Flow Cell Typing Based On High-resolution Multi-wavelength Photoacoustic Microfluidics Technology

Cancer has become one of the most threatening diseases to human beings,and the incidence and mortality of cancer have both increased rapidly in recent years.The main cause of death from cancer is the spread of malignant tumors.Therefore,early detection of tumor spread is an effective method to improve treatment effects and reduce lethality.Circulating tumor cell(CTC)is one of the "markers" of malignant tumors,thus its detection can support tumor spread determination at an early stage,which is rather important for cancer diagnostics.One classical CTC detecting method utilizes immunomagnetic beads to specifically bind CTCs and separates those using external magnetic fields.But such method is difficult t o distinguish different kinds of CTCs.Besides,optical imaging can detect CTCs even in bloods,thus it is a potential technique in cancer diagnostics.However,due to the strong light scattering effect from blood,optical imaging inevitably relies on ofte n complicated sample preprocessing such as dilution.Moreover,most optical imaging methods can still hardly identify different kinds of CTCs.Photoacoustic imaging,which combines optical high-resolution and acoustic high-penetrability,is a new type of non-destructive medical imaging technology.Especially due to the development of optical-resolution photoacoustic imaging,such method has been successfully employed in the observation and study on cells and other micro-structural samples.However,photoacoustic imaging cannot well applied in CTC detection mainly due to the following limitations :(1)The resolution of dynamic photoacoustic imaging system is still limited,which leads to difficulty in observing CTCs.(2)Because of the limited wavelengths in pulsed lasers with high repetition rate,the distinguishing capability on CTCs of the photoacoustic imaging system is also limited.To overcome these above mentioned limitations,this work designed and constructed a high-resolution photoacoustic microfluidic imaging system.Using this system,tumor cells in blood could be successfully detected and precisely distinguished,proving that such system can detect CTCs in blood,and also providing an important theoretical basis for photoacoustic imaging used in cl inical blood detection.The main content of this paper includes the following works.(1)A multi-wavelength photoacoustic cell imaging system with the resolution of 1.5 μm was designed to support high-resolution imaging of cells.Two pulsed lasers at 532 nm and 770 nm with a repetition frequency of 50 k Hz were used as excitation light in the system.Based on the chromophore emission induced by multiple excitation lights,the photoacoustic imaging system could distinguish unlabeled melanoma cells from blood cells.In addition,to further test the distinguishing capability of the system,three exogenous chromophores were introduced.Based on the absorption difference among these chromophores,this work proposed a multi-parameter chromophore identification method mainly according to photoacoustic signal amplitude,photoacoustic signal frequency and chromophore absorption ratio.Using this method,the dual-wavelength photoacoustic cell imaging system realized the sorting of four different types of cells.(2)A multi-wavelength photoacoustic microfludic cell imaging system was designed and constructed for cell sorting as well as melanoma cell detection in blood.Based on the multi-wavelength photoacoustic cell imaging system,microfluidic supporting substrate was introduced for high-resolution imaging on cells flowing in the microfluidic channel.Besides,the ultrasonic standing wave field was set in the photoacoustic system to improve the imaging quality and resolution of cells in the out-of-focus and under-focus regions.Additionally,with the optimizations on ultrasonic wave frequency,microfluidic channel size and excitation laser power,the multi-wavelength photoacoustic microfludic cell imaging system could successfully detect melanoma cells in blood,which lai d an important basis for photoacoustic imaging in blood detection.(3)A photoacoustic microfluidic cell elimination system was designed and constructed for tumor cell elimination.Based on the multi-wavelength photoacoustic microfludic cell imaging system,tumor cells could be recognized in real-time according to photoacoustic signal amplitude,cell size and photoacoustic absorption ratio,and then eliminated by triggering the high-power laser.Using the photoacoustic microfluidic cell elimination system,it is proved that the elimination efficiency of tumor cells could reach 95% when dealing with the sample mixed by melanoma cells and blood cells,and 85% when dealing with blood with melanoma cells.(4)A photoacoustic sub-cellular imaging system with the imaging resolution of0.42 μm was designed and constructed to obtain the high-resolution sub-cellular structures of the tumor cells.Such system provided high-resolution imaging of melanosomes in melanoma cells.In addition,microtubules,mitochondria and clathrin-coated pits were also observed using such system combining with the nanostructure labels.Finally,relying on the multi-wavelength illumination,multiple subcellular structures within a single cell were observed,and their intrinsic relations were quantitatively analyzed.The photoacoustic sub-cellular imaging system provides a new identification method in the sub-cellular level.