GUO Zhong-yang，YOU Qian-nan，GE Ming-feng，WANG Guo-wei，MEI Qian ，DONG Wen-fei

（1. School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine,

University of Science and Technology of China, Hefei 230026, China；2. Suzhou Institute of Biomedical Engineering and Technology,Chinese Academy of Science, Suzhou 215163, China）

* Corresponding author，E-mail: qmei@sibet.ac.cn

Abstract: In order to realize the separation and release of nucleated red blood cells from peripheral blood and develop a safe and effective non-invasive technique to separate nucleated red blood cells for prenatal diagnosis of fetal diseases, an automatic cell smear preparation system based on hydrogel material was established, and a laser focusing and microscopic imaging system for recognizing and releasing nucleated red blood cells was constructed. Firstly, the mechanical structure of cell smear preparation machine was designed, the upper computer control software was designed based on single chip microcomputer, and a hydrogel membrane substrate smear was prepared by optimizing the slide-pushing angle and speed. MXene, a twodimensional material, was introduced into temperature-sensitive hydrogel gelatin, and the near-infrared light response was realized on the surface of hydrogel membrane by using the near-infrared photothermal conversion characteristics of MXene. Then, the whole cell smear experiment was carried out on the surface of the hydrogel substrate membrane. A monolayer cell smear was prepared by optimizing the parameters of blood slide. Finally, the optical path of laser focusing and microscopic imaging was established. After the nucleated red blood cells were recognized and located, the light from an 808 nm laser source passed through a collimator lens and a convergent lens and was focused on the surface of the cell smear, which released cells under photothermal effect. A monolayer cell smear was processed and prepared, and then a photothermal effect was produced under the near-infrared light of 808 nm. After the control of the laser focusing system, a fixed cell-releasing area with a spot diameter of 300 μm was finally obtained. In this paper, the automatic slidepushing technology was applied to the preparation of a monolayer cell smear based on hydrogel membrane,

Key words: cell smear; hydrogel; prenatal diagnostics; near-infrared light response; cell release

1 Introduction

Prenatal diagnosis is essential for the early diagnosis and screening of birth defects such as Down syndrome, neural tube defects and single-gene diseases[1]. The existing prenatal diagnoses can be divided into invasive diagnoses and non-invasive diagnoses. Invasive diagnoses include amniocentesis,umbilical cord blood sampling, and chorionic puncture, etc.[2]. They are performed by invasive methods, and are often accompanied by miscarriage, amniotic fluid overflow, infection and other risks[3].Therefore, establishing a non-invasive prenatal diagnosis method is the focus and mainstream trend of the current prenatal diagnosis technology development[4]. Fetal Nucleated Red Blood Cells (NRBCs)contain the entire fetal genome and the early gestational expression (expressed in peripheral blood at the 6th week and maximized between 12thand 14thweek). They are highly distinguishable in maternal blood cell population and are hard to be confused with maternal cells. With obvious cellular morphological characteristics, they are easy to identify and are the preferred fetal cells for prenatal diagnosis[5].However, fetal NRBCs are very few in maternal peripheral blood. How to identify and separate NRBCs from peripheral blood is a major challenge in prenatal diagnosis[6].

The existing methods for separation and enrichment of peripheral blood cells include density gradient centrifugation, magnetic activated cell sorting, fluorescence activated cell sorting, microfluidic chip technology, etc[7-9]. Among these methods, the separation and detection are mainly realized by the specific binding of antibody and cells. The procedures have a complex operation process, long detection cycle and high cost. Simplifying the process of cell separation and enrichment is an urgent problem to be solved for non-invasive prenatal diagnosis.

Using the slide-pushing technique to make a good cell smear for blood cell morphology examination is critical in clinical application[10]. The traditional man-made cell smear cannot be standardized due to the uncontrollability of manual operation,which leads to cell overlap and rupture. The automatic cell smear preparation machine can standardize the preparation of monolayer cell smears and identify the NRBCs by optical imaging based on their special morphology. In order to achieve the collection of NRBCs for downstream application,the thermal response release system reported so far can release the captured cells at physiological temperature, and help the released cells maintain enough activity. This method can release cells in a large area, however, it is hard to release cells in a fixed area. Correspondingly, light-stimulated response system is considered as an ideal controllable release system due to its operability[11]. Compared with ultraviolet light, near-infrared light produces less damage to cells and has stronger penetration, so it is more suitable for the construction of light-responsive release system. By introducing a light response system into the thermal response release system, more efficient fixed-point release can be achieved. This paper proposes the use of the filmforming and photothermal properties of hydrogel[12],in which MXene two-dimensional material is introduced into thermo-sensitive hydrogel gelatin, the near-infrared photothermal effect of MXene can be combined with the thermal response of hydrogel substrate. Thus an automatic monolayer cell smear preparation machine is prepared, and a laser convergence and microscopic imaging system are established to solve the traditional problem of cell capture, separation and release depending on the specific binding of antigen and antibody. By optimizing the structure of each module and material properties,the recognition and fixed-point release of NRBCs provide a new approach for noninvasive prenatal testing.

2 Design of cell smear preparation machine system and recognition &release system

2.1 Overall design of control system

The scheme of automatic pushing and laser convergence system used to recognize and release the NRBCs is shown in Figure 1. The automatic slide pushing system consists of a mechanical motion subsystem, an adaptive smear system, a microstructural slide-loading platform and an electronic control subsystem. The movement of the XY stage is controlled by the mechanical motion subsystem,which controls the pushing speed and angle. The adaptive smear structure has two DOFs, which can adaptively fine-tune the condition of the smear and the surface in contact with it. By combining the two-dimensional motion of the motion subsystem, the three-dimensional motion of the smear and slide can be controlled. The microstructural slide-loading platform can carry the standard microscope slides(25 mm × 75 mm) for the preparation of hydrogelbased smears and cell smears. The electronic control subsystem is used to realize the communication and interactive operation among the modules of the whole smear system.

Firstly, a hydrogel membrane is prepared on a slide by using the automatic slide-pushing system.The membrane is taken as substrate, and then covered with a monolayer blood cell by spreading the blood sample. In this way, a cell smear is obtained. Secondly, the blood cell membrane is stained by Wright-Giemsa solution to examine cell morphology. As a clinical hematological detection method,this method has the advantages of good staining effect, obvious nucleo-cytoplasm contrast, easy operation and rapid staining[13]. After the staining of the cell smear, the NRBCs can be identified and located under optical microscope according to unique cell morphology. Then the cell smear is placed in a laser convergence and microscopic imaging system.According to the near-infrared response properties of hydrogel substrate, an 808 nm laser is selected for fixed-point irradiation to realize the photothermal conversion and release NRBC in the spot area.

2.2 Design of an automatic smear system

The automatic smear system includes four parts: a mechanical motion subsystem, an adaptive smear system, a microstructural slide-loading platform and an electronic control subsystem. The design principle of mechanical motion subsystem is shown in Figure 2. The system is divided into two modules: speed regulation and angle adjustment.The speed regulation module, which consists of an X stage, a carrier base and a closed-loop stepper motor, can adjust the speed within the range of 0?150 mm/s. The angle adjustment module is composed of a Y stage, a smear structure base, a closedloop stepper motor and an R-axis decelerating stepper motor. It can tune the angle within the range of 20°?50° when spreading the blood sample.

The adaptive smear system consists of smear clips and two sets of hinges that are perpendicular to each other in the pushing direction. With two DOFs,the system can adaptively fine-tune the smear while keeping it fit to the contact surface. The microstructural slide carrier is designed with a slide-loading groove and a waste liquid collection groove.

A 200 μm height difference between the slideloading groove and the slide surface is designed to ensure a hydrogel substrate membrane with uniform thickness during pushing. The waste liquid collection groove is used to collect excess hydrogel when pushing the slide to the end, so as to avoid the backflow-caused damage and contamination. The electronic control subsystem consists of an upper computer control system, a motion control card, a motor driver, a photoelectric limit switch and a power supply. The upper computer control software is programmed based on a single chip microcomputer. The serial port is connected with the motion control card to realize the interactive operation and control of pushing speed module and angle module.

2.3 Design of cell recognition and release system

According to the properties of hydrogel substrate membrane and the design principle of cell smear, the cell recognition and release system shown in Figure 3 has been designed in order to achieve the recognition and fixed-point release of NRBCs. It is composed of a laser-focusing subsystem, a bright field illumination subsystem and a microscopic imaging subsystem. According to the near-infrared response characteristics of hydrogel substrate membrane, an 808 nm laser source is combined with a collimating lens and a convergent lens(C1) into a laser convergence subsystem to irradiate a fixed area of the cell smear, which, in turn, produces the photothermal effect and releases the cells.In order to observe the cell release, a bright field illumination subsystem and a microscopic imaging subsystem are constructed on the basis of the laser convergence subsystem. The bright field illumination subsystem is composed of the bright field source and Kohler lens group. Through the application of dichromatic mirror, the bright field source and the 808 nm laser source can share an optical path. Subsequently, the microscopic imaging subsystem module composed of a microscopic objective, a reflecting mirror, a convergent lens (C2) and an imaging detector is used to observe the cell-releasing effect.

The laser convergence system is built to produce the near-infrared response, which enables the cell release at a fixed point. The laser light is focused to form a tiny light spot. A smaller light spot means a smaller spot area projected on the cell smear and higher efficiency and precision of cell release. To control the size of the light spot, the spot diameter should be reduced as much as possible so as to narrow the range of cell release at a fixed point. According to Gauss formula[14]:

whereω1is the spot diameter after focusing;Fis the focal length of the convergent lensC1,F=F1+F2,whereF1is the distance from the convergent lens to the dichromatic mirror andF2is the reflection distance from the dichromatic mirror to the cell smear;andω0is the waist radius of the laser Gaussian beam. The spot size and light intensity distribution on the equiphase planes on both sides of the convergent lensC1 are the same, soω0is the spot radius after collimation. By substituting the laser wavelengthλ(808 nm) and the collimated spot radiusω0(850 μm) into Equation (1), the spot diameter after laser convergence can be obtained as follows:

The bright field light is radiated to the cell smear surface through the Kohler lens group, and then is reflected to the detector by the imaging system composed of a microscope objective, a mirror and a convergent lens. By moving the stage, the whole cell smear can be imaged and the NRBCs can be identified and located. The laser light irradiates the identified target area through the collimating lens and convergent lens, and triggers the near-infrared response and photothermal conversion followed by fixed-point cell release. Finally, the cellreleasing performance is characterized by a microscopic imaging system.

3 Experiment and results

3.1 Preparation and optimization of hydrogel pusher

The experimental hydrogel was two-dimensional MXene composite gelatin (C102H151O39N31,gelatin) prepared by our group. The gelatin and MXene were stirred and mixed evenly at a mass ratio of 200:1 in a water bath at 38 ℃ to obtain the hydrogel. The prepared hydrogel was kept at 38 ℃to ensure the melting state through magnetic stirring, pushed and spread on the slide surface by a home-made automatic pushing machine, and then naturally cooled to form a layer of hydrogel membrane with smooth surface and uniform thickness.The prototype machine designed and fabricated based on a single-chip microcomputer is shown in Figure 4. The slide-carrier is located in a horizontal position. The pusher-loading platform has an angle to the slide carrier, but with a base parallel to the slide carrier. The preparation of hydrogel membrane was optimized by adjusting the pusher’s speed and angle. At first, a certain amount of hydrogel solution was draw with a pipette, and dropped evenly on the front end of the slide. Then the pusher was started and descended until its bottom touched the slide horizontally. The pusher was pushed forward with an angle to the slide so that a hydrogel membrane was formed on the surface of the slide.

In the experimental process, orthogonal experiments were carried out at different pushing speeds(20 mm/s, 30 mm/s and 40 mm/s), pushing angles(25°, 30° and 35°) and hydrogel amounts (200 μL,250 μL and 300 μL) to optimize the preparation parameters of hydrogel membrane. The results showed that a faster pushing speed and a larger pushing angle could cause the damage to the hydrogel membrane more easily. The hydrogel amount is mainly determined whether the hydrogel membrane could completely cover the slide. After repeated experiments, the preparation parameters of hydrogel membrane were optimized at a pushing speed of 30 mm/s, a pushing angle of 30° and a hydrogel amount of 250 μL.

The prepared hydrogel membrane was observed and characterized by Scanning Electron Microscope (SEM) and Atomic Force Microscope(AFM). As shown in Figure 5 (a, b), the hydrogel membrane had a uniform thickness of about 200 μm and an average surface roughness ofRa= 1.31 nm,providing a good surface smoothness for subsequent whole blood experiment. An ultraviolet absorption test was carried out on the slide spread with a composite gelatin. As can be seen from the test results in Figure 5(c), the prepared composite gelatin has an obvious absorption peak at about 800 nm, which indicates an achievable near-infrared response and photothermal conversion. Based on the above optimized conditions, a hydrogel film was prepared.Its photothermal properties were tested by 808 nm laser irradiation, and the characterization results are shown in Fig. 5(d), including the photothermal response curves of gelatin, composite gelatin and stained composite gelatin under the irradiation of 808 nm laser (laser power: 150 mW/mm2). Under the laser irradiation, the temperature of gelatin was almost unchanged, and no photothermal effect was produced. However, within the irradiated area of composite gelatin, the temperature increased and quickly rose to 37 ℃ within 120 s and even up to 47 ℃ within 360 s (this temperature could be applied to the subsequent cell release). To verify whether the Wright-Giemsa staining method would affect the photothermal properties of stained composite gelatin, a laser irradiation experiment was performed under the same conditions. The stained composite gelatin exposed to 150 mW/mm2laser irradiation could be heated to 37 ℃ within 20 s and up to 62.9 ℃ within 320 s. When the laser power was reduced to 100 mW/mm2, the photothermal conversion effect on the stained composite gelatin was similar as that on the unstained composite gelatin.

Fig. 1 Schematic diagram of cell smear preparation and laser response system圖 1 細胞涂片制備及激光響應(yīng)系統(tǒng)示意圖

Fig. 2 Schematic diagram of mechanical system of cell smear圖 2 細胞涂片機械系統(tǒng)原理示意圖

Fig. 3 Schematic diagram of laser focusing and microscopic imaging system圖 3 激光會聚與顯微成像系統(tǒng)示意圖

Fig. 4 Prototype of preparation machine of automatic cell smear圖 4 自動細胞涂片制備機樣機

Fig. 5 (a) Characterization by SEM; (b) surface roughness characterization by AFM; (c) ultraviolet absorption spectrum;(d) photo-thermal curves圖 5 （a）SEM表征；（b）AFM表面粗糙度表征；（c）紫外吸收圖譜；（d）光熱曲線

3.2 Preparation and optimization of a cell smear on hydrogel membrane

An automatic cell smear preparation machine was designed to carry out blood slide-pushing experiment on the surface of hydrogel membrane. The blood samples were from the peripheral blood with a hematocrit (HCT) ranging from 0.39 to 0.45 examined in a clinic. The reference value of HCT for normal females is 0.35?0.45. However, during pregnancy, the HCT value will vary within 0.33?0.46 as the mother undergoes a series of physiological changes with the growth and development of her fetus[15].

4 groups of peripheral blood samples with the HCT values of 0.39?0.45 were pushed on the surface of hydrogel substrate. According to the parameters mentioned above, the pushing speed, the pushing angle and the whole-blood volume were investigated and optimized in the process of cell smear preparation. After optimization, the pushing effect was shown in Figure 6 (Color online) (smear speed: 100 mm/s, pushing angle: 40°, blood volume:5 μL). The Fig. 6 compares the effects of a stained cell smear on slide substrate, a cell smear on hydrogel membrane substrate and a standard cell smear prepared by a fully automatic blood analyzer Mindray SC-120 which is currently used in clinical tests. The front, middle and tail ends of the three cell smears were analyzed by microscopic imaging system, repectively. Five sampling points were randomly selected from each part within the 0.3 mm2FOV (field of view) of the imaging system to obtain the cell distribution images of the smears. The cells were then counted using ImageJ software. The cell distributions on the smears with 4 groups of blood samples are shown in Fig. 7. As can be seen from the figure, the cells aggregate and overlap on the smear with slide substrate, while the cells are in a single layer format adhere to hydrogel membrane substrate and are distributed uniformly. By comparing the front and tail parts of the cell smears, it can be seen that the cell smear with hydrogel membrane substrate shares the same monolayer cell distribution with the standard smear. In addition, the average of cell density is 19.3% higher than that of the standard smear, thus improving the efficiency of the NRBC observation and detection based on cell smear.

Fig. 6 Comparison of self-made cell smear and Mindray SC-120 standard cell smear: (a-c) slide; (d-f) hydrogel membrane; (g-i) Mindray SC-120圖 6 自制細胞涂片與邁瑞SC-120標準細胞涂片效果比對分析：（a-c）玻片；（d-f）水凝膠膜；（g-i）邁瑞SC-120

Fig. 7 Cell distribution statistics圖 7 細胞分布統(tǒng)計

The performance parameters of the prototype machine proposed in this paper were compared with those of the commercial Mindray SC-120 automatic pushing machine. The comparison results are summarized in Table 1. Compared with Mindray SC-120, the proposed prototype has the following advantages: (1) The pusher-loading platform is modularized so that the various substrates (glass/hydrogel/releasable) can be made by changing the platform; (2) the scraping operation in traditional scraping-pushing process is not necessary, so the blood consumption is reduced down to only 4 μL; (3) the pusher can be a standard medical type, rather than a customized product, so the maintenance cost is reduced.

Tab. 1 Performance comparisons between the proposed prototype and Mindray SC-120 automatic pushing-staining machine表 1 本樣機與邁瑞SC-120全自動推片染色機的性能參數(shù)對比

3.3 NRBC release under near-infrared response

3.3.1 Recognition and release of NRBCs

The NRBC recognition and release based on cell smear preparation technique were achieved by using the photothermal response properties of hydrogel. The prepared cell smear was placed in the microscopic imaging system. Then the NRBC was identified and determined according to its morphological features, such as round nucleus, a nucleuscytoplasm ratio of less than 1/2, no cytoplasmic granules, and nucleus amesiality[16]. As shown in Fig. 8(a), the NRBCs in the cell layer on the smear were identified and located, and then irradiated for 90 s by an 808 nm laser source (laser power:100 mW/mm2) fixed at a distance where a 1.7 mm spot was formed. Finally, they were rinsed with deionized water and dried, and observed under a microscope.

As can be seen from the characterization results (Fig. 8(b)), almost all the cells fall off with the photothermal melting of the hydrogel membrane within the fixed area of hydrogel membrane irradiated by laser. Therefore, the fixed-point release of cells can be achieved by introducing photothermally responsive hydrogel.

Fig. 8 (a) Recognition and localization of NRBC; (b-d)comparison of cell release areas before and after laser convergence with different spot diameters.(b) D = 1 700 μm; (c) D = 600 μm; (d) D = 300 μm圖 8 （a）NRBC識別定位結(jié)果；（b-d）激光會聚前后細胞釋放區(qū)域比較：（b）光斑直徑D = 1 700 μm；（c）光斑直徑D = 600 μm；（d）光斑直徑D = 300 μm

3.3.2 Optimization of laser convergence system

The fixed-point release of NRBCs was achieved through direct irradiation of 808-nm laser.However, the spot diameter directly projected onto the cell smear surface was 1.7 mm and required further adjustment relative to the cell size. Therefore, a laser convergence system was built by connecting the laser with a collimating lens and a convergent lens to reduce the spot diameter on the smear surface. This system could not only ensure the function of cell release, but also improve the accuracy of NRBC release. By substitutingω0= 300 μm andω0= 150 μm into Equation (2) respectively, the focal lengthsF= 100 cm andF=50 cm can be obtained. SinceF2is a fixed distance (F2= 20 cm), the focus diameter can be adjusted by adjustingF1=80 cm andF1= 30 cm, whereF1is the distance between the convergent lensC1 and the dichromatic mirror. The photothermal releases generated by the converged spots of about 600 μm and 300 μm are shown in Figure 8(c) and Figure 8(d) respectively. The micro-characterization results show that the proposed laser convergence system can scale down the cell-releasing area to about 271.2 μm (in diameter), while achieving the same photothermal conversion effect.

4 Conclusion

In this paper, a monolayer cell smear based on hydrogel membrane was prepared by automatic pushing technique. The prepared hydrogel membrane had the property of near-infrared light response and showed photothermal conversion under 808 nm laser irradiation. The processing parameters of hydrogel membrane substrate and cell smear were optimized. The whole blood cells were spread into a single layer on the surface of the hydrogel membrane substrate with a uniform thickness of 200 μm. A laser convergence system and a microscopic imaging system were constructed using an 808 nm laser source and applied to cell smears. By designing and constructing the light path for laser convergence and microscopic imaging, the light source was focused on a fixed area of the cell smear to realize the cell recognition and release. The results showed that when the pushing speed was 100 mm/s and the pushing angle was 40°, the prepared cell smear had uniform cell distribution and an average cell density 19.3% higher than a standard smear. While keeping laser power at the cell-releasing level, this technique can reduce the spot diameter to about 300 μm, so as to realize the cell release and enrichment. This paper provides a new technical approach that can be combined with automatic microscanning imaging and microneedle extraction in subsequent studies to efficiently and accurately extract the NRBCs for noninvasive prenatal diagnoses.

——中文對照版——

1 引 言

產(chǎn)前診斷對于唐氏綜合征、神經(jīng)管缺陷、單基因疾病等出生缺陷的早期診斷和篩查至關(guān)重要[1]，目前產(chǎn)前診斷主要分為有創(chuàng)性診斷和無創(chuàng)性診斷。有創(chuàng)性診斷包含羊膜槍穿刺、臍血取樣以及絨毛膜穿刺等[2]，是通過侵入式方法進行的，在診斷過程中往往伴隨著流產(chǎn)或羊水溢出、感染等風險[3]。因此，開發(fā)以非侵入式進行無創(chuàng)性產(chǎn)前診斷是當前產(chǎn)前診斷技術(shù)發(fā)展的研究重點和主流趨勢[4]。胎兒有核紅細胞（NRBCs）包含了胎兒全部基因組，具有孕早期表達（6周在外周血中表達，12?14周表達量達到最高），在母血細胞群中可鑒別性高，不會出現(xiàn)與母體細胞混淆的情況，具備明顯的細胞形態(tài)學特征，易識別，是應(yīng)用于產(chǎn)前診斷的首選胎兒細胞[5]。然而，其在母體外周血中數(shù)量極其稀少，如何從外周血中對有核紅細胞進行識別分離是產(chǎn)前診斷中的一大挑戰(zhàn)[6]。

目前已有的對外周血細胞進行分離富集的方法主要有密度梯度離心富集、磁激活細胞分選法、熒光激活細胞分選法以及微流控芯片等[7-9]，這些方法多以抗體與細胞特異性結(jié)合為核心進行分離檢測，操作流程復(fù)雜、檢測周期較長且成本較高。在已有的研究基礎(chǔ)上簡化細胞分離和富集過程是當前提高產(chǎn)前診斷亟待解決的問題。

采用推片技術(shù)制作良好的細胞涂片用于血液細胞形態(tài)學檢查是臨床應(yīng)用中一種重要的細胞檢測技術(shù)[10]。傳統(tǒng)的人工制作細胞涂片受限于人工操作的不可控性，常導致細胞重疊、破裂等而無法標準化。通過自動化細胞涂片制備機可以實現(xiàn)單層細胞涂片的標準化制備，結(jié)合光學成像對有核紅細胞進行識別。為了實現(xiàn)有核紅細胞后續(xù)的分離以進行進一步測定應(yīng)用，目前如熱響應(yīng)釋放系統(tǒng)，可以在生理溫度下實現(xiàn)捕獲細胞的釋放，并使釋放細胞保存足夠的活性，這種方法雖然能夠?qū)崿F(xiàn)大面積的細胞釋放，但是無法對固定區(qū)域進行定點釋放。相對應(yīng)的，光刺激響應(yīng)系統(tǒng)由于其可操作性目前已被作為一種理想的可操控釋放體系[11]，其中，近紅外光相較于紫外光對細胞的傷害較小，穿透力更強，更適用于構(gòu)建光響應(yīng)釋放體系，通過在熱響應(yīng)釋放系統(tǒng)中引入光響應(yīng)體系，達到實現(xiàn)更高效的定點釋放目的。本文提出利用水凝膠材料的成膜以及光熱特性[12]，在溫敏水凝膠明膠中引入MXene二維材料，將MXene的近紅外光熱效應(yīng)和熱響應(yīng)凝膠基質(zhì)相結(jié)合，在此基礎(chǔ)上制備自動化單層細胞涂片制備機，建立激光會聚和顯微成像系統(tǒng)，解決了傳統(tǒng)依賴于抗原抗體特異性結(jié)合的捕獲與分離釋放的問題，通過對各模塊的結(jié)構(gòu)和材料的性能優(yōu)化，實現(xiàn)了有核紅細胞的識別與定點釋放，為無創(chuàng)產(chǎn)前檢測提供了一種新的途徑。

2 細胞涂片制備機系統(tǒng)設(shè)計和識別釋放系統(tǒng)設(shè)計

2.1 控制系統(tǒng)總體設(shè)計

圖1 所示為應(yīng)用于識別釋放有核紅細胞的自動推片與激光會聚系統(tǒng)結(jié)構(gòu)示意圖。其中，自動推片系統(tǒng)由機械運動子系統(tǒng)、自適應(yīng)涂片系統(tǒng)、微結(jié)構(gòu)玻片載臺以及電控子系統(tǒng)組成。XY軸滑臺的二維運動由機械運動子系統(tǒng)控制，進行推片速度和角度的調(diào)控。自適應(yīng)涂片結(jié)構(gòu)具備雙自由度，可實現(xiàn)涂片與其接觸表面之間的自適應(yīng)狀態(tài)微調(diào)，結(jié)合運動子系統(tǒng)的二維運動，可達到調(diào)控涂片、載玻片三維運動的目的。微結(jié)構(gòu)玻片載臺可承載（25 mm × 75 mm）標準顯微鏡載玻片，用于水凝膠基底涂片以及細胞涂片的制備。電控子系統(tǒng)用于整體涂片系統(tǒng)各模塊之間的通信與交互操作的控制。

通過自動推片系統(tǒng)在載玻片制備水凝膠膜，以此為基底膜，在其表面進行推片獲得具備單層血細胞膜的細胞涂片。其次，選用Wright-Giemsa染色法對血細胞膜進行細胞形態(tài)學染色，該方法作為臨床血液學檢測方法具備染色效果清晰、核質(zhì)對比明顯、操作簡便以及染色快速等優(yōu)點[13]。細胞涂片經(jīng)過染色可依據(jù)細胞形態(tài)于光學顯微鏡下進行NRBC的識別與定位。識別定位后的細胞涂片置于激光會聚與顯微成像系統(tǒng)下，根據(jù)水凝膠基底的近紅外響應(yīng)性質(zhì)，選取808 nm激光進行定點照射，實現(xiàn)定點區(qū)域的光熱轉(zhuǎn)換釋放有核紅細胞。

2.2 自動推片系統(tǒng)設(shè)計

自動推片系統(tǒng)包括機械運動子系統(tǒng)、自適應(yīng)涂片系統(tǒng)、微結(jié)構(gòu)玻片載臺以及電控子系統(tǒng)4部分。其中，機械運動子系統(tǒng)設(shè)計原理如圖2所示，該系統(tǒng)分為速度調(diào)節(jié)和角度調(diào)節(jié)兩大模塊。速度調(diào)節(jié)模塊包括X軸滑臺、載體底座和閉環(huán)步進電機，可實現(xiàn)0~150 mm/s范圍內(nèi)的速度調(diào)控；角度調(diào)節(jié)模塊由Y軸滑臺、涂片結(jié)構(gòu)底座、閉環(huán)步進電機以及R軸減速步進電機組成，推片時可實現(xiàn)20°~50°內(nèi)的角度調(diào)節(jié)。

自適應(yīng)涂片系統(tǒng)具備雙自由度，包含涂片夾、推進方向上相互垂直的兩組鉸鏈，其可自適應(yīng)微調(diào)涂片，與接觸底面保持貼合的狀態(tài)。微結(jié)構(gòu)玻片載臺整體設(shè)計包含玻片承載槽和廢液收集槽，其中，玻片承載槽表面與玻片表面經(jīng)由設(shè)計加工呈現(xiàn)200 μm的高度差，用于推片制備厚度均勻的水凝膠基底膜；廢液收集槽用于推片到底部時收集多余的水凝膠樣品，避免回流破壞水凝膠膜、造成污染。電控子系統(tǒng)包含上位機控制系統(tǒng)、運動控制卡、電機驅(qū)動器、光電限位開關(guān)以及供電電源。通過單片機制作上位機控制軟件，將串口與運動控制卡連接，實現(xiàn)推片速度與角度兩大模塊的交互操作調(diào)控。

2.3 細胞識別釋放系統(tǒng)設(shè)計

根據(jù)水凝膠基底膜的性質(zhì)與細胞涂片的設(shè)計原理，為了實現(xiàn)有核紅細胞的識別與定點釋放，設(shè)計如圖3所示的細胞識別釋放系統(tǒng)，分為激光會聚子系統(tǒng)、明場照明子系統(tǒng)和顯微成像子系統(tǒng)3部分。根據(jù)水凝膠基底膜的近紅外響應(yīng)特性，以808 nm激光光源為主體，結(jié)合準直鏡和會聚鏡（C1）組成激光會聚子系統(tǒng)，用于定點照射激發(fā)細胞涂片產(chǎn)生光熱效應(yīng)，從而釋放細胞。為了觀察細胞釋放情況，在激光會聚子系統(tǒng)的基礎(chǔ)上構(gòu)建明場照明子系統(tǒng)和顯微成像子系統(tǒng)。選用明場光源和科勒鏡組搭建明場照明子系統(tǒng)，通過二向色鏡達到明場光源和808 nm激光光源公路共用的目的。隨后，通過由顯微物鏡、反射鏡、會聚鏡（C2）以及成像探測器構(gòu)建的顯微成像子系統(tǒng)模塊進行后續(xù)細胞釋放效果的觀察。

構(gòu)建激光會聚系統(tǒng)實現(xiàn)近紅外響應(yīng)定點釋放細胞，激光被聚焦后形成一個微小光斑，光斑越小，投射到細胞涂片表面的面積就越小，釋放細胞的效率和精度也越高。為了調(diào)控照射光斑大小，盡可能縮小光斑直徑，從而縮小定點釋放的細胞范圍，根據(jù)高斯公式[14]：

其中，ω1為聚焦后的光斑直徑，F(xiàn)為會聚鏡C1的焦距，F(xiàn)=F1+F2，其中F1為會聚鏡到二向色鏡的距離，F(xiàn)2為二向色鏡到細胞涂片的反射距離，ω0為激光高斯光束的束腰半徑，會聚鏡C1兩側(cè)等相位面上的光斑大小和光強分布相同，因此ω0又為準直后的光斑半徑。將激光波長λ（808 nm）、準直后的光斑半徑ω0（850 μm）作為常數(shù)代入式（1）可得激光會聚后的光斑直徑為：

基于自動推片機制備的單層細胞涂片，明場光源經(jīng)由科勒鏡組照射到細胞涂片表面，再結(jié)合顯微物鏡、反射鏡和會聚鏡的成像系統(tǒng)被探測器獲取。通過位移平臺的移動，實現(xiàn)細胞涂片的全載片成像，從而進行有核紅細胞的識別與定位。激光光源經(jīng)由準直鏡和會聚鏡準直會聚照射在已識別定位的目標區(qū)域，產(chǎn)生近紅外響應(yīng)光熱轉(zhuǎn)換，從而實現(xiàn)細胞定點釋放。最后由顯微成像系統(tǒng)成像表征細胞釋放性能。

3 實驗與結(jié)果

3.1 水凝膠推片的制備與優(yōu)化

實驗用水凝膠為課題組前期制備的二維材料MXene復(fù) 合 明 膠（C102H151O39N31, gelatin）材料，將明膠與MXene以200:1的質(zhì)量比在恒溫38 ℃的水浴條件下攪拌混合均勻，制備得到水凝膠。制備好的水凝膠經(jīng)磁力攪拌后保持在38 ℃的恒溫溶融狀態(tài)，通過搭建的自動推片機推涂在玻片表面，自然冷卻形成一層表面平整且厚度均勻的水凝膠薄膜。圖4為基于單片機控制軟件設(shè)計制造的樣機結(jié)構(gòu)，玻片載臺位于水平位置，推片載臺與玻片載臺形成夾角，其底部與玻片載臺平行，通過調(diào)控推片的速度和角度對水凝膠膜的制備進行優(yōu)化。首先手動用移液器吸取一定量的凝膠溶液，均勻滴在玻片前端，啟動推片，推片向下，底部水平接觸到玻片，推片與玻片形成一定的角度，向前推動從而在玻片表面制備水凝膠薄膜。

實驗過程中，選取不同的推片速度（20 mm/s、30 mm/s和40 mm/s），推片角度（25°、30°和35°）以及凝膠用量（200 μL、250 μL和300 μL）進行正交實驗，優(yōu)化水凝膠膜制備參數(shù)。實驗結(jié)果表明涂片速度越快、涂片角度越大則越易導致凝膠膜破損；凝膠用量則主要影響凝膠膜是否能完整覆蓋玻片。經(jīng)重復(fù)實驗后，優(yōu)化水凝膠推片參數(shù)如下：涂片速度為30 mm/s，涂片角度為30°以及凝膠用量為250 μL。采用掃描電子顯微鏡（SEM）和原子力顯微鏡（AFM）觀察表征制備好的水凝膠推片，如圖5（a），5（b）所示，所制備的水凝膠膜厚度大約在200 μm左右，且厚度均勻，其表面平均粗糙度Ra= 1.31 nm，為后續(xù)全血展開提供了較好的表面平整光滑性。對復(fù)合明膠推片進行紫外吸收測試，結(jié)果如圖5（c）所示。從圖5（c）的測試結(jié)果可以看到，所制備的復(fù)合明膠在~800 nm處具有明顯的吸收峰，可以實現(xiàn)近紅外響應(yīng)產(chǎn)生光熱轉(zhuǎn)化?；谏鲜鰞?yōu)化條件制作了水凝膠推片，采用808 nm激光照射對其進行光熱性質(zhì)測試，表征結(jié)果如圖5（d）所示。圖中分別為明膠、復(fù)合明膠以及復(fù)合明膠染色后在808 nm激光照射下的光熱響應(yīng)曲線（激光功率為150 mW/mm2），可見：明膠在激光照射下溫度基本不變，無光熱效應(yīng)；復(fù)合明膠照射區(qū)域的溫度隨著激光照射時間變長而逐漸升高，在120 s內(nèi)可快速升溫到37 ℃，360 s內(nèi)最高溫度可達47 ℃，可應(yīng)用于后續(xù)的細胞釋放。同時為驗證Wright-Giemsa染色法是否會對材料的光熱特性造成影響，在相同實驗條件下對染色后的復(fù)合明膠進行了激光照射實驗?？梢?，在150 mW/mm2功率下，染色后的復(fù)合明膠在20 s內(nèi)可升溫至37 ℃，320 s內(nèi)最高溫度可達到62.9 ℃；降低激光功率到100 mW/mm2，此時光熱轉(zhuǎn)換效應(yīng)與未染色的復(fù)合明膠基本一致。

3.2 基于水凝膠膜的細胞涂片制備與優(yōu)化

采用自行設(shè)計的自動細胞涂片制備機在水凝膠膜表面進行血液推片實驗，血液樣本來自于醫(yī)院臨床檢驗的紅細胞比容(HCT)范圍在0.39~0.45的外周血樣。正常女性的HCT參考值為0.35~0.45，而妊娠期間隨著胎兒的生長發(fā)育，母體會發(fā)生一系列的生理變化，HCT指標會隨妊娠期進展的不同在0.33~0.46范圍內(nèi)變化[15]。

采用4組HCT值均在0.39~0.45內(nèi)的外周血血樣，在水凝膠基底表面進行推片，根據(jù)上述制作優(yōu)化水凝膠推片的方法，對細胞涂片制備過程中的涂片速度、角度以及全血樣本用量進行考察優(yōu)化，優(yōu)化后（涂片速度為100 mm/s，涂片角度為40°，血液用量為5 μL）推片效果如圖6（彩圖見期刊電子版）所示。圖6分別為玻片基底、水凝膠膜基底染色后 與目前在臨床檢驗中所使用的全自動血液分析儀邁瑞SC-120制備的玻璃基底標準細胞涂片的效果對比。通過顯微成像系統(tǒng)分別對這3種細胞涂片的前端、終端、尾端進行取樣分析，在該成像系統(tǒng)拍攝視野為0.3 mm2內(nèi)分別在各部分隨機選取5處作為取樣點，得到細胞涂片的細胞分布圖像，隨后利用ImageJ軟件進行細胞計數(shù)。圖7為4組血液樣本的細胞涂片細胞分布情況，從圖中可以看出，以玻片為基底的細胞涂片出現(xiàn)了細胞聚集重疊現(xiàn)象，而基于水凝膠膜基底的細胞涂片呈現(xiàn)細胞單層粘附且分布均勻緊密的形態(tài)。對比細胞涂片的前端和尾端部分可以看出，以水凝膠膜為基底所制作的細胞涂片與標準涂片具備相同的單層細胞分布形態(tài)，且平均細胞密度比標準涂片增加了19.3%，提高了以細胞涂片為基礎(chǔ)觀察檢測有核紅細胞的應(yīng)用效率。

本文搭建的樣機與目前邁瑞SC-120全自動推片機的性能參數(shù)對比結(jié)果如表1所示。本文所搭建的樣機相較于邁瑞SC-120有以下幾方面優(yōu)點：（1）推片載臺模塊化，可通過更換不同載臺制作玻璃/凝膠/可釋放等多類涂片；（2）推片模式無需先刮后推，降低用血量，最少僅需4 μL血液；（3）推片可使用標準醫(yī)用推片，無需使用配套定制推片，降低維護成本。

3.3 近紅外響應(yīng)釋放有核紅細胞

3.3.1 有核紅細胞的識別與釋放

利用水凝膠的光熱響應(yīng)特性，實現(xiàn)以細胞涂片制備技術(shù)為基礎(chǔ)的有核紅細胞識別與釋放。將所制作的細胞涂片置于顯微成像系統(tǒng)下，根據(jù)有核紅細胞的核型圓潤，核質(zhì)比小于1/2，胞質(zhì)無顆粒且細胞核偏向一側(cè)的形態(tài)學特征[16]進行識別并確定有核紅細胞區(qū)域。如圖8（a）所示，對細胞涂片細胞層中的NRBC進行了識別定位，然后使用808 nm激光光源進行照射，將激光器固定在光斑直徑為1.7 mm大小的距離，激光功率為100 mW/mm2，照射時間為90 s，最后用去離子水沖洗并干燥，在顯微鏡下進行觀察。

從表征結(jié)果（圖8（b））可以看出，激光照射的定點區(qū)域隨著水凝膠膜的光熱熔化，表面細胞也隨之脫落，基本無殘留細胞。因此，通過在細胞涂片制作中引入光熱響應(yīng)水凝膠可以實現(xiàn)細胞的定點釋放。

3.3.2 激光會聚系統(tǒng)的優(yōu)化

采用808 nm激光直接照射達到了有核紅細胞定點區(qū)域釋放的效果，然而，直射到細胞涂片表面的光斑直徑為1.7 mm，相較于細胞尺寸還需要進一步調(diào)整。通過準直鏡和會聚鏡連接激光器共同搭建激光會聚體系，縮小激光照射到細胞涂片表面的光斑直徑，在保證細胞釋放功能的同時，提升對有核紅細胞釋放的準確度。ω0分別取300 μm和150 μm，計算得到F分別為100 cm和50 cm，其中F2= 20 cm為固定距離，通過調(diào)節(jié)使F1分別為80 cm和30 cm，即會聚鏡C1到二向色鏡的距離，實現(xiàn)會聚直徑的調(diào)節(jié)。圖8（c）、8（d）分別是經(jīng)過會聚后直徑約為600 μm以及300 μm的光斑產(chǎn)生的光熱釋放結(jié)果，顯微表征結(jié)果表明所搭建的激光會聚系統(tǒng)可將細胞釋放區(qū)域縮小到直徑為271.2 μm左右，且達到了同等的光熱轉(zhuǎn)換效應(yīng)。

4 結(jié) 論

本文采用自動推片技術(shù)制備以水凝膠膜為基底的單層細胞涂片，所制備的水凝膠膜具備近紅外光響應(yīng)特性，在808 nm激光照射下將產(chǎn)生光熱轉(zhuǎn)換。優(yōu)化水凝膠膜基底以及細胞涂片的加工設(shè)計參數(shù)，在200 μm厚度均一的水凝膠膜基底表面將全血細胞進行了單層展開。基于808 nm激光響應(yīng)光源構(gòu)建了應(yīng)用于細胞涂片的激光會聚系統(tǒng)和顯微成像系統(tǒng)，通過設(shè)計和制作激光會聚和顯微成像光路，將光源聚焦到細胞涂片定點區(qū)域，以實現(xiàn)細胞的識別與釋放。結(jié)果表明，當推片速度為100 mm/s、角度為40°時制備出細胞分布均勻且平均細胞密度較標準片提升19.3%的細胞涂片。在保證激光功率達到可釋放細胞效果的同時，將光斑直徑縮小到~300 μm，從而實現(xiàn)細胞的釋放富集。本文為后續(xù)研究中結(jié)合自動顯微掃描成像技術(shù)以及顯微微針提取系統(tǒng)，高效、精準提取有核紅細胞，并應(yīng)用于無創(chuàng)產(chǎn)前診斷提供了一種新的技術(shù)途徑。