Name: deep-tissue three-photon fluorescence microscopy in intact mouse and zebrafish brain
Uploaded: 2022-01-13T14:00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain at JoVE.com

This work was supported by NSF DBI-1707312 Cornell NeuroNex Hub and NIH 1U01NS103516.

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+adult+neural+stem+cell+behavior+in+the+intact+and+injured+zebrafish+brain.">Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.</a> Science. 348 (6236), 789-793 (2015).</li><li>Dray, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+live+imaging+of+adult+neural+stem+cells+in+their+endogenous+niche.">Large-scale live imaging of adult neural stem cells in their endogenous niche.</a> Development. 142 (20), 3592-3600 (2015).</li><li>Kobat, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+multiphoton+microscopy+using+longer+wavelength+excitation.">Deep tissue multiphoton microscopy using longer wavelength excitation.</a> Optics Express. 17 (16), 13354-13364 (2009).</li><li>Wang, M., Wu, C., Sinefeld, D., Li, B., Xia, F., Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+effective+attenuation+lengths+for+long+wavelength+in+vivo+imaging+of+the+mouse+brain.">Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain.</a> Biomedical Optics Express. 9 (8), 3534-3543 (2018).</li><li>Vogel, A., Noack, J., H&#252;ttman, G., Paltauf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+femtosecond+laser+nanosurgery+of+cells+and+tissues.">Mechanisms of femtosecond laser nanosurgery of cells and tissues.</a> Applied Physics B. 81 (8), 1015-1047 (2005).</li><li>Li, B., Wu, C., Wang, M., Charan, K., Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adaptive+excitation+source+for+high-speed+multiphoton+microscopy.">An adaptive excitation source for high-speed multiphoton microscopy.</a> Nature Methods. 17 (2), 163-166 (2019).</li><li>Kobat, D., Horton, N. 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The authors declare no competing interests.

This protocol explains step-by-step procedures for setting up 3P imaging with a commercial microscope and laser source. Compared to 2PM, 3PM has an advantage in applications requiring optical access in the deeper regions such as in the mouse brain hippocampus. Although 3PM is mostly used in neuroscience, 3PM can be potentially applied in other tissues such as lymph nodes, bones, and tumors for deep-tissue observation.
It is important to verify that the imaging system performs at close to the shot-noise limit, which ensures that the detection and data acquisition electronics contribute negligible noise to the image after the PMTs. The uncertainty in the number of photons detected is fundamentally limited by photon shot noise. Shot-noise limited performance can be achieved in a typical multiphoton microscope using a high-gain photodetector (e.g., a PMT). Photon shot noise follows a Poisson statistical distribution, wherein the standard deviation of the distribution is equal to the square root of the mean of the distribution. To verify the shot-noise limited performance, follow step 1.14 in the protocol section.
To avoid light attenuation by H2O, using D2O for immersion is helpful, particularly for ~1,700 nm excitation. When D2O is used, it is essential to refresh D2O every ~10 min or use a large volume of D2O to avoid D2O/H2O exchange during imaging. One can also seal the D2O from the room environment3. If a long working distance (WD) objective lens (e.g., WD at 4 mm or longer) is used for imaging, the immersion liquid thickness can exceed 2-3 mm. The increased thickness makes H2O absorption non-negligible even at ~1,300 nm21. Therefore, D2O may be necessary even for 1,300 nm 3PM when using a long WD objective lens.
As the 3P fluorescence intensity depends on the cube of the excitation pulse energy at the focus (Eq. (1)), setting the appropriate laser power is particularly important to obtain adequate 3P fluorescence signals while avoiding thermal and nonlinear damage in living tissues. The average laser power should be kept below the thermal damage threshold. In the mouse brain, for example, to avoid thermal tissue damage, the average power on the mouse brain surface should be kept at or below ~100 mW for ~1,300 nm excitation at a depth of 1 mm and with a field-of-view (FOV) of 230 &#181;m x 230 &#181;m21. Similarly, the average power at ~1,700 nm should be kept at or below ~50 mW at ~1 mm depth and a FOV of ~230 &#181;m x 230 &#181;m (unpublished data). Further, to avoid excitation saturation and potential nonlinear damage, the excitation pulse energy should be kept at <img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />2 nJ and <img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />3 nJ for ~1,300 nm and ~1,700 nm excitation, respectively30.
Due to light absorption and scattering in tissues, the pulse energy at focus is attenuated to 1/e (~37%) after penetration of tissues by 1 EAL. The EAL varies in different tissues and with the excitation wavelengths, e.g., in the neocortex of the mouse brain, the EAL is ~300 &#181;m and ~400 &#181;m at ~1,300 nm and ~1,700 nm, respectively3,29&#160;(Figure 5). Therefore, to keep the same pulse energy at focus (e.g., 1 nJ/pulse) at a depth of n EALs, the surface pulse energy needs to be multiplied by 1 nJ &#215; en. For fast imaging of structural and functional dynamics, an excitation laser with a high repetition rate (at 1 MHz or higher) is desirable to achieve a high frame rate5,6,7,10. However, the pulse energy requirement and the average laser power limit constrain the applicable repetition rate.
For example, when we image a moderately deep region at 4 EALs (i.e., ~1.2 mm in the mouse cortex with 1,300 nm excitation), ~55 nJ/pulse at the surface is required to keep 1 nJ/pulse at focus. When the average power limitation is 100 mW, we can apply a ~2 MHz laser repetition rate. However, to image deeper at a depth of 7 EALs, ~1,100 nJ/pulse is required at the surface to maintain 1 nJ/pulse at focus. Assuming the maximum average power is 100 mW to avoid thermal damage, the laser repetition rate should be reduced to 0.1 MHz to achieve a 1,100 nJ/pulse at the surface. Table 1&#160;summarizes typical imaging conditions in the mouse brain cortex. Note that the imaging depths in Table 1&#160;assume that the EAL is uniform in the entire mouse cortex.
Moreover, because of the laser power limitation in deep-tissue 3PM, a trade-off exists between the frame rate and the image pixel size, which is particularly important for functional imaging such as calcium imaging. The maximum available laser repetition rate is decided at each depth based on the required pulse energy at focus and the applicable average laser power as discussed above, e.g., 2 MHz at a depth equivalent to ~4 EALs with 1,300 nm excitation. In general, imaging requires at least one pulse per pixel. Accordingly, the minimum available pixel dwell time is determined by the laser repetition rate, e.g., 0.5 &#181;s/pixel with 2 MHz excitation.
To keep the high spatial resolution (~1 &#181;m in lateral) in 3P images, it is ideal to set 1 pixel to an area of ~1 &#181;m2, e.g., 256 x 256 pixels for a FOV of 250 x 250 &#181;m2. Hence, to perform fast imaging with a considerably large FOV (e.g., 250 x 250 &#181;m2 with 256 x 256 pixels), 0.5 MHz, 1 MHz, and 2 MHz pulse repetition rates give theoretical maximum frame rates of ~7.6, ~15, and ~30 frames/s, respectively. Likewise, the optimization of the laser repetition rate is essential, depending on the target depth, scan speed, and FOV, to apply adequate pulse energy under the thermal damage threshold. To increase the imaging speed, an adaptive excitation source can be used to concentrate all the excitation pulses on the neurons (i.e., regions of interest) by delivering laser pulses on demand to the neurons31.
3PM is advantageous when compared to 2PM in deep imaging within living tissues and through highly scattering media such as a skull, bones, and the white matter layer (i.e., the external capsule) of the mouse brain. The longer EAL and the higher-order nonlinear excitation of 3PE benefits deep-tissue imaging. For example, to image GCaMP6 in the mouse cortex, 2P&#160;fluorescence signal with 920 nm excitation is higher than 3P&#160;fluorescence signal with 1,300 nm excitation in shallow regions at <img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />690 &#181;m (i.e., ~2.3 EALs at 1,300 nm)21. However, due to the longer EAL at 1,300 nm compared to 920 nm, 3PE gives stronger fluorescence than 2P excitation (2PE) at a depth of ~690 &#181;m and deeper21. This depth is defined as &#39;signal crossover depth,&#39; at which the fluorescence signal strengths of 2PE and 3PE are identical with the same repetition rate and the same maximum allowable average powers21. The signal crossover depth depends on the excitation wavelengths for 2PE and 3PE and the fluorophore.
In practice, 920 nm excitation allows higher average laser power than 1,300 nm excitation due to less water absorption. However, the higher average power of 2PE would push the signal crossover depth only by 0.9 EALs4. In addition, when the sample is densely labeled, 3PE has the additional advantage of much higher SBR. Therefore, even before reaching the signal crossover length, 3PM can be better for imaging than 2PM. For example, when imaging the mouse brain vasculature, which has a volume fraction (i.e., labeling density) of ~2%, 1,300 nm 3PM with 100 mW excitation power outperforms 920 nm 2PM with 200 mW excitation power at a depth of ~700 &#181;m for fluorescein.
3PM also has an advantage when imaging through a thin but highly scattering layer that may distort the point-spread function of the excitation beam and generate a defocus background4. For example, through the intact skull of the mouse brain, 2PM images suffer from the defocus background even at the shallow depth of &#60;100 &#181;m from the brain surface13. A similar defocus background was observed in 2PM with 1,280 nm excitation through the white matter in the mouse brain32. Therefore, when tissues are imaged through turbid layers, 3PM is preferable to 2PM for high-contrast imaging regardless of the labeling density.
We recently reported a beads phantom and theoretical analysis showing that the imaging depth limit of 3PM is over 8 EALs33; 8 EALs are equivalent to ~3 mm with ~1,700 nm excitation in the mouse cortex. However, the currently available laser does not have enough pulse energy to achieve 8 EALs in the mouse brain. Further development of stronger lasers will push the current imaging depth limit of 3PM.

In life science, multiphoton microscopy (MPM) techniques, such as 2PM and 3PM, have been powerful tools for deep in vivo imaging with high spatiotemporal resolution and high contrast in scattering tissues. Additionally, these methods cause less photobleaching when compared to one-photon confocal microscopy1,2,3,4. 3PM is advantageous for deeper-tissue imaging when compared to 2PM due to two major features : (i) employment of longer wavelength excitation (~1,300 nm or ~1,700 nm) reduces tissue scattering, and (ii) the higher-order excitation process (i.e., fluorescence signal depends on the cube of the excitation power in 3PM instead of the square of the excitation power in 2PM) that suppresses the unwanted background fluorescence3. Consequently, 3PM enables high-contrast imaging at deeper regions in living tissues such as the hippocampus in an intact adult mouse brain3,5,6,7,8,9,10,11&#160;and the entire forebrain of adult zebrafish12, including Ca2+ activity recording and multicolor observations. Moreover, high-contrast images have been obtained with 3PM through the intact skulls of mouse and adult zebrafish12,13.
Today, excitation laser sources suitable for 3P excitation (3PE) at ~1,300 and ~1,700 nm are commercially available. As the laser scanning system is essentially the same for 2PM and 3PM, converting an existing a 2P&#160;setup to a 3P setup is possible in biology laboratories with the installation of a commercially available laser for 3PE. The 3P fluorescence signal is dependent on the laser power, pulse duration, laser repetition rate, and numerical aperture (NA) of the objective lens. Assuming a diffraction-limited focus (i.e., the back aperture of the objective lens is overfilled by the excitation beam), Eq (1) describes the time-averaged fluorescence photon flux from the focal volume resulting from 3PE.
<img alt="Equation 1" src="/files/ftp_upload/63213/63213eq01v3.jpg" /> (1)
Where f is the laser repetition rate, &#964; is the laser pulse duration (full width at half maximum), &#981; is the system collection efficiency, &#951; is the fluorescence quantum efficiency, &#963;3&#160;is the 3P absorption cross-section, C is the fluorophore concentration, n0&#160;is the reflective index of the sample medium (e.g., water), &#955; is the excitation wavelength in vacuum, NA is the numerical aperture of the objective lens, a3&#160;is the spatial integration constant of the focal volume, <img alt="Equation 1" src="/files/ftp_upload/63213/62313eq02.jpg" /> is the time-averaged excitation photon flux (photons/s) under the objective lens, z is the imaged depth, and EAL is the effective attenuation length14. Here we have assumed that the EAL (typically &#62; 100 &#181;m) is much greater than the axial resolution of the microscope (typically &#60; 10 &#181;m). Under paraxial approximation, a3&#160;is equal to 28.114. gp(3) is the 3rd-order temporal coherence of the excitation source, and gp(3) is 0.41 and 0.51 for hyperbolic-secant-squared pulses and gaussian pulses, respectively. The collection efficiency &#981; can be estimated by considering the fluorescence collection by the objective lens, transmittance of the objective lens, the reflectivity of the dichroic mirror, the transmittance of the filters, and the detection efficiency of the detector (e.g., photomultiplier tube, or PMT). As the 3P fluorescence intensity is highly dependent on various parameters, optimization of the 3P setup is required to maximize the 3P fluorescence signals.
This protocol illustrates the optimization process of a typical 3P setup, which will be useful particularly for biology laboratories that have a 2P setup and plan to expand its capability to 3P imaging or to maintain their commercial 3P setup at optimal performance. This video article also demonstrates deep-tissue 3P imaging in living animal brains. The first section addresses the optimization of a typical 3P setup with a commercially available laser source and multiphoton microscope. The second and third sections describe zebrafish and mouse preparation, respectively, for 3PM of neuronal structures and activities. The mouse craniotomy surgery has been previously reported in protocol papers as well15,16,17. The fourth section demonstrates intravital 3P imaging in zebrafish and mouse brains.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5% Povidone-iodine</td>
			<td>Amazon</td>
			<td>NDC 67818-155-32</td>
			<td>Aceptical cleaning of surgical areas</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>CAS 64-17-5</td>
			<td>Aceptical cleaning of surgical areas</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A4718-256</td>
			<td>Preparing zebrafish chamber</td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bergamo II</td>
			<td>Thorlabs</td>
			<td></td>
			<td>Multiphoton Imaging Microscope</td>
		</tr>
		<tr>
			<td>Bupivacaine</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donut shape glass (ID4.5, OD6.5)</td>
			<td>Potomac Photonics</td>
			<td></td>
			<td>Cover glass used for craniotomy</td>
		</tr>
		<tr>
			<td>eye ointment (or topical ophthalmic ointment)</td>
			<td>Puralube Vet Ointment</td>
			<td>NDC 17033-211-38</td>
			<td>Used as a lubricant to prevent irritation or to relieve dryness of the eye during surgery and anesthesia</td>
		</tr>
		<tr>
			<td>GaAsP Amplified PMT</td>
			<td>Thorlabs</td>
			<td>PMT2100</td>
			<td>PMT detector</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heater (800 W)</td>
			<td>Finnex</td>
			<td></td>
			<td>Aquarium heater for zebrafish water)</td>
		</tr>
		<tr>
			<td>Isoflurane USP 250 mL</td>
			<td>Piramal</td>
			<td>NDC 66794-0013-25</td>
			<td>For anesthesia of mice</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td></td>
			<td>Laboratory tissue for preparing zebrafish</td>
		</tr>
		<tr>
			<td>Nanofil syringe (10 micrometer) with 36 G needle</td>
			<td>WPI</td>
			<td>NANOFIL + NF36BV</td>
			<td>Syringe and needle for injection of pancuronium bromide</td>
		</tr>
		<tr>
			<td>Optical Adhesive</td>
			<td>Norland</td>
			<td>NOA 68</td>
			<td>To stick round coverslip and donut shape glass together.</td>
		</tr>
		<tr>
			<td>Pancuronium Bromide</td>
			<td>Cornell Veterinary Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Elemental Science</td>
			<td>ESI MP2</td>
			<td>Water pump for zebrafish setup</td>
		</tr>
		<tr>
			<td>Polyethylene tubing (I.D. 0.58 mm., O.D. 0.965 mm.)</td>
			<td>Elemental Science</td>
			<td>MP2 pump tubing</td>
			<td>Tubing that goes in the mouth of the zebrafish</td>
		</tr>
		<tr>
			<td>Round Cover Slip German Glass #1.5, 5 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>7229605</td>
			<td>Cover glass used for craniotomy</td>
		</tr>
		<tr>
			<td>Spirit-NOPA</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Tunable Optical Parametric Amplifier</td>
		</tr>
		<tr>
			<td>SR400</td>
			<td>Stanford Research Systems</td>
			<td>SR400</td>
			<td>Photon counter</td>
		</tr>
		<tr>
			<td>Standard Photodiode Power Sensor</td>
			<td>Thorlabs</td>
			<td>S122C</td>
			<td>Power detector</td>
		</tr>
		<tr>
			<td>Sterilized phosphate buffered saline (PBS)</td>
			<td>Millipore Sigma</td>
			<td>SKU 806552-500ml</td>
			<td>Used during mouse brain surgery</td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Dynarex disposable towel drape</td>
			<td>4410</td>
			<td>For aceptical mouse surgery</td>
		</tr>
		<tr>
			<td>Thin strip boxing wax</td>
			<td>Corning Rubber Co., Inc.</td>
			<td></td>
			<td>Holding tubing in place in zebrafish chamber</td>
		</tr>
		<tr>
			<td>ThorImage</td>
			<td>Thorlabs</td>
			<td></td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>Tricaine (Ethyl-m-aminobenzoate methanesulfonate salt)</td>
			<td>MP</td>
			<td>103106</td>
			<td>Zebrafish anesthesia and euthanasia</td>
		</tr>
		<tr>
			<td>Tygon tubing (I.D. 1/16 in., O.D. 1/8 in.)</td>
			<td>Tygon</td>
			<td></td>
			<td>Tubing for water flow for zebrafish preparation</td>
		</tr>
		<tr>
			<td>VaporGuard</td>
			<td>VetEquip</td>
			<td>931401</td>
			<td>For recycling isoflurane</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>To glue the glass window on the mouse skull, and to glue the laboratory tissue when preparing the fish.</td>
		</tr>
		<tr>
			<td>XLPLN25XWMP2</td>
			<td>Olympus</td>
			<td></td>
			<td>Multiphoton Excitation Dedicated Objective</td>
		</tr>
	</tbody>
</table>

deep-tissue three-photon fluorescence microscopy in intact mouse and zebrafish brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.
Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

The successful completion of this protocol will result in a properly aligned microscope with optimal light parameters (e.g., pulse duration, NA) and animal preparations appropriate for in vivo 3PM. The commercially available 3P setup comprises appropriate mirrors and lenses for both ~1,300 nm and ~1,700 nm; therefore, no change in optics is required when the excitation wavelength is switched between 1,300 nm and 1,700 nm. If the lenses in a 3P setup do not have an appropriate coating for 1,300 and 1,700 nm, these need to be replaced with appropriate ones to reduce laser power loss. With the optimized 3PM and proper animal preparation, in vivo fluorescence and THG images with high contrast can be collected deep within the brains.
Figure 3&#160;shows representative 3P images of intact adult zebrafish. High-resolution, non-invasive, and deep imaging of genetically labeled neurons in the adult zebrafish brain is achieved using 3PM. Although imaging in the telencephalon region has been reported in the adult zebrafish brain using 2PM24,25,26,27, 3PM enables access to the entire telencephalon and regions that are more challenging or impossible to observe using other techniques. The distribution of cell layers in the optic tectum and cerebellum can be observed in Figure 3C. In a successful imaging session, the bone is visible in the THG channel and neurons visible in the fluorescence channel. For adult zebrafish imaging, the camera feature of the microscope was used to locate the fish (Figure 3A). This step is not necessary for mouse brain imaging as the glass window is large enough to make the brain easily accessible. High-resolution structural images of adult zebrafish brain were obtained using the system described in the previous sections. The skull is seen in the THG channel (Figure 3B), which helps navigate the brain and find the top surface. As observed in Figure 3C, neurons are distinguishable with a high signal-to-background ratio (SBR) deep in the adult brain. The tissue above the brain is visible in the fluorescence channel due to autofluorescence.
Figure 4&#160;shows multicolor 3P images of GCaMP6s-labeled neurons (green) and Texas Red-labeled blood vessels (red) together with THG (blue) signals in the adult mouse brain with 1,340 nm excitation10. The images are reproduced from previous work10. In Figure 4, the pulse energy at focus was maintained at ~1.5 nJ in the entire depth to obtain sufficient fluorescence and THG signals, and the maximum average laser power was ~70 mW. The pulse duration was adjusted to ~60 fs, and the effective NA was ~0.8. With optimization of the 3PM setup, high-contrast images were successfully obtained down to 1.2 mm from the brain surface, in the CA1 hippocampus region (Figure 4A,B). Figure 4C,D show Ca2+ activity traces of GCaMP6s-labeled neurons at a depth of 750 &#181;m for a 10 min recording session, showing high recording fidelity.
If the excitation laser is misaligned, nonuniformity in signal brightness across the field of view may be observed. Additionally, if the laser parameters, such as the pulse duration, the excitation pulse energy at focus, and the effective NA, are not optimized, the THG image from the fish skull or the craniotomy window of the mouse brain will not be visible and/or requires high excitation pulse energy (e.g., &#62;2 nJ/pulse at focus). Hence, the THG signals at the brain surface can be used as an indicator for an optimized 3PM setup before starting deep-tissue imaging.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63213/63213fig1v5.jpg" /> 
Figure 1: Schematic illustration of a 3PM setup. The wavelength of the excitation laser is set at ~1,300 nm or ~1,700 nm, output from the idler port of the NOPA. The prism-pair compressor and the Si plate compressor are used for the ~1,300 nm and ~1,700 nm laser, respectively, to prechirp the excitation laser pulse. The ~1,300 nm and ~1,700 nm laser beams can be switched with flipper mirrors. The signal port of the NOPA is used to obtain the triggering signal. A half waveplate and a PBS are used to control the excitation power. The fluorescence and THG are detected by GaAsP PMTs. Appropriate combinations of dichroic mirrors and bandpass filters are used to separate the fluorescence and THG signals. Abbreviations: 3PM = three-photon microscopy; NOPA = non-collinear optical parametric amplifier; PBS = polarizing beam splitter; THG = third-harmonic generation; DAQ = data acquisition; PMT = photomultiplier tube. <a href="https://www.jove.com/files/ftp_upload/63213/63213fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63213/63213fig2v5.jpg" /> 
Figure 2: Representative screenshots for intravital Imaging in fish (protocol section 4.1). (A) Camera mode view of the image acquisition software with 4x objective lens in place. Key features of the software are outlined and numbered as follows: 1. Imaging mode of the image acquisition software. The mode options are Camera, Multiphoton, and Multiphoton GG. For white light imaging with CCD camera, the Camera mode is chosen. 2. Clicking the Live button turns on the camera (or PMTs if in multiphoton options), and a real-time view of the microscope can be observed. 3. In Capture Setup tab, the desired imaging parameters (power, location, depth) are set. 4. In Capture tab, a folder location is assigned for the images to be saved. The imaging can be started in this tab. 5. The Z Control setting controls the depth of imaging by moving the z stage motor. 6. Representative image of a zebrafish head. The rostral side of the head is on the left. (B) Representative view of Camera mode with 25x objective lens. (C) Representative view of Multiphoton GG mode containing the THG image of image seen in (B). Abbreviations: CCD = charge-coupled device; PMT = photomultiplier tube; THG = third-harmonic generation. <a href="https://www.jove.com/files/ftp_upload/63213/63213fig2v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63213/63213fig3v5.jpg" /> 
Figure 3: Representative images of the adult zebrafish brain acquired with the image acquisition software. (A) Camera mode image of adult zebrafish head acquired with a 4x objective lens. The top of the image is the rostral direction. The OT lobes and CB are outlined. (B) Representative image acquired in Multiphoton GG mode with a 25x objective lens containing the THG image of (A). (C) Fluorescence images of adult zebrafish brain at the intersection of the cerebellum and the optic tectum where GFP is expressed in cytoplasm of neurons in various depths. Scale bars = 100 &#181;m. Abbreviations: OT = optic tectam; CB = cerebellum; THG = third-harmonic generation; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/63213/63213fig3v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63213/63213fig4v5.jpg" /> 
Figure 4: Multicolor 3PM of GCaMP6s-labeled neurons (green), Texas Red-labeled blood vessels (red), and third-harmonic generation (blue) upon 1,340 nm excitation in the mouse brain. (A) Z-stack images down to 1,200 &#181;m from the brain surface with a field-of-view of 270 x 270 &#181;m (512 x 512 pixels per frame). The laser power was varied according to the imaging depth to maintain ~1.5 nJ pulse energy at the focus. Maximum average power under the objective was 70 mW. (B) Selected 2D images at various imaging depths. (C) Activity recording site at 750 &#181;m beneath the dura with a field-of-view of 270 x 270 &#181;m (256 x 256 pixels). (D) Spontaneous brain activity traces recorded in an awake mouse from the labeled neurons indicated in (C). The frame rate was 8.3 Hz, with a pixel dwell time of 0.51 &#181;s. The laser repetition rate was 2 MHz, and the average power under the objective lens was 56 mW. Each trace was normalized to its baseline and low-pass filtered using a hamming window of 0.72 s time constant. Scale bars = 50 &#181;m. This figure and the figure legend are reproduced from 10. Abbreviation: 3PM = three-photon microscopy. <a href="https://www.jove.com/files/ftp_upload/63213/63213fig4v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63213/63213fig05.jpg" /> 
Figure 5: Effective attenuation length in the neocortex of the mouse brain. EAL (magenta line) is calculated from the tissue scattering (red line) and the water absorption in the tissue (blue line), assuming 75% water composition. The black stars indicate reported experimental data of EAL in the neocortex of the mouse brain3,21,28,29. Note that EAL varies in different tissues. Abbreviation: EAL = effective attenuation length. <a href="https://www.jove.com/files/ftp_upload/63213/63213fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Excitation wavelength 
			(nm)</td>
			<td>Immersion water</td>
			<td>Maximum laser power 
			(mW)</td>
			<td>Maximum pulse energy at focus* 
			(nJ)</td>
			<td>Typical EAL in the mouse cortex 
			(&#181;m)</td>
			<td>Imaging depth in the mouse cortex** 
			(mm)</td>
			<td>Pulse energy under the objective*** 
			(nJ)</td>
			<td>Maximum laser repetition rate**** 
			(MHz)</td>
		</tr>
		<tr>
			<td rowspan="4">1300</td>
			<td rowspan="4">H2O or D2O</td>
			<td rowspan="4"><img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />100</td>
			<td rowspan="4"><img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />2</td>
			<td rowspan="4">~300</td>
			<td>0.8</td>
			<td>~14</td>
			<td>~7</td>
		</tr>
		<tr>
			<td>1.2</td>
			<td>~55</td>
			<td>~2</td>
		</tr>
		<tr>
			<td>1.6</td>
			<td>~210</td>
			<td>~0.5</td>
		</tr>
		<tr>
			<td>2.1</td>
			<td>~1100</td>
			<td>~0.1</td>
		</tr>
		<tr>
			<td rowspan="4">1700</td>
			<td rowspan="4">D2O</td>
			<td rowspan="4"><img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />50</td>
			<td rowspan="4"><img alt="Equation 3" src="/files/ftp_upload/63213/63213eq03v2.jpg" />3</td>
			<td rowspan="4">~400</td>
			<td>0.8</td>
			<td>~7</td>
			<td>~7</td>
		</tr>
		<tr>
			<td>1.2</td>
			<td>~20</td>
			<td>~2.5</td>
		</tr>
		<tr>
			<td>1.6</td>
			<td>~55</td>
			<td>~1</td>
		</tr>
		<tr>
			<td>2.1</td>
			<td>~190</td>
			<td>~0.3</td>
		</tr>
	</tbody>
</table>
Table 1: Typical 3P excitation conditions for mouse cortex imaging. 
*With a high NA (~1.0) objective, pulse width of ~50 fs, and typical fluorophores such as fluorescent proteins (e.g., GFP and RFP). 
** With the assumption that the EAL is uniform in the entire cortex. 
*** To achieve ~1 nJ/pulse at focus, calculated from the EAL and the imaging depth. 
**** Calculated from the pulse energy under the objective and the maximum permissive laser power. 
Abbreviations: 3P = three-photon; NA = numerical aperture; GFP = green fluorescent protein; RFP = red fluorescent protein; EAL = effective attenuation length.

Watch this Scientific Journal Video about Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain at JoVE.com

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