The thesis is from the College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China
Abstract The fluorescence microscopy and fluorescence spectrometer coupling system can obtain the information of microscopic fluorescence imaging, micro-regional fluorescence spectrum and fluorescence lifetime measurement. It is widely used in the structural and functional analysis of proteins in cells and tissues, nucleic acid recognition and detection, metal ions and free radicals. Quantitative determination, as well as the hotspots of bioanalytical research such as the development of nanobioprobes.
1 Introduction <br> Fluorescence microscopy has been widely used in biology and medicine, and is a powerful tool for observing cell morphology, structure and life phenomena. As an indispensable analytical method, fluorescence microscopy is often used to qualitatively observe the spatial distribution and intensity distribution of fluorescent substances inside cells, obtain fluorescence images of cells, and study the structure of cells. However, the fluorescence microscope cannot quantitatively give the intensity value of the image luminescence, and it is impossible to study the physiological process in which the luminescence intensity of the cell image does not change much or the spatial distribution of the fluorescent substance changes slightly. Therefore, with the rapid development of biomedicine, there is an urgent need for instruments with higher sensitivity, faster operation, and more complete functions to meet the requirements of biological and medical research and development. A system instrument coupled with a fluorescence microscope and a fluorescence spectrometer can acquire microscopic fluorescence imaging and measurement information of fluorescence intensity and fluorescence lifetime of the micro region. The fluorescent probe is selected to specifically label the label, and the brightness of the fluorescent probe, that is, the fluorescence intensity, reflects the relative content of the label. After the fluorescence microscope is used for image acquisition, the fluorescence spectrometer can measure the fluorescence intensity of the image, quantify the fluorescence intensity of the acquired image, and provide more detailed data information for comprehensive analysis and study of the internal structure of the cell.
2 Fluorescence microscope and fluorescence spectrometer coupling system and characteristics <br>Application of single-arm fiber-optic connection fluorescence microscope and fluorescence spectrometer to achieve simultaneous measurement of fluorescence spectrum measurement and real-time imaging of two-dimensional signals. A high-frequency pulsed laser is used as an excitation source to excite the sample under the microscope to emit a wavelength in the visible range, presenting a fluorescent image. The signal was collected using an objective lens of a fluorescence microscope, and the coupled fluorescent signal was transmitted from a fluorescence microscope to a fluorescence spectrometer through a single-arm fiber to record a fluorescence emission spectrum. Inductively coupled device (CCD) camera system is used as a photoelectric sensor for microscopic image acquisition technology to collect cell images, and image signals are converted into electrical signals and input into a computer for data reading and image processing to obtain micro-area fluorescence spectrum scanning spectra. The detailed information of the figure, and the image acquisition, exposure adjustment, image editing in multi-color space, pre-defined image setting, auto-focusing, automatic post-processing processing adjustment, to obtain vivid, high-definition images. Using fiber-coupled fluorescence spectrometer and fluorescence microscope, combined with fluorescence image scanning microscopic fluorescence spectroscopy and determination of micro-area fluorescence lifetime (ultra-high time resolution, the fluorescence lifetime of different components in the composite can be obtained), optimized and upgraded as a single The function of the instrument. Red-sensitive photomultiplier tube (PMT) and infrared PMT switch for ultra-wide spectral range detection (200 ~ 1700 nm), suitable for the study of fluorescence spectra from ultraviolet to near-infrared, for the development of near-infrared fluorescent probes in biological systems Applied research provides strong support.
3 Applications in Bioanalysis <br>Fluorescent probes are fluorophores designed to localize or respond to specific stimuli in specific areas of biological specimens. They can detect complex biomolecules and specific cells in living cells with high sensitivity and selectivity. ingredient. Compared with conventional fluorescence detection, in the near-infrared region, the light absorption or fluorescence intensity of the biomatrix is ​​small, and the light scattering of the dense medium (such as tissue) is significantly reduced, and the penetration of the excitation light is stronger, thus autofluorescence. Background interference is significantly reduced. Due to its special photophysical and photochemical properties, fluorescent probes have the advantages of high sensitivity and wide dynamic response range, and their measurement conditions are suitable for the physiological environment of living organisms and are widely used in the field of life science research. The NIR fluorescent probe is designed to be introduced into tissues and cells, and is enriched in specific components of tissues and cells. Under the fluorescence microscope, not only fixed cells and tissue sections can be observed, but also the structure of living cells and biological macromolecules can be observed in real time. Observation and detection, combined with fluorescence spectrometer to obtain quantitative information of fluorescent images. Therefore, fluorescence microscopy and fluorescence spectrometer coupling systems will have broad application prospects in the field of bioanalysis.
Protein structure and function analysis and nucleic acid recognition detection
The physiological state of cells in living organisms is influenced by internal and external factors and can be expressed by gene expression and subsequent protein expression. Therefore, the study of the structure and function of proteins is the key to understanding the life process. Microscopic fluorescence imaging analysis of proteins in living cells is an important aspect of biomicroscopy. Dawn et al. successfully designed and synthesized a series of fluorescent molecular probes in the living cells with a N-terminal cysteine ​​protein. The fluorescent molecular probe utilizes a small molecule containing a thioester under physiological conditions to efficiently cross the cell membrane and chemically react with a protein having a cysteine ​​at the N-terminus to generate fluorescence, and to track and detect the function and interaction of the protein. Fluorescence microscopy was used to observe the microscopic fluorescence imaging of the labeled cells. The emission wavelength of the probe was determined by fluorescence spectroscopy in the near-infrared region, which is suitable for fluorescence imaging analysis of proteins in living cells.
Life activities are inseparable from the catalytic action of enzymes, and the metabolism of substances in the body is carried out in an orderly manner under the catalysis of enzymes. The enzyme is synthesized by living cells and is a protein capable of efficiently catalyzing a specific substrate. Fluorescence-based enzyme assays are widely used with the development of enzyme-based fluorescent probes, which are commonly used to detect the role of luciferase cytochemistry. In order to study the physiological processes in certain cells, fluorescence resonance energy transfer (FRET) is often combined with fluorescence microscopy. Mupam et al. used fluorescence microscopy, CCD, and fluorescence spectrometry to localize FRET in intact cells, and improved intracellular physicochemical reactions to the level of organelles. The fluorescent probe designed by Chi-wang et al. using FRET working principle includes a substrate domain specifically recognizing a known protein kinase, and a phosphorylation recognition domain that binds to a phosphorylated serine substrate structure. When the substrate structure is phosphorylated, the internal folding of the phosphorylation recognition domain and its binding occurs inside the molecule, and energy transfer occurs when the two fluorescent proteins at both ends of the probe are close to each other. Fluorescence spectroscopy provides fluorescence intensity changes before and after phosphorylation of the molecule. FRET imaging obtained by fluorescence microscopy reflects the interaction between donor protein and receptor protein, so that the activity of protein kinase can be observed periodically, quantitatively and locally in living cells.
Nucleic acids are life information materials that play an important role in protein synthesis, cell division and replication, and biological inheritance. Fluorescent probe technology has the advantages of simple method and high sensitivity. It is widely used in the quantitative analysis, structure and mechanism of nucleic acid in combination with fluorescence microscopy. As a sensitive and specific nucleic acid probe, molecular beacons determine the emission fluorescence characteristics through spatial structure change, realize the collection and quantitative detection of nucleic acid localization information in living cells, and use molecular beacon technology to metabolize biological macromolecules in vivo. Dynamic process for tracking analysis. Santangelo et al. designed a pair of novel molecular beacons complementary to the target mRNA sequence to be made into liposomes for ingestion into cells, which produced FRET when hybridized with target mRNA, effectively suppressing active errors compared to single-molecule beacons. Detection of information, sensitive and quantitative detection of target mRNA. Fluorescence microscopy was performed by injecting the living cells by microinjection, and the fluorescence intensity was measured to reflect the metabolic transfer process of living RNA in real time. (For more information, please visit Jingpu's official website)
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