Microscopy is better in color: development of a streamlined spectral light path for real-time multiplex fluorescence microscopy

Abstract

Spectroscopic image data has provided molecular discrimination for numerous fields including: remote sensing, food safety and biomedical imaging. Despite the various technologies for acquiring spectral data, there remains a trade-off when acquiring data. Typically, spectral imaging either requires long acquisition times to collect an image stack with high spectral specificity or acquisition times are shortened at the expense of fewer spectral bands or reduced spatial sampling. Hence, new spectral imaging microscope platforms are needed to help mitigate these limitations. Fluorescence excitation-scanning spectral imaging is one such new technology, which allows more of the emitted signal to be detected than comparable emission-scanning spectral imaging systems. Here, we have developed a new optical geometry that provides spectral illumination for use in excitation-scanning spectral imaging microscope systems. This was accomplished using a wavelength-specific LED array to acquire spectral image data. Feasibility of the LED-based spectral illuminator was evaluated through simulation and benchtop testing and assessment of imaging performance when integrated with a widefield fluorescence microscope. Ray tracing simulations (TracePro) were used to determine optimal optical component selection and geometry. Spectral imaging feasibility was evaluated using a series of 6-label fluorescent slides. The LED-based system response was compared to a previously tested thin-film tunable filter (TFTF)-based system. Spectral unmixing successfully discriminated all fluorescent components in spectral image data acquired from both the LED and TFTF systems. Therefore, the LED-based spectral illuminator provided spectral image data sets with comparable information content so as to allow identification of each fluorescent component. These results provide proof-of-principle demonstration of the ability to combine output from many discrete wavelength LED sources using a double-mirror (Cassegrain style) optical configuration that can be further modified to allow for high speed, video-rate spectral image acquisition. Real-time spectral fluorescence microscopy would allow monitoring of rapid cell signaling processes (i.e., Ca2+ and other second messenger signaling) and has potential to be translated to clinical imaging platforms.

Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy

“…Hyperspectral imaging approaches have been applied to microscope systems using a variety of spectral technologies: LCTFs [ 18 , 19 ], AOTFs [ 13 , 20 ], TFTFs [ 22 ], gratings [ 36 ], prisms [ 37 ], interferometers [ 54 ], spectral snapshot cameras [ 55 ], and light-emitting diode (LED) arrays [ 56 , 57 ]. Each technology has associated advantages and disadvantages, and in some cases, may be implemented in more than one way, such as for scanning of the fluorescence emission or excitation spectrum.…”

High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters

“…Approaches have also been developed to allow detection of a single, a few, or many fluorescent labels in a sample. A common theme across much of the development of fluorescence microscope technologies has been that of trade-off 1,2 , or in other words, how a microscope system can be optimized for one set of studies (for example, high-speed imaging) at the potential expense of losing the capacity to perform other studies (for example, high spatial-resolution imaging). Although trade-offs will perhaps always exist due to the fundamental physics behind fluorescence microscopy, the capability for customization and optimization of systems and components continues to allow new technologies that push the boundaries of spatial resolution, temporal sampling, and sensitivity.…”