vertical angles has to traverse less tissue (check the Labrigger post on this). We haven’t systematically tested the difference of over vs. underfilling, but it looks like the effect on achieving imaging depth is pretty negligible in our hands, partially because the sparsity of L6 firing makes z-resolution less important than it would be otherwise. Only in cases where L4/5 neurite fluorescence was an issue, overfilling significantly improved matters. We also switched to overfilling for occasional high-magnification scans of individual cells to verify that the cells appeared healthy – typical imaging resolution and PSF degradation in L6 means that the cell nucleus was almost never clearly visible.
Excitation wavelength & Pre-chirping
We’re using a Spectra-Physics Mai Tai DeepSee laser, usually at a wavelength of 980nm, which is a good choice for exciting Gcamp6, and gives us more ballistic photons than shorter wavelengths. Generally, longer wavelengths result in less scattering – this increase in mean free path length at longer wavelengths is a significant factor in deep imaging because only non-scattered photons contribute to the 2p excitation at the focal volume (see Helmchen&Denk 2005 for a review, Durr et al. 2011 also has some nice quantification of this in non-brain tissue). We observe massively increased tissue autofluorescence at the dura for wavelenghts of >1000nm, so we settled on 980nm for most deep imaging.
Here’s a plot of the available power (Lamber-Beer law, squared to account for 2p excitation power) for a few wavelengths, mean free path length estimates are taken from Jaques, 2013. Take this with a grain of salt – the estimates depend heavily on estimates of the scattering coefficients of alive neural tissue which vary substantially, but the general trend should apply in any case.
Lambert-Beer exponential decay of non-scattered photons by depth for a few wavelengths (P_0 * exp(-depth/l_s))^2. All mean free path length estimates are approximations, the literature is not fully consistent on the numbers, so the values will not match specific setups.
For deep imaging past 700μm we typically set our laser power at 980nm to ~160-180mW total with the galvos centered, which corresponds to a maximum of 70-80mW total going into tissue when scanning at ~8-10Hz with an approximate pixel dwell time of 1-2μs. We haven’t systematically tested how much further we could push the power levels. In our experience total delivered powers above 140-150mW damage the tissue, though there is evidence that higher levels could be possible without causing damage (Podgorski et al.) – the details of the surgery, duty cycle of the imaging, area over which the beam is scanned, wavelength, pulse frequency vs energy per pulse etc. seem to start to matter substantially in this regime.
We also use a pre-chirper to maximize 2p excitation. The effect of tuning the pre-chirper is much more pronounced in deep imaging than at L2/3, but it looks like most animals with good image quality should work, albeit with lower yield and requiring marginally more power without tuned pre-chirping. For tuning, we use software that displays a trace of the mean brightness of some large region of the image where we see fluorescence, and we manually select a setting that maximizes brightness.
GCaMP6s & virus expression time scale
We’re using GCaMP6s to maximize SNR – the slower kinetics of 6s are a good fit for the very low firing rates of L6 CT cells. We haven’t tested 6f yet in this preparation, but with good surgeries it seems like it should work as well, if maybe at a slightly lower yield.
It is also noteworthy that we almost always observe a sudden shift from expression levels that were too low for imaging but gave us a few barely visible cells to great expression – often from one day to the next. We’re not sure whether this is due to a nonlinearity in apparent cell brightness on top of a linear increase in indicator level, or if there’s an uptick in indicator expression somewhere ~2-3 weeks post infection.
We used AAV2/1-hSyn-Flex-GCaMP6s, and usually had to wait ~3 weeks for good expression, but in some animals the data quality still improved slightly after week 6. This is fairly typical of AAV2/1 and matches the time scale of the increase in chr2 photocurrent when using aav mediated chr2.
- Deep tissue two-photon microscopy. 2005, Nat. Methods, Helmchen Fritjof, Denk Winfried (link)
- Inﬂuence of optical properties on two-photon ﬂuorescence imaging in turbid samples. 2000, Applied Optics, Andrew K. Dunn, Vincent P. Wallace, Mariah Coleno, Michael W. Berns, and Bruce J. Tromberg (link)
- Epifluorescence collection in two-photon microscopy. 2002, Applied Optics, Emmanuel Beaurepaire and Jerome Mertz (link)
- Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy. 2004, Microsc Res Tech., Tung CK1, Sun Y, Lo W, Lin SJ, Jee SH, Dong CY. (link)
- Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues
Nicholas J. Durr, Christian T. Weisspfennig, Benjamin A. Holfeld, and Adela Ben-Yakar