Biophotonics is the interdisciplinary science dealing with generation and utilization of light and radiant energy for detection, imaging, and manipulation of biological materials. It is a noninvasive and non-destructive analytical method used to investigate molecular mechanisms in the life science field and to identify, diagnose, and treat diseases in the medical field.
The various instruments used in this field are:
A multidimensional fluorometer is a photonic device whose working principle is based on fluorescence excitation-emission matrices (EEM). It consists of a set of controlled laser sources used to acquire 3-D spectrum bands in a single measurement, as well as multidimensional spectrum bands by scanning excitation and emission wavelengths, intensity, and the frequency modulation of the excitation light.
This device is used to study the cell culture process. It is also used to analyze the effects of environmental pollutants on the spectral properties of oceanic phytoplankton.
The working principle of a STED microscope is based on excitation and depletion of fluorescence dye by a pulsed laser source. The device is used to improve the resolution of specimens below the optical resolution (nanometer scale) by involving a differentiating method of two distinct diffracting patterns.
First, molecules are colored using fluorescing dyes to sensitize them to a particular wavelength. Then the first beam is made incident on the molecules, exciting them and causing fluorescence. The second beam is a donut-shaped red pulse that brings the excited molecules to the ground state, making them to lose their fluorescence. This leaves behind only the particles unaffected by the red pulse to fluoresce, which can be clearly imaged.
The super-resolution imaging of the STED microscope is used to study nanosized structures and processes inside biological cells.
STED Confocal Super-Resolution - Leica TCS SP8 STED 3X
A fluorescence microscope is a biophotonic instrument that uses high-intensity light to energize fluorescent particles in a specimen. These particles emit a low energy light that is used to produce an enlarged image of the particles. The working principle of this instrument is similar to that of confocal microscopy.
The instrument consists of a dichroic filter with a threshold wavelength that reflects the light with a shorter wavelength and allows the light with a longer wavelength. Therefore, the fluorescence incident from the specimen is separated from the excitation light by the filter because the light produced by the specimen is with low energy and longer wavelength than the light used for excitation.
This instrument is used for imaging of the structural constituents of cells, performing viability analysis on cell populations, visualizing genetic materials, and imaging specific cells from the whole population.
Fluorescence microscope demo video – EVOS FL
Total Internal Reflection Fluorescence Microscope
Total internal reflection fluorescence microscope (TIRFM) is based on the working principle in which when light incident from an energized particle is totally internally reflected in a translucent solid at its boundary with liquid, an evanescent wave (electromagnetic field) is produced in the liquid at the boundary of solid–liquid with the same frequency as the energized light. The intensity of the evanescent wave reduces exponentially with increase in distance from the solid.
TIRF microscope is used to selectively energize the fluorophores close to the supporting cell surface with reduced fluorescence from the cellular regions. This helps to reduce the cellular photo damage and increase in the signal-to-noise ratio.
TIRFM is used to take high-contrast images of fluorophores near the plasma membrane and to obtain very low cell background and rapid exposure duration.
A laser scalpel is the device used for surgical ablating or cutting of biological tissues with blood vessels by use of laser lights. The device consists of a clad, sculptured silica fiber, and cone-shaped cutting tip. Commonly used laser sources are carbon dioxide laser, diode laser, and NdYAG laser. The radiation produced by a laser source is supplied into the probe which attains maximal power density at the exit point. The radiated energy penetrates the tissue as a cut made and thus causes an effective blood flow stability intensified by thermal energy diffusion from the nearby environment of the scalpel tip.
The hemostatic property of a laser scalpel has the advantage of improving the visibility of surgeons by reducing incisional bleeding. It also works great in treatments involving extra-bulbar tissues such as extraocular muscles, lacrimal tissue, and for facial surgery.
An optical tweezer (OT) is a device that involves light for manipulation of microstructures such as biological cells and for carrying out complex biophysical/biochemical characterizations. The process of cell manipulation has great significance in in vitro fertilizations, cellular interactions, embryology, cell biology, cell adhesion, tissue engineering, stem cell, regenerative medicine, and cell transfection. The working of the device is based on the optical trapping process. OT uses the radiation pressure exerted by a highly focused laser beam to trap tiny objects at their centers. The pressure applied on the particle by the laser beam consists of scattering force and gradient force.
OT has the advantage of applying a noncontact force to manipulate cells, accurate force resolution, and amicable nature to liquid media. Other applications of OT include positioning of cells, transportation of foreign particles into a single cell, sorting of cells in microfluidic structures, etc.