Multicolor imaging has been widely applied across various biological and medical applications,especially essential for probing diverse biological structures.However,existing multicolor imaging methods often sacrifice ...Multicolor imaging has been widely applied across various biological and medical applications,especially essential for probing diverse biological structures.However,existing multicolor imaging methods often sacrifice either simultaneity or speed,posing a challenge for simultaneous imaging of over three fluorophores.Here,we proposed off-axis spectral encoding multicolor microscopy(OSEM)with a single camera that simultaneously captures encoded multicolor signals and reconstructs monochromatic images by decoding.Based on the natural intensity modulation difference of a single illumination spot across off-axis detection positions,we adjusted the multicolor excitation beams with distinct off-axis offsets from the same detection position to achieve spectral encoding.The method achieved multicolor simultaneous imaging in a single camera without extra sacrifice of frame rate.We evaluated OSEM's imaging performance by imaging multicolor synthetic samples and fluorescent microbeads.We also demonstrated that OSEM reduced imaging time by 5.8 times and achieved 99%accuracy in classifying and counting multicolor fluorescent bacteria,outperforming sequential imaging.We obtained four-color fluorescent optical-sectioning images of a mouse brain slice at a speed of 2.85 mm^(2)∕s,demonstrating its effectiveness for high-throughput multicolor imaging of large tissue samples.These results indicate that OSEM offers a reliable and efficient tool for multicolor fluorescent imaging of large biological tissues.展开更多
The burgeoning field of computational spectrometers is rapidly advancing,providing a pathway to highly miniaturized,on-chip systems for in-situ or portable measurements.The performance of these systems is typically li...The burgeoning field of computational spectrometers is rapidly advancing,providing a pathway to highly miniaturized,on-chip systems for in-situ or portable measurements.The performance of these systems is typically limited in its encoder section.The response matrix is largely compromised with redundancies,due to the periodic intensity or overly smooth responses.As such,the inherent interdependence among the physical size,resolution,and bandwidth of spectral encoders poses a challenge to further miniaturization progress.Achieving high spectral resolution necessitates a long optical path length,leading to a larger footprint required for sufficient spectral decorrelation,resulting in a limited detectable free-spectral range(FSR).Here,we report a groundbreaking ultraminiaturized disordered photonic molecule spectrometer that surpasses the resolution-bandwidth-footprint metric of current spectrometers.This computational spectrometer utilizes complicated electromagnetic coupling to determinately generate quasi-random spectral response matrices,a feature absents in other state-of-the-art systems,fundamentally overcoming limitations present in the current technologies.This configuration yields an effectively infinite FSR while upholding a high Q-factor(>7.74×10^(5)).Through dynamic manipulation of photon frequency,amplitude,and phase,a broad operational bandwidth exceeding 100 nm can be attained with an ultra-high spectral resolution of 8 pm,all encapsulated within an ultra-compact footprint measuring 70×50μm^(2).The disordered photonic molecule spectrometer is constructed on a CMOS-compatible integrated photonics platform,presenting a pioneering approach for high-performance and highly manufacturable miniaturized spectroscopy.展开更多
基金National Natural Science Foundation of China(62325502,81827901)。
文摘Multicolor imaging has been widely applied across various biological and medical applications,especially essential for probing diverse biological structures.However,existing multicolor imaging methods often sacrifice either simultaneity or speed,posing a challenge for simultaneous imaging of over three fluorophores.Here,we proposed off-axis spectral encoding multicolor microscopy(OSEM)with a single camera that simultaneously captures encoded multicolor signals and reconstructs monochromatic images by decoding.Based on the natural intensity modulation difference of a single illumination spot across off-axis detection positions,we adjusted the multicolor excitation beams with distinct off-axis offsets from the same detection position to achieve spectral encoding.The method achieved multicolor simultaneous imaging in a single camera without extra sacrifice of frame rate.We evaluated OSEM's imaging performance by imaging multicolor synthetic samples and fluorescent microbeads.We also demonstrated that OSEM reduced imaging time by 5.8 times and achieved 99%accuracy in classifying and counting multicolor fluorescent bacteria,outperforming sequential imaging.We obtained four-color fluorescent optical-sectioning images of a mouse brain slice at a speed of 2.85 mm^(2)∕s,demonstrating its effectiveness for high-throughput multicolor imaging of large tissue samples.These results indicate that OSEM offers a reliable and efficient tool for multicolor fluorescent imaging of large biological tissues.
基金supports from National Key R&D Program of China(2021YFB2800404)Natural Science Foundation of China(62175151,62341508)+1 种基金Shanghai Municipal Science and Technology Major Project(BH0300071)a Leverhulme Trust Early Career Fellowship grant,reference ECF-2022-711.
文摘The burgeoning field of computational spectrometers is rapidly advancing,providing a pathway to highly miniaturized,on-chip systems for in-situ or portable measurements.The performance of these systems is typically limited in its encoder section.The response matrix is largely compromised with redundancies,due to the periodic intensity or overly smooth responses.As such,the inherent interdependence among the physical size,resolution,and bandwidth of spectral encoders poses a challenge to further miniaturization progress.Achieving high spectral resolution necessitates a long optical path length,leading to a larger footprint required for sufficient spectral decorrelation,resulting in a limited detectable free-spectral range(FSR).Here,we report a groundbreaking ultraminiaturized disordered photonic molecule spectrometer that surpasses the resolution-bandwidth-footprint metric of current spectrometers.This computational spectrometer utilizes complicated electromagnetic coupling to determinately generate quasi-random spectral response matrices,a feature absents in other state-of-the-art systems,fundamentally overcoming limitations present in the current technologies.This configuration yields an effectively infinite FSR while upholding a high Q-factor(>7.74×10^(5)).Through dynamic manipulation of photon frequency,amplitude,and phase,a broad operational bandwidth exceeding 100 nm can be attained with an ultra-high spectral resolution of 8 pm,all encapsulated within an ultra-compact footprint measuring 70×50μm^(2).The disordered photonic molecule spectrometer is constructed on a CMOS-compatible integrated photonics platform,presenting a pioneering approach for high-performance and highly manufacturable miniaturized spectroscopy.
