Ziyang Lin

Temporally and spectrally resolved sampling imaging with a specially designed streak camera.

We present a novel sampling imaging technique capable of performing simultaneous two-dimensional measurements of the temporal and spectral characteristics of light-emission processes by use of a specially designed streak camera. A proof-of-principle experiment was performed with a homemade multifocal multiphoton fluorescence microscope. The system was calibrated with a Fabry-Perot etalon and a standard fluorophore solution (rhodamine 6G in ethanol) and was shown to have temporal and spectral resolution of 6.5 ps and 3 nm, respectively, as well as high accuracy and reproducibility in lifetime and spectrum measurement. Temporally and spectrally resolved images of 4 x 4 foci on the sample can be obtained with a snapshot.

Development of a multispectral multiphoton fluorescence lifetime imaging microscopy system using a streak camera

We report on the development of a multispectral multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) system that is the combination a streak camera, a prism spectrophotometer, a femtosecond Ti: Sapphire laser and a fluorescence microscope. This system is versatile with multispectral capability, high temporal (10ps) and spatial (0.36μm) resolution and can be used to make 3-dimensional (3D) (x-y-z) multiphoton fluorescence intensity, spectrally resolved intensity and lifetime measurements with a single detector. The system was calibrated with a F-P etalon and a standard fluorescent dye and the lifetime value obtained was in good agreement with the value reported in the literature. Preliminary results suggest that this MM-FLIM system has integrated high temporal, spatial, and spectral resolution fluorescence detection in one microscopy system. Potential applications of this system include multiwell imaging, tissue discrimination, intracellular physiology and fluorescence resonance energy transfer imaging.

Development of a Simultaneously Time- and Spectrum-Resolved Multifocal Multiphoton Microscopy System Using a Streak Camera

We report the development of a multifocal multiphoton microscope (MMM) system using a specially designed streak camera, which is capable of providing simultaneously time- and spectrum-resolved fluorescence microscopy. Temporal and spectral resolutions of the system have been calibrated to be 6.5-200 ps and 1-3 nm respectively. We demonstrate the applicability of the system to spectrum-resolved fluorescence and lifetime imaging with fluorescent dyes, fluorescent microspheres. Potential applications of the system include the measurement of tissue pathology, intracellular physiology etc.

Ultra-broadband time-resolved coherent anti-Stokes Raman scattering microspectroscopy

In a broadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy with supercontinuum (SC), the simultaneously detectable spectral range is limited by the spectral continuity and simultaneity of various spectral components of SC in an enough bandwidth. According to our theoretical analysis and experiments, the optimal experimental conditions are obtained. The broadband time-resolved CARS microspectroscopy based on the SC with required temporal and spectral distributions is achieved. The global CARS spectrum with well suppressed nonresonant background noise can be obtained in a single measurement and used as the imaging contrast. It will be more helpful to provide a complete and accurate molecular atlas, and to exhibit a potential to understand not only both the solvent dynamics and the solute-solvent interactions, but also the mechanisms of chemical reactions in the fields of biology, chemistry and material science.

Time-resolved Two-photon Excitation Fluorescence Spectroscopy Using a Streak Camera

We present a time-resolved two-photon excitation fluorescence spectroscopy system that is based on a picosecond streak camera and a prism spectrophotometer, and can perform simultaneous fluorescence spectrum and lifetime measurement using two-photon excitation. The potential applications of the system in biomedicine have been demonstrated with tissue endogenous fluorophore (flavin adenine dinucleotide), plant specimen (aloe epidermis) and gastric smooth muscle tissue.

Data and image processing for a simultaneous time-and spectrum-resolved multifocal multiphoton microscopy

A Simultaneous Time- and Spectrum-Resolved Multifocal Multiphoton Microscopy (STSR-MMM) system has been developed for the acquisition of fluorescence lifetime information of any spectrum of the biological samples in only one measurement. The near-infrared light from mode-locked titanium: sapphire femtosecond laser is split with a microlens array into an array of beams that are transformed into an array of high-aperture foci at the sample for parallel multiphoton excitation. The Gaussian shape of the excitation beam results in non-uniformity and asymmetry of the excitation foci array and also of the reconstructed fluorescence image. A coefficient matrix has been presented for the correction of the non-uniformity and asymmetry in the fluorescence image. Data acquisition and image processing methods have also been investigated to increase the image acquisition speed and improve the reconstruction of time- and spectrum-resolved fluorescence image. The methods for obtaining fluorescence intensity image, spectrally resolved intensity image and spectrally resolved lifetime image are also presented.

A novel field of view zoom scanning protocol for simultaneous time-and spectrum-resolved multifocal multiphoton microscopy

We present a simultaneous time- and spectrum-resolved multifocal multiphoton microscopy (MMM) system using a novel field of view (FOV) zoom scanning protocol. The system employs a microlens array for producing discrete excitation spot array, a dispersive prism and a high repetition rate streak camera for simultaneous temporal and spectral resolutions. By combining a pair of galvo mirrors and a sample stage for fine and coarse scanning respectively, the resolution and FOV of the system can be changed without changing any optical elements. The system can be operated not only in low resolution and large FOV, but also in high resolution and small FOV applications, without compromising the performances of the optical elements in the system. By implementing a special system control protocol and image reconstruction algorithm, fluorescence images in different FOVs and resolutions can be obtained. This FOV zoom scanning protocol is demonstrated with two-photon excitation fluorescence imaging of a fluorescence resolution test target.

Multifocal multiphoton microscopy using a novel field of view zoom scanning protocol

We present a novel field of view (FOV) zoom scanning multifocal multiphoton microscopy (MMM) system that is based on a microlens array for producing discrete excitation spot array, and the combining of a pair of galvo mirrors and a sample stage for changing the resolution and FOV with fixed optical system and objective. The system can be operated not only in low resolution and large FOV, but also in high resolution and small FOV applications, without compromising the performances of the optical elements in the system. By implementing dedicated system control protocol and image reconstruction algorithm, fluorescence images in different FOV and resolution can be obtained according to users definition. The performance of the system is demonstrated with two-photon excitation fluorescence imaging of a fluorescence resolution test target, prepared plant stem slide and fluorescence microspheres suspension. Preliminary results indicate that the system has high spatial resolution and holds potential for providing high image acquisition rate. This FOV zoom scanning protocol can be used in our simultaneous time- and spectrum-resolved MMM system, which can provide spectral and lifetime information simultaneously in fluorescence microscopy for biomedical imaging.

Compound imaging of ocular tissues with backward second harmonic generation and two-photon excitation fluorescence

We report the implementation of compound imaging with backward second harnionic generation (SHG) and two-photon excitation fluorescence (TPEF) in a standard confocal microscope. Various biological tissues, including skin, muscle and ocular tissues (e.g. cornea. iris, choroid, and sclera) have been investigated with different sample preparation methods (fresh, fixation). The results show that collagen-rich ocular tissues, like cornea, iris, choroids, and sclera can produce strong SHG signals. In sclera, there are not only plenty of collagen, but also various endogenous fluorophores. The organization of collagen fibers in sclera is clearly distinguishable from its SHG images viewed transversely and horizontally respectively. We also find that fixation of the sample with formaldehyde has reduced the intensity of SHG intensity by almost 5O%. As the SHG and TPEF can provide complementary information about collagen and fluorophores rich biological tissues, compound imaging of SHG and TPEF presented in this study is believed to have potential applications in biomedicine and clinical diagnosis.

Multifocal two-photon excitation fluorescence sampling imaging combining lifetime and spectrum resolutions

Multifocal multiphoton microscopy (MMM) is a more efficient and powerful method for three-dimensional (3-D) fluorescence imaging with reduced acquisition time compared with conventional confocal and two-photon excitation fluorescence microscopy. We present a novel multifocal two-photon excitation fluorescence sampling imaging technique that is based on a specially designed streak camera and combines fluorescence lifetime and spectrum resolutions. A proof-of-principle experiment is performed on a standard fluorescent dye solution (Rhodamine 6G in ethanol), Time- and spectrum-resolved sampled fluorescence image of Rhodamine 6G is obtained in a snapshot. The reconstructed two-dimensional (2-D) fluorescence image of a prepared plant slide is also obtained by moving the sample laterally. The capability of this system capable of performing simultaneous 2-D measurements of temporal and spectral information has many potential applications, e.g., multi-well imaging and spectrally resolved multifocal multiphoton fluorescence lifetime imaging etc.