The research on improving the resolution of optical microscope mainly focuses on two aspects. One is to improve the spatial resolution under various conditions by using classical methods, such as SPIM technology for thick sample research, SHG technology for rapid measurement and MPM technology for living cell research. The second is to combine the latest nonlinear technology with high numerical aperture measurement technology (such as STED and SSIM technology). Biological science research can not be separated from the support of ultra-high resolution microscopy, and people urgently need to update microscopy to meet the requirements of the development of the times. Recent studies show that the resolution of optical microscope has successfully broken through the diffraction limit of 200nm transverse resolution and 400nm axial resolution. The development of high-resolution or even ultra-high-resolution optical microscope not only lies in the progress of technology itself, but also will greatly promote the study of biological samples and provide new means for subcellular and molecular research.
Optical microscope; High resolution; Nonlinear technology; Nanometer scale
Microscopy has played an important role in the development of biology, especially some important discoveries in the field of early microscopy, which directly contributed to the breakthrough development of cell biology and related disciplines. The observation of biological structure and process of fixed samples and living samples makes optical microscope an essential instrument for most life science research. With the development of life science research from the whole species to the molecular level, the spatial resolution of microscope and its ability to distinguish fine details have become a very key technical problem. The development history of optical microscope is the history that human beings constantly challenge the resolution limit. In the range of 400 ~ 760 nm of visible light, the resolution limit of microscope is about half of the wavelength of light wave, about 200nm, while the limit reached by the latest research results is 20 ~ 30 nm. In this paper, the technical principle of high-resolution optical microscope and the research progress of breaking through the diffraction limit of light in recent years are summarized from two aspects: high-resolution three-dimensional microscopy and high-resolution surface microscopy
1 Resolution of traditional optical microscope
The size of optical microscope image mainly depends on the wavelength of light and the limited size of microscope objective. The brightness distribution of an object similar to a point light source in image space is called the point spread function (PSF) of an optical system. Because of the characteristics of the optical system and the emitted light, the optical microscope is not really a linear translation invariant system. PSF is usually distributed radially symmetrically on the x-y plane perpendicular to the optical axis, but it has obvious expansion along the Z optical axis. According to Rayleigh criterion, the minimum resolvable distance between two points is approximately equal to the width of point spread function.
According to Rayleigh criterion, the resolution limit of traditional optical microscope is expressed by the following formula [1]:
Transverse resolution (x-y plane): dx, y=■
Axial resolution (along Z axis): dz=■
It can be seen that the improvement of the resolution of optical microscope is restricted by the wavelength λ of light wave and the numerical aperture N.A of microscope. The narrower the PSF, the higher the resolution of the optical imaging system. There are two ways to improve the resolution: (1) choose a shorter wavelength; (2) In order to improve the numerical aperture, materials with high refractive index are used.
Rayleigh criterion is based on the assumption of propagating waves. If the non-radiation field can be detected, it is possible to break through the limitation of Rayleigh criterion on diffraction barrier.
High resolution three-dimensional microscope
In the study of improving the resolution of optical microscope, it is of great significance to correct the aberration and chromatic aberration of microscope objective. The imaging quality of optical microscope has been improved obviously from the general lens combination to the use of diaphragm to limit non-paraxial light, from stable achromatic to hyperchromatic to hyperchromatic. Recently, Kam et al. [2] and Booth et al. [3] applied the principle of adaptive optics to carry out the related research on aberration correction of microscope. Adaptive optical system consists of wavefront sensor, anamorphic lens, computer, control hardware and special software, which is used to continuously measure the aberration of microscope system and automatically correct it. Generally speaking, the existing high-resolution three-dimensional microscopy can be divided into three categories: * * focusing and deconvolution microscopy, interference imaging microscopy and nonlinear microscopy.
2. 1 *** focusing microscopy and deconvolution microscopy The mature methods to solve the microscopic imaging of thick biological samples are * * * focusing microscopy [4] and three-dimensional deconvolution microscopy (3-DDM) [5], both of which can be used without preparing physical sections of samples.
The main feature of the * * focusing microscope is to remove the fluorescence generated by the non-* * focal plane fluorescent target by using the detection pinhole, thus improving the image contrast. * * * The PSF of the focusing microscope is square with that of the conventional microscope, and the resolution is improved by about ■ times. In order to obtain satisfactory images, three-dimensional focusing technology often needs to use high-intensity excitation light, which leads to dye bleaching and phototoxicity to living biological samples. In addition, the structure is complex and expensive, which limits the application to some extent.
3-DDM uses software to process the whole optical slice sequence. Compared with * * * focusing microscope, this technique uses low intensity excitation light, which reduces photobleaching and phototoxicity, and is suitable for studying living biological samples for a long time. Using scientifically cooled CCD sensor to detect photons in focal plane and near defocus plane at the same time has wide dynamic range and long exposure time, which improves optical efficiency and image signal-to-noise ratio. 3-DDM expands the application field of traditional wide-field fluorescence microscope and attracts extensive attention in the field of life science [6].
2.2 Selective Planar Illumination Microscopy According to the light absorption and scattering characteristics of large living biological samples, Huisken[7] and others developed the Selective Planar Illumination Microscopy (SPIM). Different from the usual way of cutting and fixing samples on glass slides, SPIM can observe larger living biological samples of 2 ~ 3 mm in an approximate natural state. SPIM forms ultra-thin layer light through cylindrical lens and thin optical window, moves the sample under the illumination of ultra-thin layer to obtain slice images, and can also scan and image the sample at different observation angles through the rotatable stage, thus realizing high-quality three-dimensional image reconstruction. Because of the use of ultra-thin layer light, SPIM reduces the damage of light to living biological samples, and enables the complete samples to continue to survive and grow, which is impossible for other optical microscopes at present. The appearance of SPIM technology provides a suitable microscopic tool for observing the instantaneous biological phenomena of large living samples, which is of special significance for developmental biology research and observing the three-dimensional structure of cells.
2.3 Structured illumination technology and interference imaging When a fluorescence microscope images a thick biological sample with an objective lens with a high numerical aperture, optical sectioning is an ideal method to obtain high-resolution three-dimensional data, including a * * * focusing microscope, a three-dimensional deconvolution microscope and a Nipkov disk microscope. The microscopy based on structural illumination reported by Neil et al. in 1997 is a new technique to realize optical sectioning by using conventional fluorescence microscope, which can obtain the same axial resolution as that of * * * focusing microscope. The application of interference imaging technology in optical microscope 1993 was first put forward by Lanni et al., and it has been further developed with the application of I5M, HELM and 4Pi microscope technologies. Compared with the fluorescence observed by conventional fluorescence microscope, the emitted fluorescence recorded by interference imaging technology carries higher resolution information. (1) structured light technology: It combines specially designed hardware system and software system. The hardware includes the grid structure skateboard and its controller, and the software realizes the control of the hardware system and image calculation. In order to generate optical slices, the original projection images corresponding to different positions of grid lines are collected by CCD. Through software calculation, a clear image without stray fluorescence from the defocusing plane is obtained, and the contrast and clarity of the image are obviously improved. By using the optical tomography technology of structured illumination, the problems of interference of defocusing surface stray light, time-consuming reconstruction and long calculation time in 2D optical tomography and 3D fluorescence imaging are solved. The optical slice thickness of structured illumination technology can reach 0.0 1nm, the axial resolution is twice that of conventional fluorescence microscope, and the 3D imaging speed is three times that of * * * focusing microscope. (2)4Pi microscope: 4Pi microscope based on interference principle is an extension of * * * focusing/two-photon microscope technology. The 4Pi microscope is equipped with 1 confocal objective lenses in front of and behind the specimen, and high-resolution imaging can be obtained in three ways: ① the specimen is irradiated by interference light generated by two wave fronts; (2) the detector detects the interference light generated by two emission wave fronts; (3) illumination and detection wavefront are interference light. 4Pi microscope uses laser as illumination source in * * * focusing mode, which can give a spatial lateral resolution less than 100nm, and the axial resolution is 4~7 times higher than that of * * * focusing fluorescence microscope technology. Using 4Pi microscope technology, ultra-high resolution imaging of living cells can be realized. Egner et al. [8, 9] used multiple parallel beams and 1 two-photon device to observe the fine details of mitochondria, Golgi apparatus and other organelles in living cells. Carl[ 10] used 4Pi microscope to measure the types of Kir2. 1 ion channels on mammalian HEK293 cell membrane for the first time. The results show that 4Pi microscope can be used to study the structure and morphology of cell membrane with nanometer resolution. (3) Image Interference Microscope (I2M): Two objective lenses with high numerical aperture and a beam splitter are used to collect the fluorescence images on the same focal plane and make them interfere on the CCD plane. 1996, gustafson and others used this double objective lens to irradiate the sample from both sides with incoherent light sources (such as mercury lamps), and invented the I3M microscope technology (incoherent, interference, illumination microscopy, I3m), which was combined with I2M to form the I5M microscope technology. In the measurement process, a series of images can be obtained by scanning the samples in the focal plane of * * * layer by layer, and then the data can be deconvolved appropriately to obtain high-resolution three-dimensional information. The resolution range of I5M is within 100nm.
2.4 nonlinear high-resolution microscope nonlinear phenomenon can be used to detect a very small amount of fluorescent or even unlabeled samples. Although some technologies are still in the physical laboratory stage, there is great room for development when combined with the existing three-dimensional microscope technology. (1) multiphoton excitation microscopy: (MPEM) is a kind of microscopy combining * * * focusing microscope and multiphoton excitation fluorescence technology, which can not only produce high-resolution three-dimensional images of samples, but also basically solve the problems of photobleaching and phototoxicity. In the process of multiphoton excitation, the absorption probability is nonlinear [1 1]. Fluorescence is produced by two or even three photons absorbed at the same time, and the fluorescence intensity is directly proportional to the square of photoluminescence intensity. Multi-photon excitation can only be carried out in the center of the sample for the oblique cone laser distribution generated by the focused beam, which has inherent three-dimensional imaging ability. By absorbing harmful short-wave excitation energy, the damage to surrounding cells and tissues is obviously reduced, which makes MPEM a powerful means for imaging thick biological samples. The axial resolution of MPEM is higher than that of * * * focusing microscope and 3D deconvolution fluorescence microscope. (2) Stimulated emission loss microscopy: Westphal[ 12] recently realized the concept of stimulated emission loss (STED) imaging proposed by Hell before 1994. STED imaging takes advantage of the nonlinear relationship between fluorescence saturation and excitation fluorescence loss. STED technology uses two pulsed lasers to ensure that the amount of fluorescence emitted in the sample is very small. The 1 laser is used as excitation light to excite fluorescent molecules; The second laser beam irradiates the sample, and its wavelength can make the luminescent substance molecules return to the ground state immediately after being excited. Those fluorescent molecules in the focus that suffer from the loss of STED light lose the ability to emit fluorescent photons, while the remaining fluorescent emission areas are limited to areas smaller than the diffraction limit, so the light spots smaller than the diffraction limit are obtained. Hell et al. have obtained a lateral resolution of 28nm and an axial resolution of 33nm [12, 13], and completely separated two molecules of the same kind with a distance of 62nm. Recently, the axial resolution of STED and 4Pi microscopes has been as low as 28nm, which proves for the first time that the axial resolution of immunofluorescent protein images can reach 50nm[ 14]. (3) Saturated structure illumination microscopy: Heintzmann et al. [15] put forward the theoretical hypothesis of saturated structure illumination microscopy, which is contrary to the concept of STED and was recently verified by Gustafsson et al. [16]. When the light intensity increases, these volumes will become very small, less than the width of any PSF. With this technology, a resolution of less than 50 nanometers has been achieved. (4) Second harmonic generation (SHG) imaging uses the frequency-doubled coherent radiation generated by the interaction between ultrafast laser pulses and media as the image signal source. SHG is generally a non-linear vibration process. Photons are only non-linearly scattered in biological samples and will not be absorbed, so there will be no accompanying photochemical process, which can reduce the damage to biological samples. SHG imaging does not need dyeing, which can avoid phototoxicity caused by using dyes. Because of its unique application value in nondestructive measurement or long-term dynamic observation of living biological samples, it has been paid more and more attention in the field of life science research [17].
3 surface high resolution microscope
Surface high-resolution microscopy refers to some microscopic techniques that can only be used for surface two-dimensional high-resolution measurement instead of three-dimensional measurement. It mainly includes near-field scanning optical microscope, total internal reflection fluorescence microscope, surface plasmon resonance microscope and so on.
3. 1 Near-field scanning optical microscope (NSOM) is an optical microscope with sub-wavelength resolution. Because the distance between the light source and the sample is close to nanometer level, the resolution is determined by the diameter of the optical probe and the distance between the probe and the sample, regardless of the wavelength of the light source. The transverse resolution of NSOM is less than 100nm, and Lewis[ 18] obtains a resolution less than 10nm by controlling sampling at a certain needle tip vibration frequency. NSOM has a very high signal-to-noise ratio and can quickly measure 100 images per second [19]. NSOM has been used for single fluorophore imaging and spectral analysis on cell membrane.
3.2 Total Internal Reflection Fluorescence Microscopy After the discovery of green fluorescent protein and its derivatives, TIRF technology has received more attention and application. TIRF can provide high axial resolution by using special sample optical illumination device. When the sample is attached to the cover glass near the prism, with the appearance of total internal reflection, the direct irradiation of light on the biological sample is avoided. However, due to the wave effect, a small amount of energy will still irradiate the sample through the interface between the glass slide and the liquid medium, and the brightness of these rays is enough to form a light hiding area of 100nm in the thin layer near the glass slide and excite the fluorescent molecules in this shallow layer [20]. The excited fluorescence is obtained by the objective lens to obtain a high axial resolution close to 100nm. TIRF has recently been applied to the research field of molecular gait dynamics with interference illumination technology, with a resolution of 8nm and a temporal resolution of 100μs[2 1].
3.3 Surface plasmon resonance * * * Surface plasmon resonance (SPR) [22] is a physical optical phenomenon. When the incident angle is incident on the interface of two different transparent media at a critical angle, total reflection will occur, and the intensity of reflected light should be the same at all angles. However, if a metal film is plated on the surface of the medium, the incident light will be coupled into surface plasmon, which will cause electrons to vibrate greatly, resulting in the reflected light being greatly weakened in a certain angle, and the angle at which the reflected light completely disappears is called the * * * vibration angle. * * * The vibration angle will change with the refractive index of the liquid flowing on the surface of the metal film, and the change of refractive index is directly proportional to the mass of biomolecules bound to the metal surface. By measuring the change of refractive light intensity on the surface of the coating, the slight change of surface refractive index can be obtained.
Since Fagerstan et al. applied it to the analysis of biospecific interaction in 1992, SPR technology has been applied to the analysis and detection of DNA-DNA biospecific interaction, the monitoring of microbial cells, the study of protein folding mechanism and the quantitative analysis of the affinity and specificity of bacterial toxins to glycolipid receptors [23]. When SPR information is transmitted through nano-scale holes [24] and excellent optical performance is provided, it is possible to develop a brand-new imaging principle microscope by combining SPR technology with nano-structure equipment.
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