Laser Phase Imaging And Its Application In Microbial Identification Investigation

With social development and environmental sanitation improvement,public health has attracted more and more attention as the upturn living standards,and thereby the increased requirements for the prevention and detection of diseases.Traditional environmental monitoring techniques and biomedical methods are complex,it is hard to meet the needs of in-depth analysis at the single cell level because of the limit of the obtained information.Therefore,developing a non-destructive,non-invasive,high-resolution and quantifiable detection method has important scientific significance and application value for the study of structural and physiological characteristics of cells and biological tissues.Based on optical microimaging technology,a laser diffraction phase microscopy imaging system with high-stability and high-precision is designed and built.Combined with the dynamic observation by microfluidic chips,the morphological characteristics and dry mass parameters of the two waterborne pathogenic parasites are quantitatively measured and investigated.The laser diffraction phase microscopy imaging system is designed and built combined off-axis interference with common path geometry,and the imaging system has detailedly carried on projection from the selection of source,grating and optical interference structure.The variation of optical path of this system is measured and the spatial noise standard deciation is quantified as 6.7 nm.The standard polystyrene spheres with diameter of 4.8 ?m are used as the calibration sample to validate the measurement accuracy of this system,the measured maximum phase shift error of polystyrene spheres does not exceed 4% compared to the expected result,the maximum phase shifts are consistent with the theoretical results.Exploiting the phase extraction algorithm based on Fourier transform and Hilbert transform.Meanwhile,considering the effects of pixel sampling errors and spatial noise during the optical field acquisition process,Goldstein?s phase unwrapping algorithm is used to unpack the wrapped phase to reduce the phase recovery errors.Finally,the phase distribution images of biological samples are recovered quickly and accurately after subtracting background images.The microfluidic chip with double layers and high capture efficiency is designed and fabricated in view of the shortcomings of parasitic ova,which are difficult to identify and capture in water.In the microfluidic chip,the U shape capture structure is 15 ?m long and 7.5 ?m wide,the depth and width of the center groove is 5 ?m and 10 ?m respectively.A gap of 2 ?m between the U-shaped structure and substratum chip is reserved,which effectively alleviates the flow pressure in the channel.Using this microfluidic chip,the parasitic ova can be well captured,addressed and positioned,so that they can be studied dynamically and in large quantities.Based on laser diffraction phase microscopy and the microfluidic chip with advantages of high efficiency for capturing and separating samples,the quantitative phase images of Giardia lamblia(G.lamblia)cysts and Cryptosporidium parvum(C.parvum)oocysts are obtained.The phase distribution images of this two parasitic oocysts are extracted from the interferograms,therefore the morphological parameters and dry mass can be obtained from the phase images.The major and minor diameters of G.lamblia cysts range from 8 to 15 ?m and 4 to 7 ?m respectively,and the dry mass is in range of 42.70~137.07 pg.The major and minor diameters of C.parvum cysts range from 4 to 6 ?m and 3 to 5 ?m respectively,and the dry mass is range of 6.13~14.00 pg.The viable and non-viable G.lamblia cysts are measured dynamically,and the average dry mass of 125 G.lamblia cysts decreases by 48.64 pg after inactivation.The above results show that laser diffraction phase microscope imaging system combined with microfluidic chip can achieve quantitative and dynamic measurement of tiny organisms and obtain morphological and physiological parameters.