Microscopy

An apparatus and methods for characterizing the response of a particle to a parameter that characterizes an environment of the particle. A change is induced in the parameter characterizing the environment of the particle, where the change is rapid on a timescale characterizing kinetic response of the particle.

The response of the particle is then imaged at a plurality of instants over the course of a period of time shorter than the timescale characterizing the kinetic response of the particle. The response may be detected by measuring a temperature jump or by measuring correlation and anticorrelation between probe parameters across pixels.

More particularly, the particle may be a molecule, such as a biomolecule, and the environment, more particularly, may be a biological cell. The parameter characterizing the environment of the particle may be a temperature, and change may be induced in the temperature by heating a volume that includes the particle, either conductively or radiatively. The volume may be heated by means of a laser, such as an infrared laser, for example, or by microwave heating.

This technology produces highly spatial and temporally resolved microscopic images of induced relaxation dynamics through the combination of microscopy with a temperature jump in materials and biosystems under observation. 

Compatible with a variety of microscopy techniques, the technology allows for fast and controlled temperature change. Initiation of dynamics can also be achieved in a variety of systems, including chemical, physical, and biological systems. 

This technology can be applied to the observation of real-time temperature dynamics inside living cells. Compared to current options in the market, the technology is cheaper to produce and better for observing the aforesaid conditions. 

The phase contrast microscope is a vital instrument in biological and medical research; it facilitates the observation of living cells by using spatial light modulation to enhance the intrinsic contrast of transparent and colorless components in a cell. It can reveal cell structure without the need of contrast agents. However, the information obtained is only qualitative.

Jones Phase Microscopy (JPM) and White Light Quantitative Phase Microscopy (PC++) of transparent and anisotropic samples are novel interferometric techniques that are quantitative and polarization sensitive. They are capable of quantifying the optical phase delays associated with live cell samples while providing information about the morphology and dynamics.

Quantifying these optical path length shifts allows for nanometer scale measurements with broad applications in nanotechnology and life sciences, e.g. from inspecting semiconductor wafers to studying processes in live cells.

Description/Details

Jones Phase Microscopy (JPM) technique is capable of extracting the spatially-resolved Jones matrix associated with transparent and anisotropic samples. It can provide the quantitative phase image of the cells as well as the intrinsic structure details and dynamics. JPM setup consists of a modified Hilbert Phase microscope; the quantitative phase image is correlated to the sample using a 2D spatial Hilbert transform. The full Jones matrix in each point within the field associated with the transparent sample is extracted and the embedded quantitative information of phase delay is used to reconstruct its image.

The White Light Quantitative Phase Microscope (PC++) interfaces with a commercial phase contrast microscope and transforms it into a quantitative instrument by performing two additional measurements (hence the name PC++). In a conventional phase microscope the additional phase delay is introduced by the phase objective, the PC image is delivered at the image plane and recorded by a CCD. However, in PC++, the optical field at the image plane is further modulated before detection which is equivalent to spatial filtering with three different phase masks and reconstruction of image by addition of fields containing information about the unscattered light (forming the uniform background) and the fine structural details (the contrast). 

Applications

• Optical microscopy and imaging of live cells and thin tissue slices, microorganisms, lithographic patterns, and nanoparticles
• Study of cell dynamics and morphology

Benefits

• These techniques provide improved contrast in unstained biological specimens and can be utilized to examine dynamic events in living cells.
• Quantitative Phase delay information
• Polarization sensitive imaging; while Jones Phase Microscope is a modified Hilbert Phase microscope, the PC++ is an add-on to a commercial phase contrast microscope.

Dr. Paul Selvin from the University of IL has developed a streamlined process for creating coverslips with built-in, nanoscale fiduciary markers using thermal NIL. Unlike 50‐100 nm fluorescent nanoparticles, these markers can be custom made to different sizes and shapes. Using this method, Dr. Selvin found that there is no resulting auto-fluorescence in the blue part of the spectrum (emission from 450 nm and farther red), allowing for more proteins to be tracked simultaneously, a major advantage in the field. Additionally, the coverslip is stable enough to culture primary neuronal cells for over 2 weeks, while other micropatterned coverslips would detach from the glass surface during that amount of time.