>Supercontinuum Laser Applications
>Biofluorescence Imaging Using Supercontinuum Laser
Although multicolor fluorescent labeled biofluorescence imagers using xenon white light as the excitation light source have occupied a large application space in the medical market, the life of xenon light sources is only about 500 hours. The detection depth is usually below 1 cm. In addition, because the optimal excitation wavelengths of the polychromatic fluorophores are different, a broad-spectrum light source with a low power spectral density cannot sufficiently and effectively excite the polychromatic fluorophores.
Super-continuous-spectrum laser (SCL), as a new type of white laser light source, has high repetition frequency, narrow pulse width, and high intensity light source characteristics. It has a greater light source intensity than traditional white light sources, and its working life is greater than 2 × 104 h. Therefore, this paper introduces a biological fluorescence imaging technique using supercontinuum laser.
In order to compare the fluorescence excitation capabilities of xenon white light and supercontinuum laser, a biological fluorescence imaging system based on SCL and xenon dual light sources was rebuilt in the experiment. The principle structure is shown in Figure 1. After the SCL (or xenon lamp) passes the fiber collimator, the light source is switched by jumping the mirror. Excitation light is irradiated on the fluorescence test sample for fluorescence excitation, and the generated fluorescence is detected and analyzed by a small animal fluorescence imaging system (Maestro EX In-Vivo Imaging System, CRI Corporation, USA). Specific process: The fluorescence information emitted by the test sample is collected by the imaging lens. After the fluorescence information is recorded using a bandpass filter and a cooled CCD, it is transmitted to the small animal fluorescence system software for subsequent fluorescence information analysis. The specific principle is shown in the gray block diagram in Figure 1.
Figure 1 Schematic diagram of biofluorescence imaging based on SCL and xenon light sources
Based on the experimental structure shown in Figure 1, a fluorescent standard test card was used to perform the fluorescence excitation experiment of the xenon light source. The xenon excitation light source is filtered to select green light with a spectral range of 503 to 548 nm, the actual excitation power is about 180 mW, and the excitation light power spectral density is 4 mW / nm. The fluorescence scanning range is 500 ~ 700 nm, the scanning step is 10 nm, and the exposure time is 80 ms. The imaging information of the fluorophore in the experimental fluorescent card based on the xenon white light excitation is shown in Figure 2. Fig. 2 (a) is a fluorescence image of an experimental sample. Under the excitation of green light, two circular fluorescent groups distributed in the left and right are excited, and the corresponding fluorescence spectrum curve is shown in Fig. 2 (b). The green line corresponds to the fluorescence spectrum of the left fluorophore with a center wavelength of 580 nm, and the blue line corresponds to the spectrum of the right fluorophore with a center wavelength of 612 nm. Figure 2 (c) is a fluorophore image under a single channel signal.
Figure 2 Green light from a xenon light source excites fluorescence information. (a) Fluorescence image; (b) Spectral curve; (c) Fluorophore
The experimental conditions under SCL excitation are the same as those of the xenon lamp experiment. The output power of the supercontinuum laser is 7.4 W, the spectral range is 480nm ~ 780nm, the actual excitation power injected into the system after filtering is 0.72 mW, and the excitation optical power spectral density is 16 μW / nm. , The exposure time is 5000 ms. The fluorophore information is shown in Figure 3. Figure 3 (a) is the fluorescence image of the experimental sample. It can be seen from the figure that the SCL light source also excites two fluorescent groups distributed left and right. Figure 3 (b) is the corresponding fluorescence spectrum. Figure 3 (c) is a fluorophore diagram under a single-channel signal. The shaded stripes on the figure are caused by the uneven coupling when the supercontinuum laser is spatially coupled into the imaging system.
Figure 3 Green light excitation fluorescence information of SCL light source. (a) Fluorescence image; (b) Spectral curve; (c) Fluorophore
Comparative analysis of fluorescence imaging information under two light sources is now performed. Figure 4 (a) is the comparison of the normalized results of fluorescence spectrum intensities under the excitation of two light sources, and Figure 4 (b) is the direct comparison of the intensities. The square label is the fluorescence spectrum excited by the SCL light source with a power spectral density of 16 μW / nm, and the round label is the fluorescence spectrum excited by a xenon light source with an excitation light power spectral density of 4 mW / nm. The hollow and solid labels correspond to the left and Right fluorophore. It can be seen from Fig. 4 (a) that although the signal backgrounds are slightly different, the fluorescence peak curves excited by the two light sources are completely consistent, and the accuracy of the peak position of the fluorescence spectrum is not affected at all. As can be seen from Figure 4 (b), under the two orders of magnitude difference in the excitation power of the light source, the fluorescence intensity of the excited left group is 2532.9 and 1431.7, and the right group is 439.6 and 207.1, respectively. The fluorescence intensity is doubled. difference.
Figure 4 Comparison of fluorescence spectra excited by xenon lamp and supercontinuum laser
In order to further compare the fluorescence excitation characteristics of the xenon light source and the SCL light source, the results of fluorescence intensity excited by different SCL powers are analyzed, as shown in FIG. 5. The injected power spectral density of SCL is from 2.67 to 15.98 μW / nm. Among them, the minimum excitation power spectral density of the 2.67 μW / nm fluorescence test group to generate fluorescence, and 15.98 μW / nm is the maximum output power spectral density of the SCL light source for experiments. It can be seen from the figure that within this power spectral density range, the central wavelength of the fluorescence spectrum excited by SCL remains stable, and the fluorescence intensity gradually increases.
Figure 5. Comparison of the fluorescence intensity of two fluorophores under different SCL power spectral density excitations. (a) the left group; (b) the right group
The fluorescence intensity at the center wavelength of the fluorescence spectrum in FIG. 5 is analyzed, and the result is shown in FIG. 6 (a). As can be seen from the figure, as the SCL excitation power spectral density increases, the fluorescence intensity of the excited group linearly increases. Therefore, a linear fitting method is used for fitting, and then the fitting result is extrapolated, and the result is shown in FIG. 6 (b). It can be seen from the figure that the left and right fluorescence intensities of a xenon lamp with a 4mW / nm excitation spectral power density are known to be 2532.9 and 439.6, respectively. However, the SCL power spectral density required to excite the same fluorescence intensity requires only 29.33 μW / nm and 37.56 μW / nm.
Fig. 6 Variation of fluorescence intensity with SCL injection power spectral density and extrapolation results
In summary, when the power spectral density of the SCL excitation light source is increased to 37.56 μW / nm, it can excite a xenon light source with a power spectral density of 4 mW / nm, which has the same intensity, the same morphology of the group, and the center wavelength of the spectrum. Consistent fluorescence. The power of the xenon light source is 106.5 times that of the SCL light source. In the above analysis, the exposure time was not calibrated the same, the exposure time for the SCL light source was 5000 ms, and the exposure time for the xenon light source was 80 ms. The exposure time of the SCL light source is 62.5 times that of the xenon light source, so the exposure time needs to be converted. The converted magnification difference is 1.7. It can be concluded that in the same exposure time, the same fluorescence intensity is excited, and the power required by the xenon light source is 1.7 times that of the SCL light source.
Fig. 7 Photon velocity information of the left fluorophore at different SCL powers
In order to further verify the above experimental results, the average photon density flow and the maximum photon density flow of the fluorophore in the experiment were analyzed. Under the pumping power of different SCL light sources, the average photon density flow avg_signal (106cm-2s-1) and the maximum photon density flow max_signal (106cm-2s-1) of the fluorophore are obtained using the left fluorophore as the standard. information. The specific results are shown in FIG. 7. The lower right side is the test shape of the standard fluorescence pattern photon density flow. No. 1 is the left fluorescent group and No. 2 is the right fluorescent group. It can be known from FIG. 7 that the photon density flow signal also increases linearly with the power of the SCL light source. The specific linear fitting formula is shown in FIG. 7.
In the experiment, the left fluorophore (No. 1 in Figure 7) under the excitation of a xenon light source with a power spectral density of 4 mW / nm produced an average photon density flow of 6.20764 × 1010 cm-2s-1, and the maximum photon density flow was 7.62065. × 1010cm-2s-1. The linear fitting extrapolation method is also used for estimation, and the required SCL light source power at the same photon density flow as the xenon light source is calculated. Using the fitting formula in Figure 7, the analysis shows that when the average photon density current is the same, the output power spectral density of the xenon lamp is 4 mW / nm, while the power spectral density required by the SCL light source is 2.24 mW / nm, which is 1.79 times. gap. At the same maximum photon density flow, the required power spectral density of the SCL light source is 2.01 mW / nm, which also has a power gap of 1.99 times. The analysis of the photon density flow information of the fluorescent group excited by the light source further proves the experimental conclusions of this paper: Because SCL has the characteristics of high brightness, high repetition frequency, and high intensity light source, its fluorescence excitation ability is much larger than that of ordinary xenon white light. It is twice as much as a xenon light source.
The use of a supercontinuum laser light source can achieve a fluorescence excitation effect completely consistent with that of a xenon light source. And SCL light source has a longer life and better light source stability. Its light source parameters are continuously and dynamically adjustable, which can meet the different needs of light source parameters in the process of fluorescence imaging research. Good theoretical and experimental basis.
From <Luo Yun, Liang Xiaobao, Li Chao, et al. The effect of supercontinuum laser on the excitation effect of biological fluorescence [J]. Progress in Lasers and Optoelectronics, 2016, 53 (12): 121401.>