Optical properties of adult Drosophila brains in one-, two-, and three-photon microscopy

: Drosophila is widely used in connectome studies due to its small brain size, sophisticated genetic tools, and the most complete single-neuron-based anatomical brain map. Surprisingly, even the brain thickness is only 200- μ m, common Ti:sapphire-based two-photon excitation cannot penetrate, possibly due to light aberration/scattering of trachea. Here we quantitatively characterized scattering and light distortion of trachea-filled tissues, and found that trachea-induced light distortion dominates at long wavelength by comparing one-photon (488-nm), two-photon (920-nm), and three-photon (1300-nm) excitations. Whole- Drosophila brain imaging is achieved by reducing tracheal light aberration/scattering via brain-degassing or long-wavelength excitation at 1300-nm. Our work paves the way toward constructing whole-brain connectome in a living Drosophila .


Microscope setups
For 1PF and 2PF imaging, the imaging was done on a commercial microscope LSM 780 (Zeiss, Germany). The built-in laser (488-nm) and photomultiplier tube was used to singlephoton excitation and signal detection. A water immersion objective was used (Olympus, XLPlan N, 25 × NA 1.05) for its high transmission in both visible and IR wavelength ranges. A pinhole with ~60 μm diameter was used to achieve optical sectioning. The image formation was done by the controlling software Zen (Zeiss, Germany).
For 2PF and 3PF in vivo imaging, the setup was similar to that done by Ouzounov et al [22]. A home-built laser-scanning microscope that is compatible to long wavelength excitation is constructed. A Ti: sapphire laser at 920-nm with an 80 MHz repetition rate, and an optical parametric amplifier at 1300-nm with a 400 kHz repetition rate, were used as excitation sources of 2PF and 3PF respectively. The same water immersion objective as single-photon microscope was used. The power levels for both lasers after the objective were limited to less than 20 mW for all imaging depths. The fluorescence and THG signals were epi-collected with a dichroic beamsplitter (Semrock, FF705-Di01-25 × 36), and then detected by a GaAsP photomultiplier tube (Hamamatsu, H7422-40) and a bialkali photomultiplier tube (Hamamatsu, R7600-200) in non-descanned configurations to maximize the collection efficiency. A 488-nm dichroic beamsplitter (Semrock, Di02-R488-25 × 36) was used to split the fluorescence and THG signals, which were further separated by a 520/60 band-pass filter (BPF, transmission at center 520-nm, FWHM 60 nm) for the fluorescence and a 420/40 BPF for the THG. A living Drosophila was fixed and placed onto a motorized stage (M-285, Sutter Instrument). A computer running the ScanImage 3.8 under Matlab (MathWorks) was used to synchronize the stage movement and image acquisition. The signal current from the detectors was converted to voltage, amplified and low-pass filtered by a transimpedance amplifier (Hamamatsu, C9999) and another 1.9 MHz low-pass filter (BLP-1.9 + , Minicircuits). Analog-to-digital conversion was performed by a data acquisition card (PCI-6115, National Instruments).

Sample preparations
All the sample preparation methods followed the protocol of previous publication on in vivo Drosophila brain imaging [23]. The samples were adult, female Drosophila between 5 and 10 days old. GFP was pan-neuronal expressed by genetic drivers (Gal4-elav.L/CyO × UAS-EGFP and Gal4-elav/UAS-mGFP). The living Drosophila was immobilized in a pipette tip with volume 100 μL after anesthetized by ice bathing. A window was cut into the head by using fine tweezers, after placing a drop of Ca 2+ -free saline on the brain to prevent desiccation, and fat bodies above the brain were removed, under a stereomicroscope. The dissection saline was then replaced with a drop of Ca 2+ -containing saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 8.2 mM MgCl 2 , 4 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 5 mM trehalose, 10 mM sucrose, and 5 mM HEPES [pH 7.5, 265 mOsm]). No cover glass was placed between the brain and the objective.
To check the optical effect caused by the tracheae structure, degassing the Drosophila brain was performed by pumping out the air inside tracheae. The degassing protocol followed the previous publication of in situ Drosophila brain imaging [24]. The degassing protocol started from immersing the Drosophila in 4% paraformaldehyde and 2% triton, expelling air in tracheae by using a vacuum chamber that was depressurized to -72-mmHg for 2.5 minutes, wait for 1.5 minutes, and then releasing to normal pressure for 2 minutes. The degassing process was completed by repeating the above procedure 4 times. After degassing, the same microsurgery preparation was performed, and observed under the same microscope.

Signal analyses
To quantify the optical properties of bio-tissues, this section explains how to derive the attenuation coefficients (μ att ), which is the inverse value of attenuation length (l a ), i.e., μ att = l a −1 . The calculation of attenuation length inside a biological tissue has been detailed in an earlier work [5]. The well-known light attenuation equation is: where I(d) is the excitation intensity at imaging depth of d, and I 0 is the intensity at tissue surface. With fluorescence excitation processes, the n-th order fluorescence intensity, F (n) (d), is related to the excitation intensity as: (2) For 1PF, 2PF and 3PF, n equals 1, 2 and 3, respectively. Combining Eqs. (1) and 2, where a is a proportional constant. F (n) (d) and d are determined experimentally, and μ att can be obtained from their dependencies. More explicitly, by taking the natural logarithmic value of both sides in Eq. (3), it becomes, ( ) ln( ( )) ln( ) -n att F d a n d μ = × × (4) By plotting the dependence of ln(F (n) (d)) on d (will be shown in Fig. 2), the decay slopes of the curves are -n × μ att . Therefore, μ att is determined by dividing the inverse value of the slopes over n. To obtain the decay slopes, it was done by linear regression fitting of the data points, and the fitting range were selected by the same criteria. They were all selected with the starting point of signal decay to the depth limit of the corresponding imaging modality. One additional note is since there are two imaging systems, 2PF in vivo experiments are performed in both systems to calibrate the attenuation value derived from different systems.

Results and discussions
Within a living brain, the 1PF images lose the image contrast at around 40 μm ( Visualization 1), as no structures are visible in the brain center where the white arrow points in the 50-60 μm panel of Fig. 1(A). The arrowheads indicate structures that located at the edge of the brain. To verify the effect of trachea, a brain is degassed, i.e. air in tracheae is pumped out. Figure 1(B) shows that 1PF in the degassed brain provides much better contrast in the center of brain at the same depth, but cannot exceed 120-140 μm. The 1PF imaging depth of the degassed brain is comparable with that in mouse brains, which is mainly limited by scattering [4]. Comparing the results in Figs. 1(A) and 1(B), it is obvious that degassing removes the additional attenuation contributed by tracheae.
On the other hand, it is well known that using long excitation wavelengths with 2PF modality efficiently improves imaging depth, approaching 1 mm in mouse brains [4]. Using the same excitation wavelength (~920-nm) and fluorescent labeling (GFP families), Fig. 1(C) shows the 2PF imaging depth in a living Drosophila brain indeed increases compared to Fig.  1(A), but reaches less than 120-140 μm, which is not adequate to penetrate the whole brain, mainly due to the tracheae. By combining 2PF modality with a degassed brain, Fig. 1(D) presents the first whole-brain imaging in a Drosophila. Reasonable contrast and resolution (see inset in the bottom panel for resolving single neuron) are maintained throughout the nearly 200 μm depth, manifesting that the trachea-induced light aberration/scattering is the major restraint for deep-brain imaging in this model animal. However, please note that the animal is no l structures, ma In order ~1300-nm wa resolution (in with an exist local processi Please note th slightly differ neuropil stru penetrability.
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Conclusions
In conclusion, we have, for the first time, characterized the optical properties of the Drosophila brain, which is filled with air, with single-photon, two-photon, and three-photon modalities. We found that the main limiting factor that impedes in vivo whole-brain singlephoton imaging is scattering, but for multiphoton imaging, light distortion from tracheae structures plays a more dominant role. The light distortion affects not only signal attenuation, but also image visibility. Although degassing enables whole-Drosophila-brain imaging by reducing trachea-induced light distortion, the only way to achieve in vivo whole-brain imaging with single cell resolution is 3PF at 1300-nm excitation, which exhibits less scattering, light distortion, and better optical sectioning. It is possible to combine with AO to further reduce light distortion [21], thus allowing deep-tissue imaging on the scale extending from a single neuron, a complete brain network, toward a whole-animal connectome [33].