Total internal reflection fluorescence microscopy (TIRFM) was used to image single molecules with evanescent waves.
The molecules were excited by an evanescent wave with different intensities. Single molecules were imaged on
low-noise high-quantum-yield charge-coupled device (CCD) cameras. Two-step photobleaching behavior was observed.
Duration from fluorescent spots appearing to disappearing was counted. The duration was decided by the speed of
photobleaching. Peak intensity was counted as exciting intensity change. The proportion relationship between the
reciprocal of duration and exciting intensity was obtained. The emitted intensity and duration of fluorescein were
compared with GFP. A single molecules emit the same number photons was proved.
Single-molecule/particle tracking technology is becoming a powerful tool to the noninvasive study of membrane
property and membrane molecular processes. Here, we used total internal reflection fluorescence microscopy combined
with oblique illumination fluorescence microscopy to investigate green fluorescence protein within living cell. Total
internal reflection illumination allowed the observation of molecules in the cell membrane of living cell since the
penetrate depth is adjusted to about 100nm, and oblique illumination is allowed the observation of molecules/particles
both in the cytoplasm and apical membrane, which suggested this combination would be promising to investigate protein
dynamics through the whole cell. Not only individual protein molecule/particle tracks have been analyzed quantitatively
to reveal the motion of protein, but also statistical analysis has been done to substantiate the observation of protein
dynamics. Our data that involved up to 700 trajectories in several hundreds cells are indicated that at least four modes of
motion are presented, that is directed motion, normal diffusion (random walk), binding motion and corralled motion.
Detailed analysis as well as statistical analysis is presented in the paper.
The combination of the confocal scanning microscopy and the defocusing imaging in the plane of imaging system was proposed. A switch lens and a pinhole with fine focusing aid are used to switch between the confocal scanning imaging mode and defocusing imaging mode. Objectives of different magnification can be used to get different field of vision. The complementary images can offer more information than any single one. Image blur or distortion caused by scanning, such as the displacement of the target and the excursion of scanning stage, can be corrected according to the comparison of two images. Location and adjustment of the target can be achieved easily and the accuracy of scanning image will be improved obviously. Some samples were tested on a home-built system.
KEYWORDS: Optical spheres, Near field, Near field scanning optical microscopy, Polarization, Optical resolution, Near field optics, Radio propagation, Statistical modeling, Super resolution, Image resolution
Using probe-sample interaction equations based on the dipole-self-consistent field theory, we studied influence of the probe tip size on resolution in near-field scanning optical microscopic system for different undulate protuberance size of the sample surface and distance from the probe to the sample surface. These parameters in the NSOM were all normalized by the probe tip size in our numerical calculation, consequently a new definition expression of NSOMs resolution was suggested. Furthermore we calculated and analyzed polarization influence of the incident light on the resolution. Using probe tip size to normalize another parameter in the calculation and analysis causes the some succinctness conclusions.
The two-photon absorption properties of a new dye have been experimentally studied using the comparable two-photon induced fluorescence method with a pulsed titanium-sapphire laser. This new dye has a two-photon absorption cross-section of 1.7 x 10-48 cm4s/photon at 790nm.
We combine the differential confocal microscopy and annular pupil filter together. The theory of differential confocal microscopy and the property of the annular pupil filter are discussed in detail. The results of computer simulation and experiment prove that this method can extend the axial dynamic range and improve transversal resolution of 3D profilometry at sacrificing axial resolution.
Three techniques for processing interference fringes were described in this paper. The first one is a phase-shifting fringe processing technique with intelligence control. The second is an inverse filtering technique to the fringe processing, and the third kind is a correlation numerical processing technique. The three processing fringe techniques are based on Mirau interferometric microscope.
Two optical sectioning imaging technologies with visible laser and near infrared laser illumination to apply industrial and biologic detection are presented respectively in this paper. One of them is based on fiber optical confocal scanning microscopy, the other is imaging-relay arrangement with small visible field. The systems have resolution of submicron at lateral direction and nanometer resolution in axial direction and wider work range about submillimeter magnitude.
Pupil filters for improving optical sectioning property of confocal microscope are presented. These pupil filters reduce the central lobe of the axial point spread function (PSF), accompanied by a sever increase of strength of the second lobes, which make them impossible to use in conventional imaging systems. While to confocal scanning microscope, an important reduction of the central lobe in axial PSF is achieved with low sidelobe.
In fiber optical confocal scanning imaging system of reflection mode, interference of the reflection light beams from terminal face of the fiber coupling tip and from the surface of the sample will always destroy the image of low reflection index samples, such as CD-R pregroove basal disc and biotic samples. A quantitative analysis was given to find out the influence of fiber terminal face reflection on the system axial response. In order to avoid the influence of the fiber reflected light, the reflectivity ratio of the fiber tip to the sample should be low. Interference noise was effectively reduced by immersing the unused fiber tip into glycerol and cleaving the fiber tip end face at an angle. The proof experimental results of axial response have been shown. Finally, good quality confocal images of the recordable CD pregroove and the chromosome were presented.
In confocal scanning imaging system, using apodization masks can improve the system resolution. A mask based on circle grating function with different spacing is presented and made, which is simple and easy to practically make and apply. The experiments of using a mask are performed in a reflection-mode fiber confocal scanning microscope. The intensity response curves in transverse and axial direction of this system are given. A sharp straight edge of a flat object is measured. And the 2D image of a biological organism is also given. The results demonstrate that adding this apodization mask does not reduce the axial resolution while improve the transverse resolution.
A novel optical system which has high resolution, large work distance and long focal depth is presented. In the system the information of transmission, reflection and scatter light can be used respectively.
KEYWORDS: Near field scanning optical microscopy, Near field optics, Image resolution, Near field, Wave propagation, Microscopy, Superposition, Fourier optics, Spatial frequencies, Optical microscopes
An expression of evanescent field of the scanning near-field optical microscope (SNOM) and a few effects on the resolving power are given. The concept about degrees of freedom of light information transmitted and resolution of SNOM are reviewed.
Here we put forward the idea of getting superresolution in confocal setup with apodization film. By this configuration we can suppress the background noise aroused by apodization film, and further avoid problem owing to non-axial points when modified lens is used solely. Also we introduce a new model based on the confocal setup to explain superior resolution mechanism in near field optical microscope. Theoretical development and skeptical comments are given in this paper.
According to the image relay concept in the optical amplification system, submicrometer resolution of a digital image processing system has been given in this paper. The accuracy and precision of the system has been examined by using a standard identification reticle, and some measurement results of a small focal spot through a computer generated holographic lens have been given.
Centroid and geometric center methods are suggested for tracing the center location of an irregular interferogram in this paper. The methods remove limitations of general software which may only analyze straight or quasi-straight fringe patterns. The F-test method is used to control accuracy of fringe analysis. The speed and accuracy of the software depend upon the quality of the fringe pattern and a number of sample points.
Scanning count is an efficient way to recognize and count the number of components in a picture . After all points in one line of a target having been scanned, one of the end points in the next line of the target can be determined,then the new line can be scanned . At the sane time , the line of another side is checked to avoid any omission .Of course, this method is easy to detect that one target has been scanned over. Each point in a target is visited approximate two times . And break points can be jumped over automatically . The number and each area of targets are then calculated . Compared with the conventional algorithms, the new one is simple and fast, and is practicable in personal computers.
It is proposed that the diffraction theory of aberration be used to evaluate approximately the optimal focused area of a high-power laser and the intensity distribution at the focused area. In one proposed method, the phase distribution of the laser beam is yielded by processing the interference fringe obtained from the front of the focal lens of the laser system. The amplitude distribution can either be assumed as super-Gaussian in shape with low frequency modulation, or can be simulated by computing the laser propagation using an FFT algorithm with real samples. In another method, the irradiation distributions are demonstrated experimentally using hologram reconstruction. This method can provide more details of the focused laser beam than digital computation.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.