Small animal living fluorescence imaging technology has become more and more popular at home and abroad. More and more researchers hope to use this technology to track and observe the growth of tumor cells and the response to drug treatment in living animals for a long time. The distribution and metabolism of fluorescently labeled polypeptides, antibodies, and small molecule drugs in the body were observed.
Compared with traditional techniques, in vivo fluorescence imaging technology does not need to kill animals, and can perform repeated tracking and imaging on the same animal for a long time, which can improve the comparability of data, avoid the influence of individual differences on the test results, and understand the markers. In the distribution and metabolism of animals, avoid many shortcomings of traditional in vitro experimental methods; in particular, the original ecological method can also be used to study the problem, that is, the research object does not need to be labeled first, and then the fluorescent marker is used to study its behavior. The observations are true and reliable.
So how do you choose the most suitable living fluorescence imaging system? This article attempts to analyze from the following points.

1. Selection of Fluorescent Labels There are three main methods for labeling in vivo fluorescence imaging: fluorescent protein labeling, fluorescent dye labeling, and quantum dot labeling. Fluorescent proteins are useful for labeling tumor cells, viruses, genes, and the like. Commonly used are GFP, EGFP, RFP (DsRed) and the like. Fluorescent dye labeling and in vitro labeling methods are the same, commonly used Cy3, Cy5, Cy5.5 and Cy7, can label antibodies, peptides, small molecule drugs. As a new labeling method, quantum dot labeling is 20 times higher than that of organic fluorescent dyes, and its stability is 100 times stronger. It has narrow fluorescence spectrum, high quantum yield, low bleaching, wide excitation spectrum and color. It is adjustable, and has many advantages such as high photochemical stability and not easy to decompose. A quantum dot is a semiconductor nanocrystal that emits fluorescence and has a size below 100 nm. It can withstand repeated excitations without the fluorescence quenching being as easy as an organic fluorescent dye.
However, the tissue penetration of different fluorescence wavelengths is different. As shown in Fig. 1, the transmittance of various wavelengths of light to various organs of mice is significantly increased at wavelengths > 600 nm. As shown in Fig. 2, in the near-infrared range of 650 nm to 900 nm, hemoglobin, fat and water maintain the absorption of light at these wavelengths at a relatively low level. Thus, selection of near-infrared fluorescent labels with excitation and emission spectra between 650 nm and 900 nm (or at least the emission spectrum is located in this interval) is more advantageous for in vivo optical imaging, especially for deep tissue fluorescence imaging. (Recommended literature: Nature Method, 2005, 2: 12 How to choose the right fluorescent protein; Science, 2009, 324: 804 Professor Qian Yongjian's research results - near-infrared fluorescent protein, very suitable for in vivo fluorescence imaging).

2, the choice of live fluorescence imaging CCD Select the appropriate CCD lens, for the visible light imaging in vivo is very important. How to choose the most cost-effective CCD for living fluorescence? The CCD has some important parameters:
1) CCD pixels. The CCD pixel determines the quality of the image to be imaged. The higher the pixel, the better the image quality. Since the fluorescent background light is strong, the generation of non-specific stray light interference is obvious, and a camera equipped with a high resolution CCD is required.
2) Front-illuminated or back-illuminated CCD. In general, back-illuminated CCDs have higher quantum efficiency, but only have obvious advantages in detecting extremely weak light signals (such as in vivo bioluminescence imaging), but there is no essential difference between the high-light detection and the front-illuminated CCD, and even more It is easy to be saturated with light, and its high cost disadvantage makes it not a conventional element of fluorescence detection.
3) CCD temperature. There are two types of refrigeration CCDs: constant low temperature refrigeration CCD and relatively low temperature refrigeration CCD. The constant low temperature refrigeration CCD has a stable background and can be subjected to background subtraction; while the relatively low temperature refrigeration CCD is generally unable to perform effective background subtraction due to background instability. The lower the CCD cooling temperature, the smaller the dark current generated. As shown in Figure 3, when the cooling temperature reaches -29 °C, the dark current generated is as low as 0.03 e/pixel/s. Since the noise generated by the instrument itself is mainly composed of dark current thermal noise and CCD read noise, the current CCD read noise can only be reduced to 2e rms; therefore, the lower temperature CCD can not significantly reduce the background noise, but the cost is Greatly improved.
4) The CCD reads noise and dark current. CCD reading noise and dark current thermal noise are the main factors that cause background noise in imaging systems, but in fluorescence imaging, the most important background noise is from fluorescent background light. The improvement in fluorescence imaging signal-to-noise ratio is mainly dependent on the effective control of fluorescent background light and background subtraction techniques (Fig. 4).

3. Autofluorescence Interference In vivo fluorescence imaging, animal autofluorescence has been plaguing researchers. Before the advent of in vivo imaging systems with multispectral spectroscopy, scientists were forced to adopt various methods to reduce autofluorescence, such as feeding mice with fluorescein-free mouse food and using nude mice. Now, a living imaging system with multi-spectral analysis of excitation light can easily separate the fluorescence signal. As shown in Figure 5, the autofluorescence of food, bladder, hair and skin can be effectively distinguished and stripped. Excitation light multi-spectral analysis can also be used for multiple fluorescent label detection, achieving a multi-labeled mouse, reducing the cost of the experiment, and effectively improving the comparability of the data.

4. Accurate positioning of fluorescent signals

As shown in Figure 6, if the signal and the target are 100% coincident, this is what scientists are pursuing; however, if the signal does not coincide with the target and is mistaken for correct positioning, this is a scientific nightmare. Perhaps, a wrongly positioned signal is worse than no signal!
The versatile in vivo imaging system with both structural imaging (such as X-ray, MRI) and functional imaging functions (such as fluorescence, luminescence, and isotope) allows you to get out of trouble and accurately locate fluorescent signals. As shown in Fig. 7, the X-ray imaging of the mouse can clearly obtain the shape and position of the gastrointestinal tract by gastrointestinal contrast, superimpose the fluorescent signal and the X-ray, and the fluorescence and the gastrointestinal overlap can accurately determine the fluorescence localization in the gastrointestinal tract.

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