Research On The Development And Applications Of A Compact Light Sheet Fluorescent Microcope System

As a versatile imaging technique,light sheet fluorescent microscopy made a remarkable progress in the last ten years.It brings the fluorescent imaging a lot of advantages,such as excellent 3D imaging,high spatial resolution to single cell,high imaging speed and low level of photo-bleaching and phototoxic effects.These properties apply the light sheet microscopy wide spread applications in the life science.Meanwhile,more and more impressive works are springing up with the help of the light sheet microscopy.However,Its drawbacks,such as poor portability,high price for commercial product,weak capability on the simultaneous multi-channel imaging,inconvenience sample manipulation and the contradiction between resolution and imaging field,bring it a lot of challenges as the researches in the life science go deeper.This dissertation mainly focuses on the improvement and optimization of the light sheet fluorescent microscopy.And as a result,a compact light sheet fluorescent microscope system which has a unique combination of capability and based on the conventional inverted fluorescent microscope have been progressed and designed.After that,various imaging applications have been conducted to illustrate the imaging performance improvement of the compact light sheet fluorescent micrioscope.First of all,based on the imaging principle of light sheet fluorescent microscopy,a compact light sheet fluorescent microscopy which was working with a conventional inverted fluorescent microscope was presented through a short light path design and hardware integration.The system got an ability of dual –channel simultaneous fluorescent imaging while adopting an imaging splitting optics path.Based on the principle of light sheet generation principle,the light sheet with a suitable thickness for different bio-samples can be produced via changing the thickness of the incident laser beam or focal length of the cylindrical lens.The charactoristics which include tunable thickness,high contract imaging and excellent axial resolution of the compact light sheet fluorescent microscopy have been demonstrated in the bio-imaging experiments which are the three-dimensional imaging of an in-vivo zebrafish embryo and high-speed dynamic imaging for a zebrafish beating heart and flowing blood.Then,the three-dimensional voxel super resolution method based on the compact light sheet fluorescent microscopy have been produced.The mathematic model for the super resolution method was built up on the base of the informations supplied by the sub-voxel shift in the three dimensional image stacks through appling the maximum likehood estimation(MLE)method on the degradation produre of the imaging process.Oblique scanning method was raised and resolution enhancement light sheet microscopy was devoloped.Super resolution experiments have been conducted for point spread function mearsurement,zebrafish embryo and transparent mouse brain block.The contrastive results indicated that 10X/0.4 objective lateral resolution can be obtained when imaged with a 4X/0.16 low magnification objective though around the whole field of view with the powerful imaging system and the axial resolution also has a 2~3 times improvement.Benifiting from this method,the contradiction between the resolution and the field of view can be partly solved.At last,the microfluidics technology was innovatively combinated into the compact light sheet fluorescent microscopy,which results in a new technology named opto-fluidics three-dimensional imaging.The self-scannnig process was investigated based on the principle of the opto-fluidics imaging technology.The manufacture process for an opto-fluidics chip was introduced and the facet flat method has been applied to the fluidics chip to reduce the light scatter brought by the unevenness chip facet and to improve the images quality.Meanwhile,the facet flat method also provided a key point for opto-fluidics imaging.The volume and the particle concentration of the micro-droplet was precisely measured based on the three-dimensional reconstruction opto-fluidics images.The results also indicate that three dimensional reconstruction imaging can be realized through the opto-fluidics imaging system.