Research Terms
Optical Engineering Biochemistry Biophysics Neurosciences Analytical Chemistry Optics
Keywords
Fluorescent Probes Neuroscience Optical Microscopy Protein Assay Rna Imaging Single-Molecule Rna Fish (Smfish) Sted Microscopy Super-Resolution Fluorescence Imaging
Industries
Researchers at the University of Central Florida have invented a fluorescence microscope that provides better real-time imaging of single molecules than conventional fluorescence technologies such as HILO (highly inclined and laminated optical sheet) microscopy. By enabling a much higher signal-to-background-ratio and a wider field-of-view (FOV), the UCF Highly Inclined Swept Tile (HIST) microscope delivers thinner illumination with greater than 40x FOV; thus providing clearer visualization of single molecules across a greater than 130µm x 130µm FOV. With such capabilities, the innovation can benefit applications such as super-resolution imaging, single-molecule tracking, and smFISH-based high-throughput gene expression profiling.
Technical Details
The UCF HIST technology is a fluorescence microscope that uses a highly inclined tile beam to scan over a biological sample object. The system spatially filters fluorescence emission from the sample through a programmable confocal slit into an sCMOS camera supporting a rolling shutter mode. The tile beam is synchronously swept with the readout of the camera to facilitate the rejection of background. The system provides for decoupling of the total imaging area from the beam thickness, which solely depends on the width of the tile beam, enabling a thinner illumination (high sectioning capability) and larger FOV for video-rate, live-cell imaging. The technology can be easily implemented onto a standard inverted microscope.
Partnering Opportunity
The research team is looking for partners to develop the technology further for commercialization.
Stage of Development
Prototype available.
Extended field-of-view single-molecule imaging by highly inclined swept illumination
Optica , Vol. 5, No. 9 September 2018A
Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging
J. Vis. Exp. , (146), e59360, doi:10.3791/59360 (2019)
Researchers at the University of Central Florida have developed a line-scanning confocal fluorescence technology that scientists can use to capture high-resolution, 3D, live-cell images while minimizing the effects of photobleaching and photodamage to target molecules. By providing a higher signal-to-noise-ratio (SNR) and lower excitation light intensity, the UCF multiple line-scanning confocal microscopy allows for longer observation time of fluorescent structures and reduces the effects of photobleaching and photodamage. Overall, the design produces faster, high-resolution images more efficiently than traditional line-scanning microscopy while maintaining deep optical sectioning capability and single-molecule sensitivity.
Technical Details
The UCF technology consists of a dual-inclined beam line-scanning (2iLS) confocal microscope apparatus and method. The microscope uses parallel excitation beams, each having a focused line shape that scans over a fluorescent sample. The sample emits fluorescence that is spatially filtered and detected by an array detector. In addition to two beams, the technology design can accommodate multiple beams (such as four, six or eight). Many applications, including tissue imaging and high-throughput imaging, can easily incorporate the technology.
As shown in the figure, the microscope design can employ parallelized dual beams with inclined illumination to lower the excitation intensity. The detector/camera assembly is a scientific complementary metal-oxide semiconductor (sCMOS) camera supporting a rolling shutter mode. The assembly includes two electrical slits to ensure the straightforward implementation of simultaneous confocal detection. Experimental results showed that the imaging method enables a two-fold longer observation time in single-molecule imaging and immunofluorescence imaging compared to traditional line-scanning microscopy.
Partnering Opportunity
The research team is looking for partners to develop the technology further for commercialization.
Stage of Development
Prototype available.
Low-photobleaching line-scanning confocal microscopy using dual-inclined beams, J Biophotonics, 2019 May 20:e201900075. DOI: 10.1002/jbio.201900075
University of Central Florida researchers have developed an easily implementable method for enhanced high-resolution, light-sheet fluorescence imaging without using a beam scanning module or a phase modulation system. The UCF single-shot, non-diffracting light-sheet generation technique provides a much larger field-of-view and higher signal-to-background ratio than current techniques requiring such equipment. In experimental results, the method generated a light sheet with a propagation distance approximately seven times longer than a typical Gaussian light sheet with a similar beam waist. Applications for the technology include single-molecule fluorescence imaging, high-throughput transcriptomics, super-resolution fluorescence imaging and tissue imaging.
Technical Details
The UCF invention employs controlled spatial coherence to generate static, non-diffracting optical light-sheets without using a beam scanning module or a spatial light modulator (SLM). For example, a one-dimensional (1D) coherent beam can be generated by increasing the spatial coherence of a light-emitting diode (LED) through an annular mask placed at the back focal plane (BFP).
As shown in the example schematic, incoherent light from an LED is collected and collimated by a condenser lens (F1) and focused by a cylindrical lens (CL). The beam passes through a narrow slit placed at the BFP of the cylindrical lens to control the spatial coherence. Then the slit is conjugated to an image plane using a relay system comprising a lens (F2) and an objective lens. An annular ring with outer and inner diameters of 2 mm and 1.75 mm, respectively, is inserted at the BFP of the objective. The beam emanating from the slit is a 1D coherent beam. It virtually produces numerous focused lines at the different positions of the BFP while illuminating the entirety of the annular mask. In the example, the beam propagation length is 6.75 mm, and its thickness is approximately 14.5 µm, comparable to a laterally scanned Bessel beam. In another example, a 1D coherent beam can also be created by decreasing the spatial coherence of a laser, making it unnecessary to scan non-diffracting beams to generate light sheets.
Partnering Opportunity
The research team is looking for partners to develop the technology further for commercialization.
Stage of Development
Prototype available.
Instantaneous non-diffracting light-sheet generation by controlling spatial coherence, Optics Communications, Volume 474, 2020, https://doi.org/10.1016/j.optcom.2020.126154.