In life science, especially neuroscience, the recording of biological dynamic activities
Photonics Research, Volume. 12, Issue 3, 456(2024)
Simultaneous dual-region two-photon imaging of biological dynamics spanning over 9 mm
Biodynamical processes, especially in system biology, that occur far apart in space may be highly correlated. To study such biodynamics, simultaneous imaging over a large span at high spatio-temporal resolutions is highly desired. For example, large-scale recording of neural network activities over various brain regions is indispensable in neuroscience. However, limited by the field-of-view (FoV) of conventional microscopes, simultaneous recording of laterally distant regions at high spatio-temporal resolutions is highly challenging. Here, we propose to extend the distance of simultaneous recording regions with a custom micro-mirror unit, taking advantage of the long working distance of the objective and spatio-temporal multiplexing. We demonstrate simultaneous dual-region two-photon imaging, spanning as large as 9 mm, which is 4 times larger than the nominal FoV of the objective. We verify the system performance in
1. INTRODUCTION
In life science, especially neuroscience, the recording of biological dynamic activities
However, due to the limited field-of-view (FoV) of commercial objectives of high NA, large-scale imaging is still challenging. To simultaneously record neural activities at different brain regions with long lateral separations, multiple mesoscale two-photon microscopes
Alternatively, with an objective of long working distance and high numerical aperture, the deflection of excitation beam after the objective can be used to extend the FoV. By placing micro mirrors after the objective lens, large imaging FoV, i.e., up to more than 6 mm in diameter, has been achieved for long-distance neural activity recording [12]. However, to switch among different imaging regions-of-interest (ROIs), the micro mirrors after the objective lens need to be rotated rapidly, in which mechanical inertia limits the maximal imaging speed.
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Here, to achieve simultaneous dual-region imaging, we take advantage of the long working distance of the objective with custom post-objective optics along with spatio-temporal multiplexing. We use a commercial objective of 8 mm working distance and a custom micro-mirror unit (MMU) to align the excitation beams to two ROIs. The excitation beams with different time delays are reflected by the MMU and focused to different transverse ROIs, while the excited fluorescence signals are detected and temporally demultiplexed. We achieve simultaneous imaging of two ROIs with a lateral interval of as large as 9 mm, which is 4 times larger than the nominal FoV (1.8 mm) of the objective. We verify the system performance by simultaneous dual-region imaging on pathological sections of human squamous epithelium and brain slices of Thy1-YFP mice. Then, we demonstrate the simultaneous dual-region recording of neural activity and cerebral vascular dynamics in mouse brain and spinal cord, respectively,
2. METHODS
A. Microscope System
The system diagram is shown in Fig. 1. We use an 80 MHz femtosecond laser (Chameleon Discovery, Coherent) as the excitation source and a Pockels cell (Pockels cell, 350-80-LA; Controller, 302RM, Conoptics, Inc.) to adjust the laser power injected on the samples. The imaging laser wavelength is fixed at 920 nm. We employ spatiotemporal multiplexing of two beams for dual-region two-photon imaging simultaneously. A halfwave plate (AHWP05M-980, Thorlabs) and a polarization beam splitter (CCM5-PBS202/M, Thorlabs) are placed after Pockels cell to adjust the excitation powers of two excitation beams, in which one undergoes a time delay of 6.25 ns relative to the other [7,9,10,13–18]. Then the two beams are combined by a pickup mirror (PFD10-03-P01, Thorlabs). Even though these two beams have the same propagation direction, they do not overlap in space. Following that, the two beams are expanded by an adjustable beam expander (BE02-05-B, Thorlabs) to match the size of the subsequent scanning galvanometer pair (8315 K & CRS 8 kHz, Cambridge Technology) and relayed to the back focal plane of an objective of 8 mm working distance and 1.8 mm FoV (XLPLN10XSVMP, NA 0.6,
Figure 1.System scheme of simultaneous dual-region imaging microscopy. Two excitation beams with a relative time delay of 6.25 ns are introduced by an optical delay line of
Then, temporal demultiplexing should be performed to split the signals from two regions. The PMT signals corresponding to two ROIs are first amplified by a high-speed current amplifier (DHPCA-100, Femto) and then split into two channels by a power splitter (ZAPD-30-S+, Mini-Circuits) [18]. Then two channels are connected to the high-speed data acquisition card (NI-5734, NI) through two cables of different lengths (
The system is controlled by ScanImage 2018 (scanimage, Vidiro). Taking advantage of spatiotemporal multiplexing, optical switching between the two regions occurs at a nanosecond-level (6.25 ns) speed. This switching speed significantly surpasses the mechanical scanning speed of the galvo scanner. Consequently, with a single complete two-dimensional scan, image acquisitions for both fields are realized. During imaging, the scanning galvanometer pair provides the same optical scanning degree to both excitation beams. Thus, as the PMT detects fluorescence signals from both ROIs, without employing spatiotemporal multiplexing and temporal demultiplexing, fluorescence signals from both fields would overlap. Temporal demultiplexing allows us to differentiate the fluorescence images from the two regions based on distinct arrival times of the fluorescence signal at the detector. Limited by the fluorescence lifetime and the bandwidth of the current amplifier, there will be some residual signal crosstalk between two channels. However, the residual crosstalk is linear and relatively stable. Thus, we perform linear demixing to eliminate this crosstalk [15].
B. Micro Mirror Unit
Drawings and a photo of the MMU are shown in Figs. 2(a) and 2(b). The design of the MMU determines the interval of two simultaneous imaging ROIs and the efficiencies of two-photon excitation and fluorescence collection. The distance between imaging ROIs depends on the interval
Figure 2.Drawings and photos of the MMU, and system resolution calibration. (a) Top and side views of the MMU. (b) Photo of MMU with
However, the system vignetting would affect actual interval of the ROIs. In the dual-region two-photon imaging system, vignetting mainly happens when the edges of the micro-mirror block some of the excitation beam, especially at larger scanning angles. Considering how the excitation beam focuses, vignetting mostly occurs at the first mirror after the excitation beam enters the MMU. Thus, vignetting stays consistent for MMUs of different
In post-objective optics, a part of the long working distance is utilized to ensure large intervals of the ROIs. Considering that the working distance of the objective is 8 mm and the side length of the mirror of the isosceles right triangle shape is 2.5 mm, the maximal effective working distance is related to the interval between the mirrors:
As examples, we design two sets of MMUs with
To facilitate the connection of the MMU and the objective lens, we fabricate an objective lens sleeve by 3D printing. Subsequently, the MMU is affixed to the end of the objective lens sleeve. The modified sleeve, housing the MMU, is then mounted onto the objective lens [Figs. 2(c) and 2(d)]. Thus, the objective lens sleeve does not block the imaging path. The MMU is typically positioned closely to the objective lens to maintain a large working distance with the biological sample. By carefully rotating the sleeve, the excitation beams with different time delays could be directed through the corresponding mirrors, thereby yielding dual-region two photon imaging. We then use the 3D motorized stage (MT3-Z8, Thorlabs) to move the biological samples for imaging at different locations.
To characterize the spatial resolution of the system, we measure the point spread function of our system by imaging 0.51 μm green fluorescent polymer microspheres (Fluoro-Max, diameter 0.51 μm, Thermo Fisher Scientific) buried in 2% agarose gel. Cross sections of green fluorescent polymer microspheres are illustrated in Figs. 2(e) and 2(f). The X, Y, and Z resolutions in full-width-at-half-maximum of two ROIs are calibrated as
C. Surgery
All experimental procedures related to animals are carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee, Tsinghua University.
1. Brain Cortex Surgery
We perform craniotomy on Ai162(TIT2L-GC6s-ICL-tTA2)-D mice (JAX: 031562) for calcium imaging. Craniotomy surgery is performed by removing the skull within range
Prior to cortex surgeries, mice are administered dexamethasone (1 mg/kg) and meloxicam (8 mg/kg) subcutaneously. After the surgery, meloxicam (4 mg/kg per day) is given subcutaneously for 3 consecutive days. Mice are allowed to recover for 4 weeks before calcium imaging experiments. During imaging, the mice are kept in darkness and not disturbed by the external environment.
For acute imaging of vascular dilation in mouse brains
2. Spinal Cord Surgery
The dorsal surface above the thoracic spinal region of the mouse is shaved and washed with iodophor twice to prevent infection. Then we perform laminectomy [23–25] at the T10-T11 level of mouse spinal cords. Anesthesia is initiated with 5% isoflurane before surgery and maintained at 1.5% isoflurane throughout the surgical procedure. Metal bars are employed to clamp three vertebrates on both sides of the imaging area to immobilize the cord. The FITC fluorescent dye is injected into the infraorbital vessels (2% mass-to-volume ratio in saline, 200 mg/kg) for staining the blood plasma.
3. Results
A. Simultaneous Dual-Region Imaging of Mouse Brain Slices
To demonstrate the imaging performance of our system, we first perform simultaneous dual-region imaging of mouse brain slices. We use coronal sections of 50 μm thickness from Thy1-YFP (H line) mouse brains, where neurons are sparsely labeled with yellow fluorescence protein in the cerebral cortex and hippocampus. The schematic diagram of the MMU during imaging is shown in Fig. 3(a). We use a custom wide field fluorescence microscope to capture the whole slide image of brain slices, as shown in Fig. 3(b).
Figure 3.Simultaneous dual-region imaging of a Thy1-YFP mouse brain section and an H&E stained pathological section. (a) 3D model of MMU. (b) Whole slice imaging of the coronal section from Thy1-YFP mouse brain. The interval between two simultaneous imaging ROIs is 7 mm. Scale bar: 1 mm. (c), (d) Two-photon fluorescence imaging results corresponding to the regions in cyan box and pink box, respectively, in (b). Scale bar: 100 μm. (e) Whole slide bright field image of the pathological slice (thickness: 3 μm). The interval between two simultaneous imaging ROIs is 9 mm. Scale bar: 3 mm. (f), (g) Zoom-in views of the regions in the cyan box and red box, respectively, in (e). Scale bar: 100 μm. (h), (i) Two-photon fluorescence imaging results corresponding to the regions in the cyan box and red box, respectively, in (e). Scale bar: 100 μm.
In Figs. 3(c) and 3(d), we demonstrate simultaneous dual-region two-photon imaging of a mouse brain slice at the positions indicated by the cyan and pink boxes, respectively, in Fig. 3(b). In this experiment, we place a
B. Simultaneous Dual-Region Imaging of H&E Stained Pathological Sections
To further demonstrate the potentials of our method, we perform simultaneous dual-region imaging of H&E stained human squamous epithelium pathological samples, with a larger interval between ROIs [Fig. 3(e)]. Here we use an MMU with
The zoom-in views of the histological section image in Fig. 3(e) are recorded in bright field mode and our two-photon autofluorescence imaging mode, as shown in Figs. 3(f) and 3(g) and Figs. 3(h) and 3(i), corresponding to the regions in the cyan and red boxes, respectively. The bright field images are well-matched with the images captured by our system, which confirms the ability of our system in cross-region imaging and image demodulation.
C. Simultaneous Dual-Region Imaging of Neuronal Activity in Mouse Cortex
We then perform
Figure 4.Simultaneous dual-region imaging of neuronal activities in mouse cortex
In this experiment, we use the MMU of
D. Simultaneous Dual-Region Imaging of Neuronal Activity in Mouse Cortex
We also perform dual-region dynamic imaging of vascular dilation in mouse brains
Figure 5.Simultaneous dual-region imaging of FITC-labeled vessels in mouse brain and in mouse spinal cord
We then use the dual-region two photon microscope to monitor vascular dynamics in mouse brains
E. Simultaneous Dual-Region Imaging of Vascular Dilation in Mouse Spinal Cord
The spinal cord relays sensory information from the body to the brain and sends motor output back to the body’s muscles and glands. Deeper understanding of the spinal cord and its functions is essential for developing new treatments for neurological disorders. Here we demonstrate simultaneous dual-region imaging of vascular dilation on mouse spinal cord
4. DISCUSSION AND CONCLUSION
We propose a simultaneous dual-region, two-photon imaging system based on custom MMUs and spatio-temporal multiplexing. We split the imaging beam and introduce a time delay to one relative to the other. These two beams are relayed to different positions of the objective back pupil and go through different paths in MMUs. The excited fluorescence signals are collected by MMUs and the objective, and are detected by a PMT for temporal demultiplexing with synchronization signals from the laser clock.
We show simultaneous dual-region imaging
Instead of expanding FoVs with custom mesolenses as in two-photon mesoscopes [7–10], our system realizes simultaneous imaging of two regions spanning over large intervals by placing post-objective MMUs. Compared to other reports, the MMU used in our system is of low costs (
Even though the excitation and collection NAs of the imaging system are reduced in the current design, resulting from the underfilling of the objective pupil by the excitation beam and the partial block of collection path by the MMU, the experiment results confirm that our system can still achieve single neuron resolutions while maintaining the optical sectioning capability. To further enhance the collection efficiency of our microscope, a potential approach is positioning customized silicon photomultipliers (SiPMs) beneath the MMU [28].
For the MMU, in order to reduce energy loss and improve photon utilization efficiency, its size should be as large as possible. Besides, in our experiment, the micro-mirror spacing is set to 3 mm or 4 mm, corresponding to the center interval 7 or 9 mm between two regions. A potential improvement is to make
In conclusion, our proposed system enables simultaneous dual-region, two-photon imaging
Acknowledgment
Acknowledgment. The authors thank Yang Lin and Kuikui Fan for help with bio-sample preparation.
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Chi Liu, Cheng Jin, Junhao Deng, Junhao Liang, Licheng Zhang, Lingjie Kong, "Simultaneous dual-region two-photon imaging of biological dynamics spanning over 9 mm
Category: Imaging Systems, Microscopy, and Displays
Received: Sep. 1, 2023
Accepted: Dec. 24, 2023
Published Online: Feb. 23, 2024
The Author Email: Lingjie Kong (konglj@tsinghua.edu.cn)
CSTR:32188.14.PRJ.504895