Abstract::Fluorescence microscope and fluorescence spectrometer coupled system can be used to obtain microscopic fluorescence imaging, micro fluorescence spectrum and the measurement information of fluorescence lifetime. It’s widely used in structure & function analysis of cell, tissue protein, recognition & detection of nucleic acids, metal ions, determination of radicals quantitative, as well as some hot fields such as research &development of nano-biological probes.
1. Introduction
Fluorescence microscope has been widely used in the field of biology and medical science, and it is a powerful tool for the observation of cell’s morphology, structure and the life phenomenon. Fluorescence microscope, as an indispensable analytical means, is commonly used in the qualitative observation of spatial distribution and intensity distribution of intracellular fluorescent material. It acquires cell fluorescence image to study the structure of a cell. However, fluorescence microscope is unable to quantitatively offer the value of image luminous intensity and study the physiological process with subtle change of luminous intensity or fluorescent substance’s spatial distribution in cell image. Thus, with the rapid development of biomedicine, there is an urgent need for greater sensitivity, more functional agility and more feature-complete biological and medical equipment to meet the requirements of research & development in biology and medicine research. Fluorescence microscopy and fluorescence spectrometer coupled system can be used to acquire measurement information of fluorescence imaging, micro fluorescence intensity and fluorescence lifetime.When using fluorescent probes for specific markers, the brightness, which is fluorescence intensity, will reflect the relative content of the markers. After acquisition of the fluorescence microscope done to the image, fluorescence spectrometer can be used to measure the fluorescence intensity of the image, quantitatively obtaining the fluorescence intensity and providing more detail data information for comprehensive analysis and research of the inner structure of the cell.
2. Fluorescence microscope & fluorescence spectrometer coupled systems &characteristics
Application of fluorescence microscopy and optical fiber connector single arm fluorescence spectroscopy，to achieve real-time fluorescence imaging measurements and cell simultaneous acquisition of two-dimensional signals. High frequency pulsed laser as the excitation source excitation microscope sample to emit in the visible range of wavelengths, showing the fluorescence image. use the fluorescent microscope objective lens signals to collect, through the single arm optical fiber coupling fluorescence signal from the fluorescence microscope to fluorescence spectrometer, recorded the fluorescence emission spectrum .CCD camera system as photoelectric sensors for image acquisition technology to collect microscopic cell image, and the image signal is converted into an electrical signal input into the computer for data acquisition and image processing, access to micro-fluorescence scan spectra details and images collection, exposure adjustment, multi-color space image editing, predefined image set, auto-focus, after auto-adjusting post-treatment process to get colorful, high definition images. Application of fiber-coupled fluorescence spectroscopy and fluorescence microscope, with fluorescence image scanning microscopy fluorescence spectra and for micro-fluorescence lifetime measurement (high temporal resolution can be obtained complexes in different components of the fluorescence lifetime), upgrading the function as a single instrument .Red sensitive photomultiplier tube (PMT) and infrared PMT switch using meet ultra wide spectral range detection (200 ~ 1700 nm), suitable for UV to the near infrared band Fluorescence Spectra Study on the Characteristics, for the development and application of near-infrared fluorescent probes in biological system research provides strong support
3. The application in biological analysis
Fluorescent probes are designed to be positioned within a specific area of biological samples for specific stimulation or fluorophores.It can be used to analysis the specific contents in complex biological molecules and living cells with high sensitivity and selective detection. Compared with normal fluorescence detection, in the near infrared region, bio-based light-absorbing or fluorescence intensity is small, the dense medium (such as tissue)obviously reduced scattering of light, and the excitation light is more penetrating, and thus the auto-fluorescence background interference is much lower .Due to its special photo physical and photochemical characteristics ,fluorescent probe gets the advantages of high sensitivity, wide dynamic range advantage, and their living conditions are suitable for measuring physiological environment. With all these into consideration, it is widely used in the field of life science research. fluorescence microscope and fluorescence spectrometer coupled system can be used to design near-infrared fluorescent probe into tissues and cells, enriched in specific components of tissues and cells, it is not only possible to observe fixed cells, tissue sections, but also on the structure of living cells, biological macromolecules other real-time observation and testing under the fluorescent microscope, and with the fluorescence spectrometer it can also be possible to obtain quantitative information of the fluorescence image. Therefore, fluorescence microscope and fluorescence spectrometer coupled system have wide application prospect in the field of biological analysis.
Structure &function analysis of protein and identification &detection of nucleic acid
Physiological state of biological cells is influenced by internal and external factors, and the influence can be observed via gene expression and the subsequent protein expression. Therefore, the study of protein structure and function is the key to understanding life processes. The analysis of protein microscopy fluorescence imaging in living cells is an important aspect of biological microscopy techniques. Dawn has successfully designed and synthesized a series of fluorescent molecular probes of whose Site-Directed Label N-terminal is cysteine protein in living cells. Small molecules of sulfurous acid ester can effectively go through the cell membrane and generate chemical reaction selectively with N-terminal material of which are the cysteine protein under physiological conditions.via this way, this kind of fluorescence molecular use fluorescence to track and detect function and interaction of proteins probe. With fluorescent microscope, we observe microscopic fluorescence imaging of cell labeled by the probe, determining that the wavelength radiated by probe is near infrared wave range according to the of fluorescence spectrum, and it is suitable protein fluorescence imaging analysis and test within living cells.
Life can’t go on without the catalysis of enzyme; methodical metabolism within the body undergoes with the catalysis of enzymes. Enzymes are synthesized by living cells, and they are proteins which have efficient catalytic effect on specific substrate. Enzyme analysis method based on fluorescence is widely used due to the development of enzyme fluorescent probe; a fluorescence microscope is often used to detect chemical effect of fluorescence enzyme. In order to study physiological process of certain cell, we often combine fluorescence resonance energy transfer (FRET) with fluorescence microscopy technology. Mupam positioned location of FRET which happened within complete cells through a fluorescent microscope, CCD and fluorescence spectrometer, and elevated the physical and chemical reactions research from the level within cell to the level of organelles. Chi - wang and his fellows use the FRET work principle to design a fluorescent probes, including a known protein kinase domain specificity of recognition of the substrate structure and a combined phosphorylation of serine phosphorylation substrate structure identify structural domain. If the substrate structure is phosphorylated. phosphorylation recognition domain structure will combine with it within molecular and cause internal folding, probe on both ends of two fluorescent protein move close to each other and energy transfer occurs. fluorescence spectrum provide changes of fluorescence intensity molecules before and after the phosphorylation, fluorescence microscope obtain FRET imaging which reflects interaction between protein and the receptor protein, and achieve timing and quantitative , positioning protein kinase activity change observation within live cell.
Nucleic acid contains life information, and plays an important role in protein synthesis, cell division, replication and biological inheritance. Fluorescent probe technique method has the advantages of simplicity, high sensitivity, etc. Combined with fluorescence microscope, it was increasingly widely applied to quantitative analysis, structure and mechanism of action research of nucleic acid. Molecular marker, as a sensitive, specific nucleic acid probe, launches fluorescence of different characteristics via spatial structure change, and realizes positioning information collection and quantitative detection of nucleic acids within the living cells ,thus ,by using molecular marker technology , biological macromolecular in vivo metabolic dynamic process is able to be traced and analyzed.
Santangelo and his fellows design a pair of new molecular marker complemented with the target mRNA sequence to be tested. They make it into liposome and make it absorbed by cells, then new molecular marker will generate FRET when hybridizing with the target mRNA. Compared with the single molecular marker, this new molecular marker will effectively restrain the active detection of error information, sensitively and quantitatively detect target mRNA. We adopt micro injection to living cells and obtain fluorescence microscopy imaging, reflecting transferring
Metabolism process of living RNA in real time with detection of fluorescence intensity. (please visit JPLY for more details)
