Observation of brain activities in freely moving animals has become an important approach for neuroscientists to understand the correlation between brain function and behavior. We describe an extendable fiber-optic-based multi-modal imaging system that can concurrently carry out laser speckle contrast imaging (LSCI) of blood flow and optical intrinsic signal (OIS) imaging in freely moving animals, and it could be extended to fluorescence imaging. Our imaging system consists of a multi-source illuminator, a fiber multi-channel optical imaging unit, and a head-mounted microscope. The imaging fiber bundle delivers optical images from the head-mounted microscope to the multi-channel optical imaging unit. Illuminating multi-mode fiber bundles transmit light to the head-mounted microscope which has a mass of less than 1.5 g and includes a gradient index lens, giving the animal maximum movement capability. The internal optical components are adjustable, allowing for a change in magnification and field of view. We test the system by observing hemodynamic changes during cortical spreading depression (CSD) in freely moving and anesthetized animals by simultaneous LSCI and dual-wavelength OIS imaging. Hemodynamic parameters were calculated. Significant differences in CSD propagation durations between the two states were observed. Furthermore, it is capable of performing fluorescence imaging to explore animal behavior and the underlying brain functional activity further.
© 2013 OSA
Neuroscientists have made great efforts to understand information processing in the brain by observing brain activity during animal behavior . In recent years, brain functional imaging of awake and freely moving animals has been developed to understand the correlation between behavior and brain activity, providing one of the most representative views of brain functionality. With the development of new microscopy techniques , several miniaturized imaging devices have been used with freely moving animals. For example, neuronal recording in freely moving animals has been achieved through miniaturization of two-photon microscopes [3–7], confocal microscopes and other fluorescence microscopes [8–10] based on fiber-optic endoscopic imaging with high spatial resolution and a small field of view (FOV), which allow researchers to observe local field neuron activities in behaving animals. Voltage-sensitive dye imaging has also been used to observe barrel cortex activity in freely moving animals through fiber-optic endoscopy . Laser speckle contrast imaging, employing a miniature complementary metal-oxide semiconductor camera, has also been used with freely moving animals .
These techniques facilitate different approaches for neural observation and recording in freely moving animals, providing researchers with new insights into brain functionality . However, thus far, no system has been able to provide multiple observations of brain activity in freely moving animal with high resolution and a wide FOV, which is important for neuroscientists to understand the behavior and the underlying dynamic representation of neural activity. In this study, we achieve a fiber-optic-based multi-modal imaging system with high resolution, a wide FOV, and an extendable multi-channel image acquisition framework, providing laser speckle contrast imaging (LSCI) and multi-wavelength optical intrinsic signal (OIS) imaging concurrently during self-determined animal behavior; this system can be extended to fluorescence imaging.
Functional imaging, including LSCI, OIS, and wide-FOV fluorescence imaging, has played an important role in the study of neurovascular coupling [13, 14], pathology , brain functional circuits and connectivities , and cortical column functions [17–19]. LSCI has increasingly been used in recent cortical hemodynamic studies because it can provide high-resolution blood-flow images over the entire field [20–22]. By measuring cortical reflectance changes at different wavelengths, OIS imaging  can provide multiple signals of the cortex during neural activities with second temporal resolution and micron spatial resolution . These functional imaging methods have recently been combined into multi-modal imaging by various techniques [25–28], providing numerous insights into the neurovascular coupling of cerebral cortex [27, 29]. Obviously, it is significant to conduct multi-modal brain functional imaging in freely moving animals to investigate neurovascular coupling and cortical column functions.
We developed a multi-modal imaging system with a fiber-optic-based microscope that can be mounted on an animal's head to perform stable image recording during movement. A gradient index (GRIN) objective lens is equipped inside the head-mounted microscope that is less than 1.5 g, giving the animal maximum capability for free movement. The optical components of the head-mounted microscope are flexibly configured for high spatial resolution, different FOVs, and acceptable spatial resolution at different observation levels. The liquid crystal tunable filter (LCTF) and filter components form the extendable framework for different kinds of functional imaging. The fiber-bundle-coupled multi-source illuminator provides light sources for different functional imaging.
We tested the spatial resolution and proved the stability of the imaging system during freely moving state. We demonstrated the system by monitoring the hemodynamic responses during KCl-induced cortical spreading depression (CSD) in freely moving and anesthetized rats using LSCI and OIS imaging. CSD is considered to be an important disease modal for migraine pathogenesis , which consists of a propagating wave of neuronal depolarization that spreads slowly across regions of gray matter and increases energy metabolism . Hemodynamic changes during CSD propagations between freely moving and anesthetized animals were compared: oxy-hemoglobin (HbO), deoxy-hemoglobin (HbR), and total hemoglobin (HbT) were calculated from dual-wavelength  OIS at 550 and 625 nm, and cerebral blood flow (CBF) was visualized by LSCI. We found a significant difference in CSD duration between freely moving and anesthetized subjects.
2. Materials and methods
2.1 Multi-modal imaging setup and fiber-optic-based microscope design
The scheme of the imaging system is shown in Fig. 1(a) . The entire system consists of three parts: a fiber-coupled multi-source illuminator, a multi-channel optical imaging unit, and a head-mounted microscope. The light sources of the illuminator consist of a laser diode (660 nm, 30 mW) and a high-power white light-emitting diode (LED) (5 W, CREE, U.S.), with both of beams shaped to a small divergence angle by a secondary optical design lens. After passing through a short-pass filter (650 nm, FF01-650/SP-25, Semrock, U.S.) the LED beam is mixed with the 660-nm laser beam by a dichroic beamsplitter (640-nm edge, FF640-Fdi01-25 × 36, Semrock, U.S.). Then, both beams are focused into the 1.5-m-long illuminating fiber bundle (multi-mode, 12-µm core, 0.8-mm diameter, Chunhui, China) through an aspherical focal lens (f = 90 mm). The filter components inside the illuminator are changeable to provide different illuminating wavelengths for different functional imaging. The mixed light transmits through the illuminating fiber bundle and the head-mounted microscope and irradiates the cortex at a 60° incident angle at the end, as shown in Fig. 1(b). The cortex is imaged at the far-end of the imaging fiber bundle (13-µm core, 2-mm diameter, Chunhui, China) by a GRIN objective lens, as shown in Fig. 1(c). The near-end surface of the imaging fiber bundle is located at the focal plane of the microscope objective.
Optical signals are separated into two parts by a dichroic beam-splitter (640-nm edge, FF640-Fdi01-25 × 36, Semrock, U.S.). The first charge-coupled device camera (CCD1, 480 × 640 pixels, PCO Computer Optics, Germany) is used to record laser speckle images through a 660-nm laser filter and tube lens. The dual-wavelength OISs are recorded by the second camera (CCD2) through the LCTF (VariSpec, Cambridge Research and Instrumentation, US) which sequentially select the wavelengths for the OISs at 550 and 625 nm. Transmission of the LCTF is 40% at 550-nm work mode and is 35% at 625-nm work mode. In both conditions the out-of-band transmittance is 0.01%. The LCTF is driven to alternate the wavelength between 550 and 625 nm in synchronization with CCD2 during each acquisition for OIS imaging. The maximal sampling rate of CCD2 is 20Hz which is limited by the 50-ms response time of the LCTF. The sampling rate of CCD1 can reach 50Hz.
The detailed design of the head-mounted microscope is shown in Fig. 1(b) with a 2-mm scale bar. It consists of imaging components and a base. The base holding the illuminating fiber bundles is placed on the craniotomy window and fixed to the skull with dental cement. The GRIN lens is held by the front-end of the post and is fixed with silicon glue. The imaging fiber bundle is inserted into the back-end of the post and is firmly fixed by a set screw. The set screw can be loosened and the imaging fiber bundle can be held by a precise motion stage and moved to a certain distance from the GRIN lens. Therefore, the image distance can be adjusted by changing the position of the imaging fiber bundle to vary the magnification and field of view. The post is then screwed into the base to be adjusted to a suitable object distance and is fixed firmly by a screw. Such fixations can ensure the stability of the head-mounted microscope during the animals’ movements.
The optical scheme of the head-mounted microscope is shown in Fig. 1(c). The GRIN lens has a pitch length of 0.25 and a numerical aperture (NA) of 0.6. The object distance and the image distance are indicated in Fig. 1(c). In experiment of hemoglobin measurements during CSD, the image distance is set to an optical magnification of approximated 2.0 × . The system resolution is around 26µm due to the 13-µm fiber-core which formed the imaging fiber bundle. The base is filled with artificial cerebral spinal fluid (ACSF; composition in mmol/L, NaCl 125, KCl 3, MgCl2 0.6, CaCl2 1.25, NaHCO3 25, and urea 6) to increase the system NA. The cortex is projected by the GRIN lens onto the far-end of the imaging fiber bundle. We can also achieve different magnifications and resolutions by adjusting the image distance as mentioned above.
2.2 Laser speckle contrast analysis
In this study, an imaging fiber bundle was used to transmit laser speckle images [32, 33]. Therefore, the effects of nonuniform intensity distribution caused by the grid structure of the fiber bundle should not be ignored. To reduce this effect, we used a preprocess normalization method before processing the data for laser speckle contrast analysis, as was demonstrated in a previous study . The normalization preprocess is performed as Eq. (1) shown:35], in which the speckle contrast matrix is computed by laser speckle temporal contrast analysis (LSTCA)  from M frames of normalized speckle images and is subsequently spatially averaged by a sliding Ns × Ns window. This method obtained an acceptable signal-to-noise ratio by using fewer frames than that used by LSTCA. In our study, the normalization frame length N and the temporal contrast analysis frame length M are both set to 10 and the spatial window Ns × Ns is set to 3 × 3, and all procedures were carried out in parallel in the GPU to obtain online blood flow images.
2.3 Hemoglobin measurements
To calculate the changes in hemoglobin concentration, we used dual-wavelength OIS imaging at 550 and 625 nm. The basic formula according to the Lambert-Beer law  is shown as follows:Eq. (2) R0 is the reflectance of the light measured by the CCD camera at the baseline moment and Rt is the reflectance at time t. D(λ) is the differential path length factor that can be determined by Monte Carlo simulation . The factors εHbO(λ) and εHbR(λ) are the molar extinction coefficients at the wavelength λ of HbO and HbR, respectively. We assumed the baseline concentrations of HbO and HbR to be 60 µM and 40 µM, respectively, according to previous studies .
2.4 Animals used and head-mounted microscope installation
All the animal protocols were approved by the Care and Use of Laboratory Animals Committee of the Huazhong University of Science and Technology. Adult male Wistar rats weighing 220 ± 20 g were used in the study. The rats were anesthetized with isoflurane (1.5%) and were placed in a stereotaxic apparatus during surgery. The animal body temperature was maintained at 37 ± 0.5°C using a rectal probe and feedback-controlled heating blanket throughout anesthesia.
The skull of the rat was exposed and cleaned, a craniotomy window (4 mm dia.) and a smaller craniotomy window (1mm dia.) were opened at the location shown in Fig. 2(a) , and the dura was kept. Silicon glue was very carefully daubed around the edge of the craniotomy window, and the base plate was then placed above this area and attached to the skull. Dental cement was used to strengthen the base plate and the skull. The base was filled with ACSF, and the imaging post was then attached to the base and fixed by the screw.
After the microscope was firmly mounted on the skull, images could be acquired by the CCD cameras. Figure 2(b) and Fig. 2(c) show the 550 and 625 nm OISs. The image of the blood flow is shown in Fig. 2(d).
2.5 Experimental procedures and data analysis
The adult male Wistar rats were anesthetized with isoflurane (1.5%) during the surgery and installation of the head-mounted microscope. Then, we tested the images to insure the imaging components were securely fixed and accurately focused on the cortex. During the measurement the CSD induction window was exposed. By using a micro-injector we dropped 1µl KCl (0.5 M) into the induction window to generate only one wave CSD each time. We eliminated the first CSD owing to its different characteristics from the subsequent CSDs  and only recorded the subsequent CSDs. For each rat, CSD was induced in both freely moving and anesthetized states for one time. Considering that the CSD might affect the cerebral metabolism [39, 40], the sequence of CSD inductions under freely moving and anesthetized states was randomized in different rats. The time interval between two CSDs was as long as 1 h to minimize the influence of the previous CSD [41, 42].
The OIS images and blood flow image sampling rate was set to 1 Hz. Raw speckle images were acquired at the rate of 50 Hz with a 10-ms exposure time for each frame. Only 10 of the 50 raw speckle images acquired each second were used to generate a blood flow image. To analyze the temporal evolutions of OISs and blood velocity during CSD, we calculated the average intensity within the regions of interest (ROIs) selected on the cortex for each frame.
The spatial changes of CSD in the OIS images were calculated following Eq. (3):
The spatial changes of CSD in blood flow images were performed as Eq. (4) shown:
3.1 Performances of the head-mounted microscope
The head-mounted microscope was less than 1.5 g, so it could be easily carried by an adult Wistar rat, as shown in Fig. 3(a) . We tested the spatial resolution of the head-mounted microscope under different conditions. A 1951 U.S. Air Force (USAF) resolution test negative board was imaged by the microscope at high magnification as Fig. 3(b) shows, in which the line marked by the arrow is 5.52 µm wide. The system can achieve a spatial resolution of 5.5µm. Figure 3(c) shows the test under a wide FOV. A pattern of 14 line-pairs per millimeter on a positive board was used in the test, and the bar represents 1mm. The grid structure of the imaging fiber bundle is visible. The rat cortex was imaged by the head-mounted microscope shown in Fig. 3(d). The same area was also imaged by an Olympus microscopy with a 0.2-NA objective at the same magnification shown in Fig. 3(e). Two zoomed-in images in Fig. 3 (f) showed ROI1 and ROI2 in Fig. 3(d) and 3(e). Compared with the traditional microscope system, the results indicated that the spatial resolution of the fiber-optic-based imaging system depended on not only the GRIN-lens objective but also the diameter of the cores inside the imaging fiber bundle. The spatial resolution of the entire system equaled to the core diameter of the fiber bundle divided by the magnification of the GRIN lens.
We used a MEMS-accelerometer (ADXL335, Analog Devices, U.S.) which was a small, light, complete 3-axis accelerometer to measure the acceleration of the rat’s head during the image recording. The voltage outputs of the accelerometer were connected to a data acquisition board (PCI-6221, National Instrument, U.S.) and were converted to digital signals to be recorded. We fixed the accelerometer on the rat’s skull beside the head-mounted microscope and achieved simultaneously recording of OIS images and the head acceleration during the resting and freely moving states. In Fig. 4(b) acceleration of the head was shown during resting and freely moving states. The 3-axis outputs were added together to represent the motion in all directions. Six states were pointed out in the acceleration graph. Points of I, II and VI were in resting state while points of III, IV and V were in freely moving state. Two measure points marked by the white arrows shown in Fig. 4(a) were chosen based on the vessel cross-structures and the geometric centers of the image was set as the origin of coordinate. We detected the image motion artifacts which might be caused by the rat’s movement by measuring the relative locations from the measure points to the origin of coordinate. Figure 4(c) showed the detection results of the image motion artifacts. At the time point of IV and V the relative locations from the two measure points to the origin of coordinate in x-axis and y-axis shifted within two pixels away from the location at time point of I. At time point of VI the locations came back. The pixel size of the core in the imaging fiber bundle was between 3 and 4 pixels in these images. The pixel shifts of the images during the freely moving state were within the pixel size of the core in the imaging fiber bundle, which indicated that the pixel shifts caused by the motion could be ignored and the slight image motion artifacts would not affect the results of the signals analysis.
We proved that the movement of the illuminating fiber bundle (12-µm core, 0.8-mm diameter) did not cause artifacts of the CBF relative changes during the rat’s freely moving state. From Fig. 5(a) we didn’t see much difference of the CBF relative changes between the resting state and freely moving state. We replaced the illuminating fiber bundle with an ordinary multi-mode fiber (800µm dia.) for illuminating in another experiment to see whether the relative change of CBF was stable. From Fig. 5(b) we didn’t see abnormal CBF relative changes in resting state. However, in freely moving state large artifacts of CBF relative changes were caused by the fiber motion, which indicated that it was not suitable to use a multi-mode fiber with a big diameter for illumination because the motion of such a fiber could affect the CBF measurement and caused large artifacts in relative changes.
3.2 Dynamic changes in cortical hemodynamics during CSD under freely moving and anesthetized conditions
To validate the system, we observed hemoglobin changes during CSD under freely moving and isoflurane-anesthetized states. Figure 6 shows typical temporal evolutions of CSD-related responses of hemodynamic parameters, which were from single CSD of each state of one rat. The first three graphs represent dual-wavelength OISs and CBF, and the last three graphs represent HbO, HbR, and HbT, which were calculated from OIS at 550 and 625 nm. CSD was induced 200 s after the start of the recording. There were significant differences in the durations of CSD-related CBF and OIS-550 nm response between the anesthetized and freely moving state. The CSD durations under anesthesia were longer, and the durations of changes in HbO and HbT were also longer during anesthesia. However, in the OIS 625-nm signal and HbR, we did not see such difference.
3.3 Spatial patterns of cerebral hemodynamic parameters during CSD under freely moving and anesthetized conditions
Figure 7 shows the spreading patterns of CSD related responses including 550-nm OIS, 625-nm OIS, and CBF under anesthetized and freely moving conditions during the time period marked by the two vertical dotted lines shown in Fig. 6. The upper two rows in Fig. 7 indicate the CSD wavefronts at 550- and 625-nm OISs, which were calculated by the subtraction of the two adjacent images at an interval of 4 s as was mentioned before. The CSD-related response to the 550-nm wavefronts seemed to be more extended under anesthesia than under freely moving, indicating that the CSD duration was longer under anesthesia, while responses at 625 nm did not show such difference. Meanwhile, there was no significant difference in the CSD propagation speed between the freely moving and anesthetized states according to the spreading patterns of OISs. The third row in Fig. 7 shows the relative changes in CBF during CSD in both states. Time intervals in the anesthetized state were longer than that in the freely moving state, which indicated the CSD duration of CBF was longer.
3.4 CSD durations in freely moving and anesthetized states
We induced CSD under both freely moving and anesthesia states in eight rats with random CSD inductions sequences. The duration of each signal during CSD was calculated using half-maximal or half-minimal amplitude. The durations of the eight CSDs in freely moving state and the eight CSDs in anesthetized state from the eight rats were statistically analyzed as Fig. 8 shown. The durations of CBF, 550-nm OIS, HbO, and HbT were significantly longer under anesthesia than under freely moving conditions. There was no significant difference in the durations of 625-nm OIS and HbR between the freely moving and anesthetized states. The CSD duration data are presented as mean ± SD. The 625-nm OIS emphasized the change in HbR, and the 550-nm OIS emphasized HbT. As a result the duration data looked similar between 550-nm OIS and HbT, and 625-nm OIS and HbR. The durations of each group of different signals were analyzed using a t-test to determine significant difference. The groups marked by a * had significant differences (p < 0.05).
We developed an extendable fiber-optic-based multi-modal imaging system and observed cerebral activities in freely moving animals. The image system was proved to be stable during the rat’s movement and was demonstrated to be able to perform OIS imaging and LSCI simultaneously. The system comprises a fiber-bundle-coupled multi-source illuminator, a multi-channel optical imaging unit, and a head-mounted microscope, and is extendable. Compared with the traditional wavelength selecting unit, LCTF provides richer wavelengths for selection and have a very short response time. Cooperating with the multi-source illuminator, it provides us with an extendable framework and can perform multi-wavelength OIS imaging and several kinds of fluorescence imaging for further studies such as voltage-sensitive dyes imaging and calcium imaging. With this framework we can achieve comprehensive observations of the cortex in freely moving animals. Furthermore, the magnification and field of view was adjustable by setting the image distance between the GRIN lens and the imaging fiber bundle. With the adjustable head-mounted microscope, high spatial resolution can be achieved which is significant to cortical functional column observation [17, 19], and a wide FOV is also possible which would allow wide-field brain functional connectivity studies [16, 43] in freely moving animals. By the use of fiber bundle for illumination, the system could avoid the artifacts of CBF relative changes caused by the motion of the fiber during LSCI of blood flow in freely moving animals. Compared with present microscope systems used in freely moving animals, it provides an extendable framework to perform various kinds of functional imaging as well as adjustable observation levels. Although this system cannot achieve such a high spatial resolution as the two-photon microscope, it can bring us new insight into the cortical activation and brain area connections with multiple views in freely moving animals.
Using this system, we recorded multiple signals during CSD under anesthesia and freely moving. These results suggest that anesthesia by isoflurane might somehow induce suppression of CSD. In the typical result shown in Fig. 6 we can see the maximum amplitude peaks are nearly simultaneous, noting that the CSDs are all induced at the same time. Additionally, in Fig. 7 we can see that the positions of the spreading stripes related to the dual-wavelength OIS signals during CSD were similar between the anesthetized and freely moving subjects. Previous studies have shown that different anesthetics (isoflurane, α-chloralose, urethane), do not alter the propagation speed of CSD . It has been shown that there are no changes in the propagation speed between equithesin anesthesia and the awake state in rats . However, Guedes and Barreto found that CSD propagation speed is significantly higher for rats in awake condition than under urethane and chloralose anesthesia . The different results may be related to different anesthetics.
In Fig. 6 we also found that the isoflurane anesthetic at the present concentration did not alter the amplitude of signal changes in CSD propagation compared with the freely moving state. It has been found that volatile anesthetics (halothane, isoflurane, and sevoflurane) at different concentrations (0.5, 1.0, and 2.0 MAC) do not alter the amplitude of a direct current (DC) signal during CSD . Similarly, it has been shown that equithesin anesthesia does not affect the amplitude of a DC signal [44, 47] and CBF  during CSD.
Figure 8 shows the differences in durations of CSD-related OISs, CBF, HbO, HbR, and HbT signals between isoflurane anesthesia and freely moving conditions. There are significant differences in the durations of 550-nm OIS, CBF, HbO, and HbT signals. We used half-maximal or half-minimal to calculate the duration of the signals during CSD. Based on this calculation method we didn’t see significant differences in the durations of HbR signal between anesthetized and freely moving conditions. In the HbR signal graph of Fig. 6 we could see that the half-minimal of CSD propagation wave did not have much difference between the freely moving and anesthetized condition which was not consistent with the results of HbO and HbT signals as Fig. 8 shown. However, in the HbR signal graph of Fig. 6 the full width of the propagation wave in HbR signal was longer in anesthetized state than freely moving state, which was consistent with the difference in HbO and HbT signals as Fig. 6 shown. A previous study  showed that isoflurane anesthesia at a high concentration (2.0 MAC) can significant extend the duration of DC potential more than a low concentration (0.5 MAC and 1.0 MAC). According to previous studies, inhaled anesthetics (halothane, isoflurane and sevoflurane) extend the CSD duration as well as reduce CSD frequency, which indicates that these anesthetics can somehow suppress CSD [38, 46].
Developing an imaging system that can provide simultaneous observation of brain functional activation and animal behavior is important to further understanding of the underlying brain functions. In this study we presented an extendable fiber-optic-based microscope that can provide multi-modal optical imaging in freely moving animals. The imaging resolution of the microscope was tested under different magnifications and FOVs. The extendable framework was capable of supporting fluorescence imaging sufficiently. We test the stability of the imaging system during the animal’s movement. We used the system to monitor the hemodynamic response concurrently during CSD under freely moving and isoflurane anesthetized conditions. HbO, HbR, and HbT were calculated from dual-wavelength OIS imaging at 550 and 625 nm, and CBF was visualized by LSCI. We analyzed the CSD propagation parameters of the two states and found that the CSD propagation duration in the isoflurane-anesthetized state was significantly longer, which suggested that isoflurane anesthetic can suppress CSD by inhibition of the NMDA receptor. Overall, the new imaging system realized stable imaging in freely moving animals and can help us further exploit hemodynamic response under new physiological condition. It will become an extendable and powerful tool for neuroscientists to study the brain function and neural activities further.
This work is supported by Science Fund for Creative Research Group of China (Grant No. 61121004), the National High Technology Research and Development Program of China (Grant No. 2012AA011602), and the National Natural Science Foundation of China (Grant Nos. 30970964, 30800339).
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