| Optical microscopes are often used in biological and chemical laboratories due to their advantages of no contact detection.However,existing fluorescence imaging methods usually have a pair of insurmountable contradictions: On the one hand,in order to achieve high spatial and temporal resolution,a high-power light source needs to be used to excite fluorescent probes so as to collect sufficient fluorescence signals in a short time;On the other hand,the fluorescent bleaching effect of intense light sources limits the ability to perform real-time imaging over long periods of time.This conflict is particularly prominent in the field of STED superresolution imaging.Therefore,the development of new fluorescent probes and more scientific imaging methods are of great significance.This thesis started with the multifunctional modification of the microscope,carried out a series of studies on the construction and application of the STED microscope and the multi-mode dark field microscope.The specific contents are as follows:(1)Due to the limitation of the diffraction limit,traditional far-field optical microscopes can only achieve a resolution of 200 nm under visible light illumination.Scientists have proposed a variety of super-resolution microscopy techniques for this issue.However,in many super-resolution microscopy techniques,only STED obtains super-resolution images directly at the physical level without extensive data analysis.In this thesis,the STED illumination system was built using a supercontinuum pulsed laser and a monochromatic 592 nm continuous laser as the light sources.After the external light source is introduced into the Leica SP5 scanning fluorescence confocal microscope,three STED imaging modes can be realized: continuous-excitation/continuous-depletion imaging mode,pulsedexcitation/continuous-depletion imaging mode,and pulsed-excitation/pulsed-depletion imaging mode.In addition,we used the Leica DMI8 microscope frame to complete the setup of the STED microscope through NI(National Instrument)as the signal input and control.(2)Organic semiconductor polymer quantum dots(Pdots)have good application prospects for long-term observation of STED due to its good biocompatibility and excellent fluorescence performance.However,due to the wide excitation spectrum,Pdots will be simultaneously excited by the excitation beam and the depletion beam during the STED imaging.This has made it impossible to achieve STED superresolution imaging.In this thesis,multiple images of the depletion beam are used to solve this problem.Through a scientific algorithm,the signals generated by the depletion beam excitation in the STED imaging are deducted quantitatively to obtain a high-fidelity super-resolution signal.(3)Noble metal nanoparticles(MNP)were widely used as imaging probes in bio-imaging area due to their localized surface plasmon resonance(LSPR)phenomenon.Compare to fluorescence probes,the noble metal nanoparticles imaging exhibits high sensitivity,outstanding signal noise ratio,extremely stability,while the particle has good photostability,both of which make the MNP probe the perfect candidates for long term imaging.Currently the most popular way to imaging and analysis noble metal nanoparticles is employing dark field microscope with a spectroscope.Since most of the dark field microscopes use halogen lamp as their illumination source,its intensity and wavelength spectrum limited spectroscopy’s temporal resolution and spectrum detective range.For example,small Au nanoparticles with diameters lower than 30 nm can not be detected with current commercial devices.In this paper we present a custom made multi-mode dark field microscope with super continuum laser as its illumination source.In its focus mode,the full spectrum of a single noble metal particle can be collected within 1ms.And it also contains a light-sheet mode to support fluorescence imaging on the same sample by switching filters,which can be co-localized with the dark field imaging mode for cell imaging. |
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