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Wide-field Multiphoton Microscopy
Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning ...
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Nonlinear Optics
Nonlinear optics (NLO) is the branch of optics that describes the behaviour of light in ''nonlinear media'', that is, media in which the polarization density P responds non-linearly to the electric field E of the light. The non-linearity is typically observed only at very high light intensities (when the electric field of the light is >108 V/m and thus comparable to the atomic electric field of ~1011 V/m) such as those provided by lasers. Above the Schwinger limit, the vacuum itself is expected to become nonlinear. In nonlinear optics, the superposition principle no longer holds. History The first nonlinear optical effect to be predicted was two-photon absorption, by Maria Goeppert Mayer for her PhD in 1931, but it remained an unexplored theoretical curiosity until 1961 and the almost simultaneous observation of two-photon absorption at Bell Labs and the discovery of second-harmonic generation by Peter Franken ''et al.'' at University of Michigan, both shortly after the constru ...
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Two-photon Excitation Microscopy
Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light d ...
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Second-harmonic Generation
Second-harmonic generation (SHG, also called frequency doubling) is a nonlinear optical process in which two photons with the same frequency interact with a nonlinear material, are "combined", and generate a new photon with twice the energy of the initial photons (equivalently, twice the frequency and half the wavelength), that conserves the coherence of the excitation. It is a special case of sum-frequency generation (2 photons), and more generally of harmonic generation. The second-order nonlinear susceptibility of a medium characterizes its tendency to cause SHG. Second-harmonic generation, like other even-order nonlinear optical phenomena, is not allowed in media with inversion symmetry (in the leading electric dipole contribution). However, effects such as the Bloch–Siegert shift (oscillation), found when two-level systems are driven at Rabi frequencies comparable to their transition frequencies, will give rise to second harmonic generation in centro-symmetric systems. ...
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Multiphoton Fluorescence Microscope
Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light d ...
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Optical Amplifier
An optical amplifier is a device that amplifies an optical signal directly, without the need to first convert it to an electrical signal. An optical amplifier may be thought of as a laser without an optical cavity, or one in which feedback from the cavity is suppressed. Optical amplifiers are important in optical communication and laser physics. They are used as optical repeaters in the long distance fiberoptic cables which carry much of the world's telecommunication links. There are several different physical mechanisms that can be used to amplify a light signal, which correspond to the major types of optical amplifiers. In doped fiber amplifiers and bulk lasers, stimulated emission in the amplifier's gain medium causes amplification of incoming light. In semiconductor optical amplifiers (SOAs), electron-hole recombination occurs. In Raman amplifiers, Raman scattering of incoming light with phonons in the lattice of the gain medium produces photons coherent with the inco ...
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Multiphoton Fluorescence Microscopy
Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light d ...
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Regenerative Amplification
In laser science, regenerative amplification is a process used to generate short but strong pulses of laser light. It is based on a pulse trapped in a laser resonator, which stays in there until it extracts all of the energy stored in the amplification medium. Pulse trapping and dumping is done using a polarizer and a Pockels cell, which acts like a quarter wave-plate. Operating principle When a pulse with vertical polarization is reflected off the polarizer, after a double pass through the Pockels cell it will become horizontally polarized and will be transmitted by the polarizer. After a double pass through the amplification medium, having the same horizontal polarization, the pulse will be transmitted by the polarizer. If a voltage is applied to the Pockels cell, a double pass through it will change the polarization of the pulse to vertical, so the pulse will be reflected off the polarizer and will exit the cavity. If no voltage is applied, then a double pass through the Pocke ...
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Sapphire Laser
Sapphire is a precious gemstone, a variety of the mineral corundum, consisting of aluminium oxide () with trace amounts of elements such as iron, titanium, chromium, vanadium, or magnesium. The name sapphire is derived via the Latin "sapphirus" from the Greek "sappheiros", which referred to lapis lazuli. It is typically blue, but natural "fancy" sapphires also occur in yellow, purple, orange, and green colors; "parti sapphires" show two or more colors. Red corundum stones also occur, but are called rubies rather than sapphires. Pink-colored corundum may be classified either as ruby or sapphire depending on locale. Commonly, natural sapphires are cut and polished into gemstones and worn in jewelry. They also may be created synthetically in laboratories for industrial or decorative purposes in large crystal boules. Because of the remarkable hardness of sapphires 9 on the Mohs scale (the third hardest mineral, after diamond at 10 and moissanite at 9.5) sapphires are also u ...
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Penetration Depth
Penetration depth is a measure of how deep light or any electromagnetic radiation can penetrate into a material. It is defined as the depth at which the intensity of the radiation inside the material falls to 1/e (about 37%) of its original value at (or more properly, just beneath) the surface. When electromagnetic radiation is incident on the surface of a material, it may be (partly) reflected from that surface and there will be a field containing energy transmitted into the material. This electromagnetic field interacts with the atoms and electrons inside the material. Depending on the nature of the material, the electromagnetic field might travel very far into the material, or may die out very quickly. For a given material, penetration depth will generally be a function of wavelength. Beer–Lambert law According to Beer–Lambert law, the intensity of an electromagnetic wave inside a material falls off exponentially from the surface as : I(z) = I_0 \, e^ If \delta_p d ...
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Optogenetics
Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. On the level of individual cells, light-activated enzymes and transcription factors allow precise control of biochemical signaling pathways. In systems neuroscience, the ability to control the activity of a genetically defined set of neurons has been used to understand their contribution to decision making, learning, fear memory, mating, addiction, feeding, and locomotion. In a first medical application of optogenetic technology, vision was partially restored in a blind patient. Optogenetic techniques have also been introduced to map the functional connectivity of the brain''.'' By altering the activity of genetically labelled neurons with light and using imaging and electrophysiology techniques to record the activity of other cells, researchers can identify ...
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