Device enables high-resolution, real-time, gentle imaging of single-molecule RNA in mammalian cells.
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.
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 Figure 1, 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.
Figure 1. Example schematic diagram of the UCF 2iLS technology: SMF is a single-mode fiber; L1-4 are lenses; CL is a cylindrical lens; M1-4 are mirrors; GM is a galvo mirror; Obj is an objective lens; DM is a dichroic mirror; TL is a tube lens, sCMOS is a scientific complementary metal-oxide semiconductor camera.
The research team is looking for partners to develop the technology further for commercialization.
Stage of Development
- Provides higher signal-to-noise-ratio and less photobleaching/photodamage
- Single-molecule fluorescence imaging, high-throughput imaging, super-resolution imaging, tissue imaging
- High-resolution fluorescence microscope for biomedical applications