流式細胞計的基本結構流式細胞計主要由四部分組成。它們是:流動室和液流係統;激光源和光學係統;光電管和檢測係統;計算機和分析係統。
(1)流動室和液流係統:流動室由樣品管、鞘液管和噴嘴等組成,常用光學玻璃、石英等透明、穩定的材料製作。設計和製作均很精細,是液流係統的心髒。樣品管貯放樣品,單個細胞懸液在液流壓力作用下從樣品管射出;鞘液由鞘液管從四周流向噴孔,包圍在樣品外周後從噴嘴射出。為了保證液流是穩液,一般限製液流速度υ<10m/s。由於鞘液的作用,被檢測細胞被限製在液流的軸線上。流動室上裝有壓電晶體,受到振蕩信號可發生振動。
(2)激光源和光學係統:經特異熒光染色的細胞需要合適的光源照射激發才能發出熒光供收集檢測。常用的光源有弧光燈和激光;jiguangqiyouyiyalizijiguangqiweipubian,yeyoupeihekelizijiguangqihuoranliaojiguangqi。guangyuandexuanzezhuyaogenjubeijifawuzhidejifaguangpuerding。gongdengshizuichangyongdehuguangdeng,qifasheguangpudabufenjizhongyu300~400nm,很適合需要用紫外光激發的場合。氬離子激光器的發射光譜中,綠光514nm和藍光488nm的譜線最強,約占總光強的80%;氪離子激光器光譜多集中在可見光部分,以647nm較強。免疫學上使用的一些熒光染料激發光波長在550nm以yi上shang,可ke使shi用yong染ran料liao激ji光guang器qi。將jiang有you機ji染ran料liao做zuo為wei激ji光guang器qi泵beng浦pu的de一yi種zhong成cheng份fen,可ke使shi原yuan激ji光guang器qi的de光guang譜pu發fa生sheng改gai變bian以yi適shi應ying需xu要yao即ji構gou成cheng染ran料liao激ji光guang器qi。例li如ru用yong氬ya離li子zi激ji光guang器qi的de綠lv光guang泵beng浦pu含han有youRhodamine 6G水溶液的染料激光器,則可得到550~650nm連續可調的激光,尤在590nm處(chu)轉(zhuan)換(huan)效(xiao)率(lv)最(zui)高(gao),約(yue)可(ke)占(zhan)到(dao)一(yi)半(ban)。為(wei)使(shi)細(xi)胞(bao)得(de)到(dao)均(jun)勻(yun)照(zhao)射(she),並(bing)提(ti)高(gao)分(fen)辨(bian)率(lv),照(zhao)射(she)到(dao)細(xi)胞(bao)上(shang)的(de)激(ji)光(guang)光(guang)斑(ban)直(zhi)徑(jing)應(ying)和(he)細(xi)胞(bao)直(zhi)徑(jing)相(xiang)近(jin)。因(yin)此(ci)需(xu)將(jiang)激(ji)光(guang)光(guang)束(shu)經(jing)透(tou)鏡(jing)會(hui)聚(ju)。光(guang)斑(ban)直(zhi)徑(jing)d可由下式確定:d=4λf/πD。λ為激光波長;f為透鏡焦距;D為激光束直徑。色散棱鏡用來選擇激光的波長,調整反射鏡的角度使調諧到所需要的波長λ。為(wei)了(le)進(jin)一(yi)步(bu)使(shi)檢(jian)測(ce)的(de)發(fa)射(she)熒(ying)光(guang)更(geng)強(qiang),並(bing)提(ti)高(gao)熒(ying)光(guang)訊(xun)號(hao)的(de)信(xin)噪(zao)比(bi),在(zai)光(guang)路(lu)中(zhong)還(hai)使(shi)用(yong)了(le)多(duo)種(zhong)濾(lv)片(pian)。帶(dai)阻(zu)或(huo)帶(dai)通(tong)濾(lv)片(pian)是(shi)有(you)選(xuan)擇(ze)性(xing)地(di)使(shi)某(mou)一(yi)濾(lv)長(chang)區(qu)段(duan)的(de)光(guang)線(xian)濾(lv)除(chu)或(huo)通(tong)過(guo)。例(li)如(ru)使(shi)用(yong)525nm帶通濾片隻允許FITC(Fluoresceinisothiocyanate,異硫氰熒光素)發射的525nmlvguangtongguo。changbotongguoerxiangsexingfanshejingzhiyunxumouyibochangyishangdeguangxiantongguoerjiangcibochangyixiadelingyitedingbochangdeguangxianfanshe。zaimianyifenxizhongchangyaotongshitanceliangzhongyishangdebochangdeyingguangxinhao,jiucaiyongerxiangsexingfanshejing,huoerxiangsexingfenguangqi,laiyouxiaodijianggezhongyingguangfenkai。
(3)光電管和檢測係統:經熒光染色的細胞受合適的光激發後所產生的熒光是通過光電轉換器轉變成電信號而進行測量的。光電倍增管(PMT)最為常用。PMT的響應時間短,僅為ns數量級;光譜響應特性好,在200~900nm的光譜區,光量子產額都比較高。光電倍增管的增益從103到108可連續調節 ,因此對弱光測量十分有利。光電管運行時特別要注意穩定性問題,工作電壓要十分穩定,工作電流及功率不能太大。一般功耗低於0.5W;最大陽極電流在幾個毫安。此外要注意對光電管進行暗適應處理,並注意良好的磁屏蔽。在使用中還要注意安裝位置不同的PMT,因為光譜響應特性不同,不宜互換。也有用矽光電二極管的,它在強光下穩定性比PMT好。
從PMTshuchudedianxinhaorengranjiaoruo,xuyaojingguofangdahoucainengshurufenxiyiqi。liushixibaojizhongyibanbeiyouliangleifangdaqi。yileishishuchuxinhaofuduyushuruxinhaochengxianxingguanxi,chengweixianxingfangdaqi。xianxingfangdaqishiyongyuzaijiaoxiaofanweineibianhuadexinhaoyijidaibiaoshengwuxuexianxingguochengdexinhao,liDNAceliangdeng。lingyileishiduishufangdaqi,shuchuxinhaoheshuruxinhaozhijianchengchangyongduishuguanxi。zaimianyixueceliangzhongchangshiyongduishufangdaqi。yinweizaimianyifenxishichangyaotongshixianshiyinxing、陽性和強陽性三個亞群,它們的熒光強度相差1~2個數量級;而且在多色免疫熒光測量中,用對數放大器采集數據易於解釋。此外還有調節 便利、細胞群體分布形狀不易受外界工作條件影響等優點。
(4)計算機和分析係統:jingfangdahoudedianxinhaobeisongwangjisuanjifenxiqi。duodaodedaoshushihedianxinhaodemaichonggaoduxiangduiyingde,yeshiheguangxinhaodeqiangruoxiangguande。duiyingdaoshunianzongzuobiaotongchangdaibiaofachugaixinhaodexibaoxiangduishumu。duodaofenxiqichulaidexinhaozaijingmo-數shu轉zhuan換huan器qi輸shu往wang微wei機ji處chu理li器qi編bian成cheng數shu據ju文wen件jian,或huo存cun貯zhu於yu計ji算suan機ji的de硬ying盤pan和he軟ruan盤pan上shang,或huo存cun於yu儀yi器qi內nei以yi備bei調tiao用yong。計ji算suan機ji的de存cun貯zhu容rong量liang較jiao大da,可ke存cun貯zhu同tong一yi細xi胞bao的de6~8個ge參can數shu。存cun貯zhu於yu計ji算suan機ji內nei的de數shu據ju可ke以yi在zai實shi測ce後hou脫tuo機ji重zhong現xian,進jin行xing數shu據ju處chu理li和he分fen析xi,最zui後hou給gei出chu結jie果guo。除chu上shang述shu四si個ge主zhu要yao部bu分fen外wai,還hai備bei有you電dian源yuan及ji壓ya縮suo氣qi體ti等deng附fu加jia裝zhuang置zhi。
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