Analytical Instrumentation : Microscopy

Analytical Instrumentation Portfolios


Algorithms to Classify T Cell Activation by Autofluorescence Imaging

Building on award-winning work, UW–Madison researchers have discovered that autofluorescence intensity images of NAD(P)H can accurately classify T cells as activated or not activated (‘naïve’ or ‘quiescent’), and have developed algorithms to classify T cell activation based on the images. Specifically, adapting pre-trained convolutional neural networks (CNNs) for the T cell activity classification task, T cells can be classified with 92 percent accuracy. These pre-trained CNNs perform better than classification based on summary statistics (e.g., cell size) or CNNs trained on the autofluorescence images alone.

This invention provides a way to non-invasively detect T cell activation by imaging NAD(P)H intensity. These algorithms can be applied to NAD(P)H images taken with commercial imaging flow cytometers / sorters, and fluorescence microscopes. If increased accuracy of T cell activation is needed for a specific application, additional measurements of the other NAD(P)H and FAD fluorescence endpoints can be obtained and used for classification.

Method and Device to Screen and Sort Cancer Immunotherapy Cells

UW–Madison researchers have developed a highly accurate label-free method to non-invasively detect T cell activation by detection of free-NAD(P)H fraction, NAD(P)H α1. NAD(P)H α1 can be measured by fluorescence lifetime imaging or spectroscopy systems. The device could also sort T cells based on NAD(P)H α1. If increased accuracy of T cell activation is needed for a specific application, additional measurements of the other NAD(P)H and FAD autofluorescence endpoints can be obtained and used for classification.

All-Glass Optical Microresonator for Single Molecule Spectroscopy and More

Building on their previous work, the researchers have developed all-glass microtoroidal resonators with improved sensitivity (i.e., superior Q/V ratio). Unlike their SiO2 on Si counterparts, the new resonators can be made chip scale – a significant advantage. Moreover, the use of glass in place of silicon makes the platform more desirable for applications including label-free sensing due to optical transparency in the visible region. Additionally, glass is a robust, chemically inert material and more biocompatible than silicon.

The new fabrication method follows the same general scheme as the previously developed oxide-on-silicon toroids, but the materials are inverted. This results in a silicon toroid atop an oxide pillar, followed by thermal oxidation to form an all-glass structure in the final step.

Microcavity Method for Single Molecule Spectroscopy

Specifically, the researchers have developed a new microcavity-based method for single molecule/particle spectroscopy. In essence, when an individual molecule or particle lands on the microcavity surface, it absorbs energy from a free space pump laser beam and generates heat. The heat is transferred to the microcavity, causing a shift in resonance frequency and therefore detectable changes in the light (e.g., power or intensity).

The superb sensitivity of the method enables detection, identification and real-time analysis of single molecules and particles. This is exciting because current spectroscopy techniques are limited to matter in the 10 to 100 nanometer size range, such as nanoparticles and viruses.

Multidimensional Imaging with Improved Contrast

UW–Madison researchers have developed a new coherent multidimensional spectroscopy (CMDS) technique that enhances the image contrast by using multiple frequencies that provide 3-D contrast. The method uses three coherent light pulses (intense light beams) with three different frequencies to interact with multiple functionalities within the molecules (e.g., C-H bonds) to create coherent images that are highly characteristic of specific molecules within sample substructures.

Tuning Optical Microcavities

The researchers have developed a tuning method for ultrahigh-Q toroidal optical microcavities capable of rapid modulation and resonance position control.

In the new configuration, a free-space pump laser beam illuminates the pillar supporting the microcavity, which warms ups and transfers heat to the microcavity. This induces a shift in resonance frequency. The support pillar is made of silicon or other suitable material.

The intensity of the free-space laser beam can be adjusted by changing the power and/or focal spot of the beam. Different intensities are used to achieve the desired shift in resonance frequency.

Monitoring Tissue Fluorescence in Bright Light

UW–Madison researchers have developed a fluorescence imaging process that can be used in surgical suites and other brightly lit environments. Specifically, the imaging process coordinates with rapidly switched ambient room light, which turns off and on at a speed imperceptible to the human eye. Alternatively, research locations such as bioimaging facilities that are traditionally dark can be illuminated – improving productivity and safety. During the periods of darkness, fluorescence signals from microscopes can be detected and imaged without background light interference.

Multiphoton Flow Cytometer for High Throughput Analysis of Multicellular Aggregates Like Pancreatic Islets

UW-Madison researchers have developed a system that combines the high throughput characteristics of flow cytometry with the capabilities of MPLSM. This multiphoton flow cytometry system (MPFC) enables deep, high resolution images of large diameter cells and aggregates. 

The multiphoton laser can excite intrinsic cellular fluorophores such as NADH, FAD and collagen, allowing both spectral and lifetime data to be acquired. This information then can be used to reveal information on cellular processes like metabolism, viability and the functional potential of cells, pancreatic islets, embryoid bodies and other entities.

Optical System for Correction of Tissue-Induced Aberration

A UW-Madison researcher has developed a new mono-chromatic method of correcting for the optical distortion caused by intervening tissue. Variations in the optical properties of the tissue lead to aberrations in the wavefront of the beam of light from the microscope system. This method is based on a reflective correction of the wavefront error. A computer-generated reflection hologram is projected in real time via a micro-mirror array, which adjusts the phase of the light to produce the exact opposite of the wavefront errors expected from the sample. The two cancel each other out, producing images corrected for aberrations.

Method of Producing Short-Wavelength Quantum-Entangled Light Beams

UW-Madison researchers have developed a new approach to generating quantum-entangled light beams using second harmonic generation to provide entanglement at short wavelengths. The science is in its infancy, but as it develops, entanglement generated at shorter wavelengths may be important for applications in quantum measurements, quantum-enhanced lithography and ultra-secure quantum communication. Entangled light could also lead to more sensitive medical diagnoses, more powerful computer chips and scalable quantum computing.

Microscope Probe Having an Ultra-Tall Tip

UW-Madison researchers have developed a method for fabricating a microscopic coaxial tip with a height greater than 30 micrometers. A coaxial tip and a coplanar waveguide are formed on a silicon substrate. The first layer of the tip shaft is made of a conductive material. Over the first layer, a layer of non-conducting material is deposited, followed by a layer of a different conducting material. An oxide etch exposes the first conductive layer at the tip.

Microcoaxial Probes Made from Strained Semiconductor Bilayers

A UW-Madison researcher has developed a microcoaxial probe tip that incorporates atomic force, scanning tunneling and microwave microscopy into one instrument to more quickly provide a high resolution image. A strained semiconductor bilayer with a conducting strip patterned to the inner surface is attached to a supportive layer. When the supportive layer is removed, the strain causes the bilayer to coil about the conducting strip, creating a tube with an atomically sharp tip. The conducting strip serves as a channel for STM, the bilayer can be attached to a cantilever for AFM, and the tube also serves as a microwave resonator, thus providing three probes on one microcoaxial tip. One computer can collect information from all three probes at once and quickly compile it into a more accurate graphic image.

Batch Fabrication of High Aspect Ratio Micromechanical Probe Tips

UW-Madison researchers have developed a multi-step batch process for manufacturing silicon probe tips with superior tip height and high aspect ratio for atomic force microscopy. First, an etchant protective island is formed on the surface of a silicon substrate with the substrate exposed around the island. The substrate is etched isotropically around the island by reactive ion etching (RIE) to partially undercut the substrate beneath the island. Next, the silicon surrounding the island is anisotropically etched using deep reactive ion etching (DRIE) to define a tip shaft of the desired height. The sidewall of the shaft is smoothed by a wet chemical clean and RIE to remove characteristic scalloping features from the DRIE step. At the same time, the RIE creates the desired shaft diameter. Finally, the protective island is removed from the tip and the top of the tip shaft is sharpened to an apex.

Mechanical Force Detection of Magnetic Fields Using Heterodyne Demodulation

UW-Madison researchers have developed a new, high-frequency magnetic field detector that uses mechanical force to directly detect a magnetic field. The device incorporates a coil-on-cantilever design, with a conductive loop placed on a vibrating cantilever beam with one fixed end. The frequency of the alternating current in the loop is chosen so the magnitude of the cantilever’s mechanical vibration at its mechanical resonant frequency reflects the magnitude of the magnetic field. The vibration of the cantilever is then detected by reflecting a laser beam off a reflective portion at the cantilever’s free end.

Plastic Cantilevers for Atomic Force Microscopy

UW-Madison researchers have developed a robust batch process for quickly and economically producing polymer-based cantilevers for atomic force microscopy. First, a master cantilever with a tip is used to create a mold. Next, the tip cavity within the mold is filled with a tip material, such as polystyrene or a magnetic metal. The remainder of the mold cavity is filled with plastic, preferably polystyrene, to form a plastic cantilever with a tip of the desired material. Multiple master cantilevers can be used to form multiple molds, so that the desired number of plastic cantilevers can be produced. At least one surface of the plastic cantilever can be coated with a reflective material, such as gold.

Heterodyne Feedback System for Scanning Force Microscopy

Now, two UW-Madison radio engineers who are also experts in SFM technology have created a technique for down-converting the signal from high-frequency SFM probes so it can be sensed by standard detection electronics. The technique uses a heterodyne receiver to down-convert the signal, an approach that is well known in radio frequency engineering but has not been previously applied to measurement instrumentation.

Photon-Sorting Spectroscopic Microscope System

A UW-Madison researcher has developed a novel spectroscopic microscope system that is well suited for multi-photon spectral imaging for analysis of specimens containing one or more types of fluorophores. The microscope is projected to cost a fraction of what current commercial MPELSM do because it takes advantage of new digital components and integrates the controls between many parts of the system.