H High-Thrroughpu ut Fluoreescence Correlat C tion Specctroscop py with SPAD Arrays A A Tosi1, F. Villa A. V 1, F. Gueerrieri1, S. Tisa1, S. Bellissai1, F. Zappaa1, G. Scaalia2, R. Colyyer2, S. Weisss2, and X. Michalet2 1) Dipaartimento di Elettronica e Informazio one, Politecn nico di Milanno, Italy 2) Chemistryy & Biochem mistry Dep., UCLA, U Los Angeles, A USA A A ABSTRACT A novel appproach to higgh-throughpu ut Fluorescennce Correlattion Spectrosscopy (HT-F FCS) has been n developed. HT-FCS m makes it possible to mo onitor simulttaneously different loca ations with different reeactions andd observing fa fast evolvingg dynamic syystems. In thhe present ap pproach, we exploit a Liiquid Crysta al on Siliconn (LCOS) to generate a pattern p of exxcitation spoots and a Siingle-Photon Avalanche Diode (SPA AD) array too detect the fl fluorescence light of the emission patth. The capa ability of thiss system at ssingle-molecu ule level hass been demonnstrated usingg R6G and Cy3B C has sam mple. 1. IINTRODUCTION Single–Mollecule Fluoreescence Specctroscopy (S SMFS) metho ods have perrvaded scienntific domain ns as diversee as super-ressolution imagging, structu ural biochemiistry or single-protein traacking in livve cells, yield ding insightss into outstannding fundam mental biolog gical questionns. Since they have to opeerate at the single-moleccule level, th hey generallly require loong acquisitiions to havee adequate staatistics. Therrefore, an in ncreased throoughput in FCS is desirable mainly ffor two reaso ons: i) manyy different reactions can be monitoreed together aat the same time thanks to the multti-spot geom metry; ii) fastt evolving dyynamic systems can be observed by acquiring th he same kind d of data from m different lo ocations andd pooling them m together too obtain a go ood statistic iin a short am mount of timee (Michelet eet al. 2010). 2. EXPE ERIMENT TAL SETUP A novel aapproach to High-Throughput Fluoorescence Correlation C Spectroscopyy (HT-FCS S) has beenn developed iin a confocall geometry, in i which a m microscopic volume v in a solution is illluminated with w a tightlyy focused laseer beam. In the preseent approachh, we exploitt a Liquid C Crystal on Sillicon (LCOS S) to generatte a pattern of o excitationn spots and a Single Photton Avalanche Diode (S SPAD) array to detect th he fluorescennce light of the t emissionn path. The L LCOS patternn is focused on the sampple plane by the t microsco ope objectivee (UPlan Apo, Olympus,, Center Vallley, PA; 600X, NA = 1.2) 1 generatiing diffractio on limited spots. The paattern re-em mitted by thee sample is m magnified in order o to ensu ure a perfect alignment with w the detecctor (Figure 11a). SPAD array Figure 1: a)) Optical setu up: LCOS, miicroscope andd SPAD arrayy. b) Focal sp pot generatioon: the interfe ference imagee generated byy the LCOS crreates a spot in i the focal pllane. Liquid Cryystal on Siliccon (LCOS) The LCOS (X10468-01, Hamamatsu u, Bridgewatter; NJ) is a spatial s frequency phase m modulator th hat relay on a pixel-by-pixxel basis. Ouur approach uses u the LCO OS in the reaal-space dom main to generrate a real-sp pace array off spots at an iintermediate focal plane in front of thhe LCOS, as shown in Figure 1b. S it is possibble to adjust the In the LCOS t spot num mber, size and d distance. Single-Photon Avalancche Diode arrray The SPAD array (desiggned and deeveloped by Dipartimentto di Elettro onica e Inforrmazione, Po olitecnico dii Milano) is a monolithicc array of “smart pixels””, laid out in n 32 rows by y 32 columnss and manuffactured in a standard higgh-voltage 0.35µm 0 CMO OS technologgy. Every pixel comprisees a 20-µm ddiameter SPA AD, a front-end electronnics and a prrocessing circcuitry for couunting photo ons (Figure 2). 2 Photo Dettection Efficiency (PDE)) tops 43% aat 5-V exceess-bias, Darrk Count R Rate (DCR) is about 4 kcps (countts per secon nd) at room m temperaturee and the inttegral afterpu ulsing probabbility is about 3% when n an hold-offf of 200 ns is i employed.. The maxim mum frame-raate depends on the systeem clock: wiith 100 MHzz system cloock, we achiieved a free-running speeed of 100 kfframe/s from m all 1024 pixxels working g (Guerrieri et e al., 2010).. The 32x32 SPAD arrayy fulfills the main requirrements of HT-FCS: H larrge number of pixels, hiigh sensitiviity (down to o the single-photon leveel) and very high acquissition speed (i.e. either high h frame-rrates or veryy short integ gration time-slots). Figgure 2: Simpllified scheme of the SPAD array and on ne pixel in dettail. 33. METHO ODS Fluorescencce Correlatioon Spectrosco opy is a techhnique that analyzes a the fluctuations f in fluorescen nce intensityy recorded froom a samplee, due to ch hanges in thhe number off particles en ntering or leeaving the fo ocal volume.. FCS is gennerally perfoormed at nan nomolar conncentrations that is a go ood comprom mise betweeen obtainingg enough signnal during the t finite tim me of the m measurementt and being able to obsserve separaate bursts off fluorescence light abovee the noise (C Colyer et al. 2010). In order to estimate thee diffusion constant c (D) and the con ncentration (C) of the saample, the in ntensity timee trace and thhe Auto Correelation Function (ACF) oof the lumineescence signal are compuuted. From th he time tracee it is possiblle to detect the t bursts an nd compute tthe transit tim me (d) of thee molecule in the excitattion volume.. The transit ttime is relateed to the difffusion coefficcient (D) by means of thee following fformula: (1) where is the standard deviation n perpendicuular to the op ptical axis off the Point SSpread Function (PSF) off the excitatioon volume. In order to compute allso the samp ple concentrration, the ACF A of the measured ssignal is fitteed with thiss formula: is the A ACF and 1 (2) a parameter proportional p to the conceentration (C): (3) where is the excitatioon volume and a is a paarameter thaat depends on o the ratio bbetween the backgroundd intensity annd the total inntensity of th he time trace.. Since it is ddifficult to measure m the parameters p known D0 aand C0. and , th hey are estim mated using a reference sample withh In HT-FCS,, the ACF cuurves computted from diff fferent pixels can be pooled together iin order to obtain a goodd statistic in a short time. Before pooling all the daata together, a calibration n of the ACFFs is necessaary to rescalee the curves. In fact, the parameters extracted intterpolating ACFs A of diffferent pixels are usually very spreadd due to the nnon uniform mity in the ex xcitation spoot size, in thee alignment and in the ppixel perform mance. Moree details abouut this proceddure and its validation v aree reported in n Colyer et all., 2010. 44. RESUL LTS Preliminaryy measuremeents with an 8x1 and 8xx8 spots exciitation patterrn has be doone with Rhodamine 6G G (R6G) and C Cyanine 3B (Cy3B) as saample in 2000mM NaCl buffer b with diifferent conccentrations off sucrose. An examplee of time trace is reporteed in Figure 3. The baselline of 10 kccps is due too the dark co ounts and thee backgroundd, the bursts correspond c to o a moleculee that has cro ossed the excitation volum me. Figuree 3: Example of time trace obtained from m R6G 1nM in 200mM NaCl buffer withhout adding sucrose. s In the inset,, a zoom of th he time trace. An examplee of fitted AC CF curve is reported r in F Figure 4. No ote that the high correlatioon in the firsst part of thee curve is an artifact due to the afterp pulsing (phootodetector noise n correlatted with the signal), and d therefore itt has to be takken into accoount in the fiitting. Figure 4: ACF obtaiined with R6G G 1nM in 200 0mM NaCl bu uffer without ssucrose. Diffusion, afterpulsing and completee fitting are allso showed. 5. CONCLU USION The feasibiility of singgle molecule HT_FCS hhas been pro oved with small s sub-arr rray (8x8 pixels) of thee detector. Neew measurem ments with 16x16 pixels aare in progreess. In the futurre, we will exxpand the ex xcitation patttern in orderr to use the entire e SPAD array, choossing the bestt spacing betw ween spots inn order to reduce the bacckground in each e pixel. 6. REFEREN NCES Michalet X,, Colyer R A, A et al., High h-throughputt single-moleecule fluoresccence spectrooscopyusing g parallel detection, P Proceeding SPIE, S Vol 760 08, 2010. Guerrieri F,, Tisa S, Tosi A, Zappa F, F Two-Dimeensional SPA AD-Imaging Camera for Photon Cou unting, IEEE Photonic Joournal, 20100. Colyer R, S Scalia G. et all., High-throughput FCS using an LC COS spatial light modulattor and an 8x x1 SPAD array, acceppted paper foor BioOptics, 2010.
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