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Fluorescent Probe and Laser Scanning Confocal Microscopy Imaging Techniques

Confocal laser scanning microscopy is a powerful tool for in situ observation and analysis in cell biological research involving fluorescent probes. There is a class of dyes in biological stain that can be excited by ultraviolet or blue-violet light and other short-wavelength light and emit fluorescence, which is called fluorescent dyes, also known as fluorescent pigment. Now some applied dyes can also be excited by longer wavelength light and produce longer wavelength fluorescence, these fluorescent dyes are often referred to as fluorescent probes. Fluorescent staining has the most significant advantages of high sensitivity in observation. The concentration of non-fluorescent dye usually needs to be 1 % or higher to make the cells to carry with visible color. Fluorescent dyes, such as fluorescent yellow stained with a concentration of 10-5M, can stimulate visible fluorescence with ultraviolet excitation. Fluorescent dyes can produce a desired dyeing effect with concentration of from 10-4M to 10-5M.

Fluorescent dyes may be divided into three categories according to their chemical reactivity of fluorescent probes: (1) Alkalinity fluorescent dye, which contains alkaline chromophore ionized in acidic solution, with fluorescent color being cationic ions. Acridine dyes such as acridine yellow can stain cells by binding to DNA and RNA, mainly through embedded in the DNA double helix structure. Ethidium bromide stains by embedding in DNA, emitting red fluorescence under irradiation of ultraviolet light. (2) Acid fluorescent dyes that contain acidic chromophore, ionized in alkaline solution, while fluorescent color ions being anions. (3) Neutral fluorescent dye, a compound dye that is a mixture of acidic and alkalinity fluorescent dye.

Depending on different applications, the fluorescent probes can further be divided into many categories, including cell active probes, probe organelles, membrane fluorescence shoving needles, accounting probe, the membrane potential probes, ion probe, pH probe, reactive oxygen probes, immunofluorescence probes, probe caged compound, cytoskeletal protein fluorescence probes. It is essentially important to choose a fluorescent probe according to requirements of analysis and application conditions.

On the other hand, there has been a new nano-material called “quantum dots”, which have gradually become the latest fluorescent probes for their remarkable optical efficiency and exhibited a unique advantage in the field of cellular imaging technology. Study scope of quantum dots involves multiple disciplines. Even its name varies in different field of research, For example, colloid chemists attributed it to the colloidal particles (colloid particle), and material scientist named it nanocrystals (nanocrystal); while the solid-state physicists named it quantum dots due to confinement of electrons in a region of tens nanometer scale. Common semiconductor quantum dots are divided into quantum dots of iv Group (Si, Ge), iii-v Group (lnAs, GaSb), and Group II-VI (ZnTe, CdSe, CdS, Zn0). iv Group and iii-v Group quantum dots are fabricated using photolithography, selective epitaxial growth and self-assembly method. Group ii vi diselenide, e.g. CdSe, has excellent fluorescence properties because of its wide bandgap and direct band gap characteristics. Fluorescent probe label plays an important role in cell microscopic imaging, as such, ii-vi Group quantum dots have attracted wide attention in this field [4, 7, 9, 22, 33, 35, 36, 38, 49, 52, 58].

Group ii-vi quantum dots have remarkable advantages over conventional organic fluorescent dyes on the following aspects: (1) as a multi-electron system, absorption coefficient of the quantum dots is much higher than that of a single molecule, with a magnitude of absorption coefficient reaching 105 L * mol-1 * cm-1 under visible light or ultraviolet light excitation, which makes its fluorescence emission intensity much higher than the organic dye; (2) by changing the material ratio and size of the quantum dot, the fluorescence emission wavelength can cover a wide spectral range from 400 nm to 2 ^m; (3) in contrast to organic dye molecules that have a narrow excitation spectrum, quantum dots have a wide and continuous excitation spectrum and there is a quantum confinement emission peak (the longest wavelength can excite quantum dot to emit fluorescent light), any light whose wavelength is shorter than the quantum confinement peak can efficiently excite the quantum dots, so it is possible to use a single wavelength light source to excite quantum dot with different composition and sizes, making them emit different colors of fluorescence for simultaneous monitoring or distinguishing bio-processes; (4) fluorescence emission peak of quantum dots in an organic dye has a narrow and symmetrical peak, with a half-width of only 1/3 of that of fluorescent dyes, and has no tail in long-wave side; (5) the fluorescence lifetime is longer (about several hundred nanoseconds), fluorescence bleaching rate is only 1/100 of rhodamine 6G (a popular red fluorescent dye), therefore it can be used for a long lifetime fluorescence microscopy experiments with a great potential to substitute conventional organic dyes as a new biological fluorescent probe.

Two papers by Alivisatos and Nie respectively published in the same issue of Science in 1998 debuted the application of quantum dots in the biomedical fields, firstly reporting quantum dots in cell imaging as a fluorescent probes instead of conventional dyes [9]. The usage of quantum dots for highly sensitive cellular imaging has found major advances over the past decade [51]. The improved photostability of quantum dots, for example, allows the acquisition of many consecutive focal-plane images that can be reconstructed into a high-resolution three-dimensional image [59]. Another application that takes advantage of the extraordinary photostability of quantum dot probes is the real-time tracking of molecules and cells over extended periods of time [13]. Antibodies, streptavidin [27], peptides [3], DNA [18], nucleic acid aptamers [16], or small-molecule ligands [33] can be used to target quantum dots to specific proteins on cells. Researchers were able to observe quantum dots in lymph nodes of mice for more than 4 months [6].

Semiconductor quantum dots have also been employed for in vitro imaging of pre-labeled cells. The ability to image single-cell migration in real time is expected to be important to several research areas such as embryogenesis, cancer metastasis, stem cell therapeutics, and lymphocyte immunology.

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