High throughput snapshot spectral encoding device for fluorescence spectral microscopy
Inventors
Cutrale, Francesco • Wang, Pu • Fraser, Scott E.
Assignees
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Publication Number
US-12196679-B2
Publication Date
2025-01-14
Expiration Date
Abstract
Systems and methods are provided for multi-spectral or hyper-spectral fluorescence imaging. In one example, a spectral encoding device may be positioned in a detection light path between a detection objective and an imaging sensor of a microscope. In one example, the spectral encoding device includes a first dichroic mirror having a sine transmittance profile and a second dichroic mirror having a cosine transmittance profile. In addition to collecting transmitted light, reflected light from each dichroic mirror is collected and used for total intensity normalization and image analysis.
Core Innovation
The document discloses a snapshot spectral encoding fluorescence microscopy approach that uses two dichroic mirrors with spectral transmittance and spectral reflectance curves shaped as periodic waveforms. The first dichroic mirror uses sine wave profiles for its transmittance and reflectance, while the second dichroic mirror uses cosine wave profiles. By positioning the spectral encoding device between an imaging objective and an imaging sensor, the system generates spectrally encoded light channels from fluorescence signals without mechanical filter switching.
The spectral encoding device generates transmitted light channels and reflected light channels produced by the first and second dichroic mirrors. The system simultaneously captures multiple encoded channels on imaging sensors, including four encoded light channels formed through the transmitted and reflected portions of the two dichroic mirrors. The document describes using reflected anti-sine and anti-cosine channels to normalize total intensity, with the stated goal of improving signal-to-noise ratio (SNR) and throughput.
The document further describes integrating the spectral encoding device with light-sheet, wide-field fluorescence, and confocal microscopes. It also describes optical routing and focusing components for directing the encoded channels to different portions of the imaging sensor, and optional pre-filtering using long-pass, short-pass, and notch filters to suppress unwanted excitation or emission. For processing, the document describes using registered multi-channel data for phasor analysis, including G and S Fourier coefficients and phasor plots, and optionally performing linear unmixing to generate multi-spectral or hyper-spectral outputs.
Claims Coverage
The provided independent claims are clm-00001, clm-00015, clm-00024, and clm-00028. Across these, the inventive features center on periodic-waveform dichroic mirrors, generation of multiple transmitted and reflected encoded channels, and imaging with controller-based normalization and hyper-spectral or multi-spectral image generation or phasor-based unmixing.
Sine and cosine profiled dichroic mirrors
A first dichroic mirror having a first spectral transmittance curve and a first spectral reflectance curve with sine wave profiles, and a second dichroic mirror having a second spectral transmittance curve and a second spectral reflectance curve with cosine wave profiles.
Periodic-waveform spectral encoding for microscope integration
A spectral encoding assembly configured to encode emission light into a plurality of encoded light channels for integration with a microscope, where the encoding portion includes at least two dichroic mirrors and each dichroic mirror generates encoded light having a periodic waveform.
Four periodic-waveform encoded channels from fluorescence
An imaging system with an imaging objective acquiring fluorescence from a sample, an imaging sensor, and a spectral encoding device positioned between them, where the spectral encoding device includes first and second dichroic mirrors and generates four encoded light channels via the first and second dichroic mirrors, and each of the four encoded light channels has a periodic waveform.
Transmitted and reflected periodic-waveform spectral fluorescence imaging
A method for spectral fluorescence imaging that receives fluorescence signal at a spectral encoding device, generates at least two transmitted light channels and at least two reflected light channels via the spectral encoding device, and images the at least two transmitted and the at least two reflected light channels at an imaging sensor, where the spectral encoding device includes at least two dichroic mirrors each having a transmittance and reflectance profile resembling a periodic waveform.
Overall, the independent claims focus on snapshot spectral encoding using dichroic mirrors whose transmittance and reflectance profiles resemble periodic waveforms, including sine and cosine, producing multiple transmitted and reflected encoded channels, including four channels, that are imaged on an imaging sensor for fluorescence spectral imaging.
Stated Advantages
Improved signal-to-noise ratio (SNR) and throughput via normalization using reflected anti-sine and anti-cosine channels.
Documented Applications
High frame rate multiplexed spectral fluorescence imaging, including example in vivo zebrafish embryo imaging using a snapshot spectral encoding approach.
Integration with microscopy types including light-sheet microscopy, wide-field fluorescence microscopy, and confocal microscopy for fluorescence spectral microscopy.
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