We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.
Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.
Two-photon Ca2+ imaging is a useful technique for the assessment of neural activity. It can be used to identify not only the neural activity required for behavior and memory in normal animals1,2 but also an abnormal neuronal activity that occurs in mouse models of neuropsychiatric disorders3,4. The technique has been used to elucidate the neural basis of brain functions. However, although it can provide high-resolution and high-quality images, its temporal resolution is lower than that of the electrophysiological method1,3.
Optogenetics has helped to innovate the way neuroscientists understand brain function5. Given the technical limitations, the majority of optogenetic research has used activation schemes with low spatial resolution, thus limiting the types of manipulations of neural activity that can be performed accordingly. However, manipulating neural activity at finer spatiotemporal scales can potentially be useful for a more complete understanding of neural computation and the pathogenesis of neuropsychiatric disorders. Spatially precise holographic technology that can shape femtosecond near infrared laser beams promises to overcome this challenge and opens up several new experimental classes that were previously impossible6,7. This technology enables neuroscientists to uncover the fundamental aspects and pathologies of sensory, cognitive, and behavioral neural codes that have been beyond reach.
Holographic projection involves the generation of desired light patterns to access individual cells and functional networks selectively. In vivo experiments require optimal light transmission to target cells in the living brain. Infrared light penetrates deeper into living tissue and can be used for nonlinear two-photon excitation (2PE)8,9,10. Thus, two-photon holographic microscopy, which combines holographic projection and 2PE, can be used to evaluate and manipulate neural activities to probe cellular and functional networks in vivo. Recent biological applications of two-photon holographic microscopy have elucidated the neural activity and circuitry required for learning in the visual cortex11,12, olfactory bulb13, and hippocampus14.
Numerous laboratories worldwide have reported exciting results and improvements using their holographic stimulation systems15,16,17,18,19,20,21,22,23. In the system described here, the holographic stimulation system can be built as an add-on device for a conventional microscope. The phase-only spatial light modulator (SLM) is the key device for modulating a plane wavefront to any shape, and the interference effect is used to control the intensity and location of the foci. Figure 2 shows holographic stimulation and imaging light paths. The first light path is for point-scanning imaging mode and consists of a scan head and image detectors. The second light path is for holographic stimulation with a 1040 nm wavelength and consists of an SLM1. The third light path is for holographic illumination with 920 nm wavelength and consists of an SLM2 and an image sensor. The holographic imaging mode can record intensities from multiple regions of interest by illuminating multiple points in the sample. In this way, the recording speed can be increased to few hundred of frames per second. To achieve point scanning imaging or holographic illumination imaging, the 920 nm laser was split into two paths by a beam splitter with a fixed ratio of 3:7. All the optical elements were aligned on an optical breadboard with dimensions of 600 mm × 600 mm. The modulated light entered through the light port on the side of the microscopic body, while the point scanning imaging light entered through the scan head at the top of the microscopic body. These lights were integrated just above the objective lens and created foci in the sample plane. In addition, the custom-made software enabled the regular workflow to be simple and consistent.
In this article, a complete protocol is presented for the use of holographic stimulation or illumination to measure neural activity and assess the functional connectivity between neurons. For demonstration purposes, we describe here a brain surgery targeting the hindlimb area of the primary somatosensory cortex (S1HL) of the mouse brain and a method to assess and manipulate neural activity using two-photon holographic microscopy. The experimental procedure is divided into four parts. First, the head plate was fixed to the skull of the mouse using dental cement. Second, a viral vector expressing jGCaMP8f or GCaMP6m-P2A-ChRmine was stereotactically injected into the S1HL. Third, the holographic stimulation or illumination system was calibrated. Fourth, after postoperative recovery and expression of these two proteins, in vivo Ca2+ imaging was performed to assess the neural activity and functional connectivity between neurons with two-photon holographic microscopy.
All experimental protocols were approved by the Animal Care and Use Committees of Nagoya University Graduate School of Medicine (approval number: M220295-003).
1. Head plate implantation (Figure 1A)
2. Surgery and adeno-associated virus (AAV) injection (Figure 1B)
3. Preparation for the holographic stimulation or illumination system (Figure 3)
4. Ca2+ imaging using an image sensor with holographic illumination (Figure 4)
5. Two-photon imaging (point scanning mode) with optogenetics using a holographic microscope (Figure 2)
6. Image analysis and assessment of functional connectivity (Figure 4)
Representative recordings obtained using the method described here are presented. In vivo Ca2+ imaging using holographic microscopy requires 2-4 weeks to complete from head plate implantation and AAV injection to data acquisition. Therefore, to obtain stable results, it is important to reduce motion artifacts in the brain. The head plate implantation (step 1.5) and the placement of the cranial window (step 2.9) are very important steps in this process. Furthermore, it is also important to select an AAV (AAV2/8-CaMKII-GCaMP6m-P2A-ChRmine-Kv2.1) that simultaneously expresses calcium indicator and opsin in a single neuron (step 2.8).
Figure 4A shows a spot for the holographic illumination of an acquired neuron image using a custom-made MATLAB script. If holographic illumination successfully illuminated the neurons expressing jGCaMP8f, Ca2+ traces could be obtained with an image sensor (Figure 4B), as shown in Figure 4C.
Functional connectivity between neurons was evaluated using holographic stimulation (Figure 4D), as shown in Figure 4E. Because functional connectivity between neurons is one of the neural circuitry properties that is altered in the pathogenesis of a pain model mouse24, we describe a simple procedure to evaluate it. Figure 4F shows a typical image of L2/3 neurons in S1HL visualized using GCaMP6m. When one neuron (orange circle) was holographically stimulated, another neuron (red circle) was simultaneously active; thus, the number of functional connectivities between neurons was one (Figure 4G).
Figure 1: Schematic outline of the experimental procedure. (A) Fixation of the head plate to the skull. (B) Stereotactic injection of AAV into the hind paw area of the primary somatosensory cortex (S1HL). (C) Implantation of the cranial window. To assess and manipulate neural activity, in vivo Ca2+ imaging is performed in awake mice (D) with holographic stimulation (E). Flash marks indicate holographic stimulation or illumination. Please click here to view a larger version of this figure.
Figure 2: System used for the holographic microscope. (A) Images of the holographic stimulation and illumination light paths (left) near the microscope (middle) with an image sensor (right). (B) These are magnified images of holographic stimulation and illumination light paths around respective SLMs (left and right) and a point scanning light path around a scan head (middle and right). (C) A schematic of the stimulation and imaging optical paths. Phase-only SLMs are used to display digital holograms, and a beam expander (a combination of L1 and L2) and a 4f relay system (a combination of L3 and L4 for holographic stimulation and L4 and L5 for holographic illumination) are placed before and after respective SLMs to make sure each digital hologram is imaged at the exit pupil of a water immersion objective lens, with slightly under-filled image size. To suppress residual zero-order components, a beam block is placed at the intermediate plane. Please click here to view a larger version of this figure.
Figure 3: Flowchart of how to calibrate the holographic stimulation or illumination system. This flowchart describes steps to calibrate a holographic stimulation or illumination system to the sample space and imaging system. Please visit step 3, preparation for the holographic stimulation or illumination system, for detailed instructions and download a sample program. Please click here to view a larger version of this figure.
Figure 4: Representative results of imaging and functional connectivity using a holographic microscope. (A) To illuminate specific neurons expressing jGCaMP8f or GCaMP6m-P2A-ChRmine, the image of neurons is captured, and then a spot is formed on the neuron using a custom-made MATLAB script. (B) The setting of the image sensor (exposure time, imaging area, and binning). (C) Representative image and traces of neurons expressing jGCaMP8f in 100 Hz imaging with holographic illumination and an image sensor. (D) This graph shows the neural response to holographic stimulation (1,040 nm) at each laser power (data from GCaMP6m-P2A-ChRmine expressing neurons at 2 Hz imaging frame rate [n = 16]). Error bars indicate the standard error of the mean. (E) Representative Ca2+ traces during holographic stimulation (blue vertical lines) of 10 different neurons at 2 Hz (left) and 30 Hz (right) imaging frame rates. In 2 Hz and 30 Hz Ca2+ traces, the same color indicates the same neuron. (F) Schematic diagram evaluating functional connections between neurons. When the orange neuron is stimulated, the red neurons respond at the same time, indicating that there is functional connectivity between these neurons. (G) A typical image of S1HL neurons expressing GCaMP6m in WT. Scale bar = 10 µm. (H) Typical Ca2+ traces during holographic stimulation (blue vertical lines) at 2 Hz (upper) and 30 Hz (lower) imaging frame rates. The stimulated neuron is circled in orange, responding neurons are circled in red, and non-responding neurons are circled in grey. Neural responsiveness to holographic stimulation can be detected at both 2 Hz and 30 Hz imaging speeds. Please click here to view a larger version of this figure.
Our set-up 6 | Prakash, R. et al. 25 | Marshel, J. H. et al. 12 | Robinson, N. T. M. et al. 14 | |
Lateral resolution | 1.2 μm | 1.27 μm | ― | 2.22 μm |
Axial resolution | 8.3 μm | 56.86 μm | 15.5 μm | 10.26 μm |
Objective lens/Numerical Aperture | 25x/1.1 | 20x/0.5 | 16x/0.8 | 16x/0.8 |
Table 1: Summary of previous reports and this system for the spatial resolution of holographic stimulation. The lateral resolution, axial resolution, and the objective lens used during the measurement are described.
To understand brain function, it is necessary to accurately assess the neural circuitry underlying the brain function by extracting the dynamics of neural activity. Furthermore, it is essential to identify how this neural circuitry is altered to elucidate the pathogenesis of neuropsychiatric disorders. Indeed, it is known that neural activity is elevated in mouse models of Alzheimer's disease4 and fragile X syndrome26 and mice with impaired white matter function3. Moreover, in a mouse model of inflammatory pain, increased synchronization of neural activity and functional connectivity between neurons is associated with symptoms24. Two-photon holographic microscopy allows us to simultaneously observe the activity of individual neurons and the functional connections between neurons, which is necessary for understanding neural circuitry. We used a 25x objective lens with numerical aperture = 1.1 with wavelengths of 1,040 nm. The theoretical point spread function is a Gaussian distribution with full width at half maximum of 0.5 µm laterally and 1.7 µm axially. However, the actual numerical aperture is less than 1.1, and the measured spot size on a fluorescence bead is 1.2 µm laterally, and 8.3 µm axially. Given that the neuron diameter is approximately 15 µm and the calibration error is within 3 µm, the targeting is generally good. However, cells in the axial direction may be affected by the longer spot size6. Here, we described the viral injection, surgery, calibration of holographic stimulation or illumination systems, and imaging protocols for evaluating and manipulating neural activity in live mice using our microscopy system.
It takes 2-4 weeks to complete all experimental procedures, from head plate implantation and virus injection to data acquisition for in vivo Ca2+ imaging using holographic microscopy. The process is complex and laborious, and the ultimate success of the experiment depends on multiple factors, including the condition of the cranial window, which is affected by postoperative inflammation, the proper choice of Ca2+ indicator and opsin, and whether motion artifacts in the acquired images can be corrected. In particular, two steps are important for a successful outcome. The first concerns head plate fixation and surgery; it is crucial that the head plate be firmly fixed to the mouse head with dental cement. Furthermore, it is important to repeatedly clean bone fragments and clotted blood using cold ACSF during surgery. Since adherence to this procedure reduces inflammation, we successfully observed the dynamics of microglia, the cells responsible for the brain's immune system, without activating their processes and spines or the microstructures of neurons27,28. The second issue is the evaluation and manipulation of neural activity. We chose AAV2/8-CaMKII-GCaMP6m-P2A-ChRmine-Kv2.1 to express the Ca2+ indicator and opsin in one neuron simultaneously. This is because it is difficult to efficiently infect one neuron with different AAV types. Another reason for this choice was that ChRmine can efficiently activate neural activity at 1,040 nm using a two-photon laser12. Recently, it has been reported that ChRmine, by mutating its structure based on structural information obtained by cryo-electron microscopy, improves its function29, which is considered useful for target function analysis in the field of neuroscience. In light of these issues, it is necessary to share effective methods for evaluating and manipulating neurons when reading neural activity and writing information using holographic microscopy.
Recent advances in imaging and optogenetics have revealed the detailed neural activity involved in brain functions such as learning and memory, and it is possible to artificially manipulate this neural activity to express brain functions30. However, conventional methods for the manipulation of neural activity are highly invasive on account of the insertion of optical fibers in the brain and because a group of opsin-expressing cells is simultaneously stimulated, making it impossible to manipulate neural activity with temporal and spatial precision. Our method can manipulate neural activity by stimulating only specific neurons in the brain, thus enabling manipulation of neural activity with specific stimulation patterns and high spatiotemporal resolution. Furthermore, it is important to note that functional connectivity between neurons can only be evaluated in a small number of neurons using brain slice experiments31; however, this technique allows simultaneous evaluation of multiple neurons in living animals.
One of the major limitations of current holographic microscopes is the need to fix the mouse head, which limits the mouse's behavior. Recently, a miniaturized two-photon microscope was developed32, and with the further miniaturization of the device, in vivo Ca2+ imaging with holographic stimulation may be possible in free-moving mice. In addition, the potential of this microscope could be expanded by improving the temporal resolution of imaging and combining it with highly sensitive voltage-sensitive fluorescent proteins33.
The authors have nothing to disclose.
This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas (19H04753, 19H05219, and 25110732 to H. W.), Grants-in-Aid for Transformative Research Areas (A) (20H05899 to H. W., 20H05886 to O. M., and 21H05587 to D. K.), Fostering Joint International Research (B) (20KK0170 to H. W.), Grant-in-Aid for Scientific Research (B) (18H02598 to H. W.), Grant-in-Aid for Scientific Research (A) (21H04663 to O. M.), Grant-in-Aid for Early-Career Scientists (20K15193 to X. Q.), JST CREST Grant Number JPMJCR1755, Japan, and JST A-STEP Grant Number JPMJTR204C.
25x Objective | Nikon | N25X-APO-MP | Objective |
A1MP | Nikon | A1MP | Microscope |
AnesII | Bio machinery | AnesII | Anesthesia delivery system |
C2 plus | Nikon | C2 plus | Microscope |
DECADRON Phosphate Injection | Aspen | 21N024 | Avoid cerebral edema |
Dental Drill | Jota | C1.HP.005 | Dental drill |
Electric Microinjector | NARISHIGE | IM-31 | Pressure injection system |
FEATHERS | FEARGER | FA-10 | Shaving |
G-CEM ONE ADHESIVE ENHANCING PRIMER | GC | 2110271 | Resin cement primer for dental adhesion |
G-CEM ONE neo | GC | 43093 | Resin cement for dental adhesion |
Glass Capillary with Filament | NARISHIGE | GDC-1 | Glass capillary |
Image Detector | Hamamatsu | H10770PA-40 | GaAsP photocathode photomultiplier tube |
Imaging Software | Nikon | NISelements | Imaging software |
Isoflurane Inhalation Solution | Pfizer | 229KAR | Anesthetics |
iXon EMCCD Camera | Andor | iXon Life 888 | Image sensor |
Ketamine | daiitisannkyou | s9-018506 | Anesthetics |
Leica-M60 | Leica | M60 | Stereoscope |
Linicon | Linicon | LV-125 | Vacuum pump |
Mode-locked Ti:sapphire Chameleon Ultra II laser | Coherent | Chameleon Discovery NX | Femtosecond laser |
Mos-Cure | U-VIX | mini 365 | Portable LED UV Light Source |
PEN Bright | SHOFU INC. | PEN Bright | Dental light curing unit |
Puller | SUTTER instaument | P-97 | Puller |
Stereotaxic Instrument (for Mice) | NARISHIGE | SR-6M-H | Stereotaxic instrument |
Stereotaxic Micromanipulator | NARISHIGE | SM-15R | Stereotaxic micromanipulator |
Super-Bond CATALYST V | SUN MEDICAL | 8070 | Dental adhesive resin cement |
Super-Bond Dental Adhesive Monomer | SUN MEDICAL | 8071 | Dental adhesive monomer |
Super-Bond Teeth Color Polymer Powder | SUN MEDICAL | 145052000 | Teeth color polymer powder |
Tarivid Ophthalmic Ointment 0.3% | Santen Pharmaceutical | TRN3952 | Eye ointment |
UlTIMATE XL | NSK | Y141446 | Dental laboratory micromotor control unit |
UV Curing Optical Adhesives | THORLABS | NOA61 | UV Curing Optical Adhesives |
Xylazine | Bayer | KP0F2BK | Anesthetics |