2.1.

LSCI System with CW and Nanosecond Pulse Laser Sources

The proposed system achieved imaging of blood flow under a combination of the 632.8- and 532-nm cw lasers as well as a 532-nm cw and pulse wave for the first time through optical path design, as shown in Fig. 1. The He–Ne laser (HNL150L-EC, Thorlabs) emits a 632.8-nm cw laser. The nanosecond pulse laser (VGEN-G-20, Spectra Physics) emits a 532-nm nanosecond pulse laser. High-performance green laser (MGL-III-532, CNI, China) emits a 532-nm cw laser. The 632.8-nm cw and 532.8-nm cw/nanosecond pulse lasers are controlled by shutters and reflected by mirrors and then pass through the dichroic mirror into the optical path system. The light then passes through an attenuator and is irradiated onto a beam expander (GBE15-A, Thorlabs), after which it reaches the brain window of the mouse. The brain window is a square of $6\times 6{\mathrm{mm}}^2$ in size, and the spot through beam expanding needs to meet the actual experimental requirements.

Fig. 1

Schematic of the LSCI system with cw and nanosecond pulse laser sources.

2.2.

Experimental Process

Male Institute of Cancer Research mice (25 to 30 g) were purchased from the Animal Experiment Center. All animal experimental procedures were conducted in accordance with the guidelines under Institution Animal Care and Use Committee-approved protocols at Nanjing University of Aeronautics and Astronautics.

The mice were anesthetized with 5% chloral hydrate by intraperitoneal injection at $400\mathrm{mg}/\mathrm{kg}$ (body weight). After the anesthesia was completed, the head and limbs of the mice were fixed on the stereotaxic brain locator in a recumbent position to facilitate craniotomy and reduce the noise caused by movement after the mice recovered. Because of the symmetry of the head of the mice, we drilled a rectangular area on one side (about 1 to 2.5 mm from the front halogen point and 1 to 4 mm from the middle line) to open the upper skull with a dental drill and use tweezers to open the meninges as the imaging area. Light reflected from the mouse is collected by an optical tube (12X Zoom, Navitar), collected by a 12-bit CCD camera (GS3-U3-51S5M-C, PointGrey, Canada), and stored in a computer. Considering the quality of image contrast and the velocity of blood flow, the exposure time of the CCD is chosen as 20 ms, and the blood flow images could be calculated using 30 frames of raw speckle images. Therefore, it would consume at least 600 ms to obtain one frame of blood flow image in each time point.

First, the cerebral vascular image of the mouse under white light was collected by CCD. Then the blood flow image of the mouse under the combination of a 632.8-nm cw laser with the 532-nm cw laser or with the 532-nm nanosecond pulse laser was obtained. After that, experimental data were collected and analyzed. The system achieves the combined measurements of 632.8- and 532-nm lasers blood flow as well as 532-nm cw and nanosecond pulse waves for the first time through optical path design.

2.3.

Speckle Pattern Calculation

When a laser radiates on the cerebral cortex of the mice, the scattered light is collected by a CCD and raw speckle images will be formed. The motion of these scattered particles will lead to blurring in the raw speckle images. The level of blurring is quantified by the speckle contrast.^17,18 This blurring degree is usually quantified by calculating speckle contrast $K$ defined as

2.4.

SNR Calculation

When the experiment is completed, the signal-to-noise ratio (SNR) of the blood flow image obtained by the system is calculated, and the quality of the blood flow images under 532-nm cw and 532-nm nanosecond pulse lasers are compared. The method of calculating the SNR is: (1) select the region of interest (ROI) in the blood flow image and (2) obtain the mean and standard deviation of the ROI. The ratio of the mean and standard values is regarded as the SNR.

3.1.

Regional Cerebral Blood Flow Image with Different Wavelength CW Laser Source

As shown in Fig. 2(a), the brain blood vessels of the mouse can be clearly seen under the white light. Under the irradiation of 632.8 nm cw laser, rCBF image of the thick blood vessels located at the surface of the mouse brain was shown in Fig. 2(b). However, Fig. 2(c) showed clear branches of the mouse brain and the trunk was clear. The blood vessels were not observable. At the same time, by comparing the images under white light, it was found that the 532-nm nanosecond pulse laser showed a good blood flow image on the blood vessels of the mouse brain at deeper depth. This experimental result obtained the brain blood flow images of mice at different wavelength lasers. According to the imaging characteristics of different wavelength lasers, an image fusion algorithm can be used to fuse the two images to obtain more abundant blood flow information.

Fig. 2

rCBF image with different wavelength cw laser sources: (a) white-light photo of blood vessels in the mouse brain, (b) rCBF image with 632.8-nm cw laser source, and (c) rCBF image with 532-nm cw laser source.

3.2.

Regional Cerebral Blood Flow Image with 532-nm Laser Source of Different Types

Figure 3(c) shows the blood flow of equal thickness vessels in the main cerebral vessels of mice. Figure 3(a) shows the clear branches of the mouse brain and the clear trunk. In Figs. 3(b) and 3(d), the corresponding raw speckle images used to compute rCBF are provided. Comparing images of two modalities of the 532-nm laser, we found that the 532-nm nanosecond pulse laser exhibits blood flow change with higher resolution at a pulse width of 3 ns, capturing more effective blood vessel detailed information than the 532-nm cw laser (refer to Fig. 7 of the SNR results). The 532-nm cw laser showed more noise in the deep blood vessels of mouse brain. The research based on this image can only be carried out after denoising. According to the imaging characteristics of different types of laser sources, using an image fusion algorithm to fuse the two images will overcome their shortcomings.

Fig. 3

rCBF image with 532-nm laser source of different types: (a) cw laser source, (b) raw speckle images used to compute (a), (c) nanosecond pulse laser source (3 ns and 1200 kHz), and (d) raw speckle images used to compute (c).

3.3.

Regional Cerebral Blood Flow Images of Different Repetition Rates with 532-nm Nanosecond Pulse Laser Source

The experiment of rCBF imaging in mice with 532-nm cw and 532-nm nanosecond pulse laser acting alone was carried out. The main parameters of 532-nm nanosecond pulse wavelength laser are pulse width and repetition frequency. The effect of the experiment is more obvious. The pulse width is set to 3 ns. Figure 4 shows a pseudocolor image of rCBF in the mouse under the action of 1200, 800, 300, 100, and 10 kHz repetition frequencies, respectively. At the same time, we performed blood flow imaging experiments with various pulse widths of 5, 7, 10, 15, and 20 ns, respectively. The experimental results showed that the information of rCBF in mice can be obtained effectively using a nanosecond pulse laser, which reflects the development potential of the PAI system in blood flow imaging.

Fig. 4

rCBF image of pulse width 3 ns with different repetition rates with 532-nm nanosecond pulse laser source: (a) 1200 kHz, (b) 800 kHz, (c) 300 kHz, (d) 100 kHz, and (e) 10 kHz.

3.4.

Regional Cerebral Blood Flow Image with 532-nm Nanosecond Pulse Laser Source of Different Pulse Widths

At the same time, we kept the repetition frequency of the nanosecond pulse laser unchanged and obtained the experimental results of Figs. 5 and 6 by changing the pulse width. The results showed that the change of pulse width had little effect (it can be ignored) on the experimental results of rCBF imaging in mice, and all the results showed similar information of blood vessel and blood flow. After calculating the SNR (refer to Fig. 8), it is found that all the images have the same magnitude range. The experimental results in Figs. 4Fig. 5–6 further showed that the repetition frequency and pulse width of the nanosecond pulse laser have little effect on blood flow imaging in mice.

Fig. 5

rCBF images of 100 kHz with different pulse widths: (a) 3 ns, (b) 5 ns, (c) 7 ns, and (d) 10 ns.

Fig. 6

rCBF images of 3 kHz with different pulse widths: (a) 5 ns, (b) 7 ns, (d) 10 ns, and (f) 20 ns.

3.5.

Calculation Results of Signal-to-Noise Ratio

The SNRs of the ROIs of rCBF [see the box lines in Figs. 3(a) and 4] are calculated and shown in Fig. 7. The SNR of the 532-nm cw ($\mathrm{SNR}=2.04$) ROI is significantly smaller than that of 532-nm pulse laser ($\mathrm{SNR}=2.36$, repetition rate of 100 kHz, and pulse width of 3 ns), which demonstrates that the 532-nm nanosecond pulse laser can present a better imaging effect. When the pulse width is 3 ns, the SNR is highest at the repetition frequency of 300 kHz, which indicates that the blood flow imaging is sharper and the noise is less.

Fig. 7

The SNR with 532-nm cw (1) in the ROI and pulse width of 3 ns with a repetition rate of 1200 kHz (2), 800 kHz (3), 300 kHz (4), 100 kHz (5), and 10 kHz (6).

The ROI in Fig. 3 is selected for the same processing as above, and the resulting SNR is shown in Fig. 8. Under the same repetition frequency, differences in SNR among the images with various pulse widths are very small (SNR is maintained between 2.3 and 2.4), which indicates that different repetition frequencies and pulse widths have little effect on the experimental results of laser speckle blood flow imaging.

Fig. 8

The SNR with the repetition rate of 100 kHz and pulse width of 3 ns (1), 7 ns (2), 10 ns (3), and with a repetition rate of 3 kHz and the pulse width of 7 ns (4), 10 ns (5), and 20 ns (6), respectively.