Ultra high-throughput single molecule spectroscopy with a 1024 pixel SPAD

Ryan A. Colyera*, Giuseppe Scaliaa, Federica A. Villab, Fabrizio Guerrierib, Simone Tisac,

Franco Zappab, Sergio Covab, Shimon Weissa, Xavier Michaleta* aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA

bDipartimento di Elettronica ed Informazione, Politecnico di Milano, Milano, Italy cMicro Photon Devices, Bolzano, Italy

ABSTRACT Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness, conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition speed using a custom-CMOS 8x1 SPAD array. Here we present preliminary results with a 64X improvement in throughput obtained using a liquid crystal on silicon spatial light modulator (LCOS-SLM) and a novel standard CMOS 1024 pixel SPAD array, opening the way to truly high-throughput single-molecule spectroscopy. Keywords: single-molecule, photon counting, fluorescence, FCS, LCOS, CMOS, SPAD array, high-throughput.

1. INTRODUCTION

Single–Molecule Fluorescence Spectroscopy (SMFS) methods have found application in scientific domains as diverse as super-resolution imaging, structural biochemistry, and single-protein tracking in live cells, yielding insights into outstanding fundamental biological questions [1].

Since they have to operate at the single-molecule level (i.e, low concentrations), they generally require a long acquisition time (several minutes) to have adequate statistical accuracy. Therefore, an increased throughput in SMFS is desirable mainly for two reasons: i) many different reactions can be monitored together at the same time thanks to the multi-spot geometry; ii) fast evolving dynamic systems can be observed by acquiring the same kind of data from different locations and pooling them together to obtain good statistical accuracy before the dynamic system has changed [2].

In order to increase the single molecule throughput, data should be collected in parallel by means of a multi-spot excitation, fast multi-pixel detection, algorithms to handle many channels and suitable software to process all the data.

A novel approach to High-Throughput Fluorescence Correlation Spectroscopy (HT-FCS) has been developed in a

confocal geometry, in which multiple microscopic volumes in a solution are simultaneously illuminated with a tightly focused laser beam. Each spot of the excitation pattern is mapped in a pixel of a photon detector array. HT-FCS has been presented previously by us using 8 excitation spots with an 8x1 photon detector array [3-4]. In this article we present a description of the developed methods, and we present data obtained with a 32x32 photo detector array, showing a remarkable improvement in terms of throughput and parallelization. The experimental results show a factor of 64 improvement in FCS throughput, demonstrated with both beads and freely diffusing Cy3B in sucrose.

* ryancolyer@yahoo.com, michalet@chem.ucla.edu, Phone: 1-310-794-6693, Fax: 1-310-267-4672

Invited Paper

Single Molecule Spectroscopy and Imaging IV, edited by Jörg Enderlein, Zygmunt K. Gryczynski, Rainer Erdmann,Proc. of SPIE Vol. 7905, 790503 · © 2011 SPIE · CCC code: 1605-7422/11/$18 · doi: 10.1117/12.874238

Proc. of SPIE Vol. 7905 790503-1

Fluorescence Correlation Spectroscopy is a technique that analyzes the fluctuations in fluorescence intensity recorded from a sample, due to changes in the number of particles entering or leaving the focal volume. FCS is generally performed at nanomolar concentrations, which provides a good compromise between obtaining enough signal during the finite time of the measurement and being able to observe separate bursts of fluorescence light above the noise [5].

In order to estimate the diffusion constant (D) and the concentration (C) of the sample, the intensity time trace and the Auto Correlation Function (ACF) of the luminescence signal are computed. From the ACF of the time trace it is possible to detect the bursts and compute the transit time (d) of the molecule in the excitation volume. The transit time is related to the diffusion coefficient (D) by means of the following formula:

,4 (1)

where , is the beam waist (-1/e to 1/e) perpendicular to the optical axis (xy plane) of the Point Spread Function (PSF) of the excitation volume, s indicates a specific sample and k the single channel (excitation spot and corresponding detector pixel).

In order to compute the sample concentration, the ACF of the measured signal is fitted with the following formula:

1 1 (2)

is the ACF and is a parameter proportional to the concentration ( ), that depends on the sample and on the channel:

(3)

where is the excitation volume and is a parameter that depends on the ratio between the background intensity and the total intensity of the time trace per each channel.

Since it is difficult to measure the parameters and , , they are estimated using a reference sample with known D0 and C0, fitting and from the experimental data. In fact we have:

(4)

, 4 (5)

In HT-FCS, the ACF curves computed from different pixels can be merged together in order to obtain a good statistic

in a short time. However, the parameters extracted from ACFs of different pixels (i.e., different channel k) are usually very spread due to the non-uniformity in the excitation spot size, in the alignment, and in the pixel performance. Therefore, before merging all the data together, a calibration of the ACFs is necessary to rescale the curves More details about this procedure and its validation are reported in [3].

In the present approach, we exploited a Liquid Crystal on Silicon (LCOS) to generate a pattern of excitation spots and

a Single Photon Avalanche Diode array (SPADa) to detect the fluorescence light of the emission path. A schematic of the overall setup is illustrated in figure 1. The LCOS pattern is imaged into the objective by means of a recollimating lens and focused on the sample plane by the microscope objective (UPlan Apo, Olympus, Center Valley, PA; 60X, NA = 1.2) generating diffraction limited spots. The pindot just before the objective is a spot of metal, which blocks all the unmodulated light. The pattern re-emitted by the sample is magnified in order to ensure a perfect alignment with the detector.

2. METHODS AND EXPERIMENTAL SETUP

Proc. of SPIE Vol. 7905 790503-2

FiguGreein anobjeinsetcan

2.1 Liquid C

The LCOSis commonlyapproach usefront of the Ldirections, thdestructive infor the first t

Figsam

This method distance and possible to roLCOS patter

ure 1. Schematicen lines represenn intermediate i

ective lens, whicht: The LCOS paalso be adjusted

Crystal on Sili

S (X10468-01,y used in a ses the LCOS inLCOS, as showhus with a pronterference [7]ime in [3].

gure 2. Schematme total phase dela

can be replicaangle. It make

otate the opticarn generation, c

of the experiment the emission liimage plane in fh focuses it into

attern degrees of.

icon (LCOS)

Hamamatsu, Bspecial-frequenn the real-spacewn in Figure 2.oper phase shif]. The applicat

tic of the Huygeay.

ated in order to es the alignmenal pattern and mcustom C and L

ental setup usingight path towardfront of the LCO the sample. CS

f freedom are co

Bridgewater; Nncy domain ane domain to ge According to ft pattern it is tion of this ma

ens-Fresnel princ

create a multi-nt procedure ofmove single spLabVIEW softw

g an LCOS-SLMds the detector (SOS. A recollima: Coverslip, Obj

ontrollable by so

NJ) is a phase mnd requires anerate a real-spthe Huygens-Fpossible to bu

athematical me

ciple: rays interfe

-spot excitationf the excitation

pots without touware has been

M. Blue lines reprSPAD array). A fating lens sends : objective lens,ftware. The patt

modulator that iterative and

pace array of sFresnel principuild a fixed pathod in a micr

ere constructively

n pattern with n spots with theuching the phydeveloped.

resent the excitafirst array of spothis pattern to t FM: flippable mtern pitch and nu

relay on a pixd time-consumspots at an interple light rays prattern exploitinroscopy setup h

at a focal point w

adjustable spote array pixels eysical setup. In

ation light path. ots is generated the back of an mirror. Top left umber of spots

el-by-pixel basming computati

rmediate focal ropagate outwa

ng the construchas been demo

when they have the

t number, size,easier, since it iorder to manag

sis [6]. It ion. Our plane in

ard in all ctive and onstrated

e

, is ge the

Proc. of SPIE Vol. 7905 790503-3

2.1 Single

What is rframe rate. performancebeen already

A SPAD mode, becauphoton. A neavalanche cacurrent by locircuitry reseshow single detected or w

Silicon Sof “smart pix

The measSPADlabs atcounting monumber of phLoad Quenchchip dimensithe compact it avoids theprovides this

Figcome

Measuredtemperature 300ns dead tglobal shuttephoton counperformed ddepending orunning speeaddressing anfrom a single8x8 portion o

e-Photon Avala

required in parFor both requs in compact m

y demonstrated is essentially a

use of the analoearby circuit prarrier multiplicowering the revets the SPAD photon sensiti

when a carrier iPADs can be i

xels” have alreasurements repot Politecnico diode, i.e. pulses hotons countedhing Circuit [1ions are 3.5x3.active area of

e need to sepas clipping of ou

gure 3. 32 x 3omprehensive of emory.

d Photo Detecis less than 5ktime (time afteer mode and rnts are stored during the inten the system

ed of 100,000 fnd reading oute pixel, i.e. a pof the 32x32 de

anche Diode a

rallel HT-FCS uirements Sinmicroelectroni[3,8,9].

a p-n junction ogy with gas crovides the ava

cation process verse voltage bfor the next divity and no reis thermally geintegrated withady been manuorted in this pi Milano, in a sarriving withi

d during conse13], an 8 bit co5 mm. Figure the entire chip

arately align a ut-of-focus ligh

2 pixel Single f a 20 µm-diame

tion Efficiencykcps (counts peer each detecti

readout is perfinto the latch

egration time clock of the rframes/s from t smaller sub-pphoton countinetector.

array

is an array of gle Photon Aics chips. The

biased above counters of ionalanche quenchis triggered welow breakdowetection cycle eadout noise,

enerated, thus lh the front-end ufactured [11,1aper have beenstandard high-vin a set integracutive time fraounter and a la3 shows the la, this array is ppinhole array.

ht.

Photon Avalanceter SPAD, a Var

y (PDE) tops er second) for ion during wh

formed with noes (one for eaof the follow

readout board:the whole 102

pixel sets of thng time-tagging

f detection chanAvalanche Diod

convenience o

breakdown fornizing radiationhing and recha

within the SPADwn. This preven

by raising thesince they proeading to a so circuitry in CM

12]. n obtained by voltage 0.35µmation time are ames. Every pixatch memory. Tayout of the entparticularly ide. The small ac

che Diodes arrariable-Load Que

43% at 5V ex70% of the pix

hich the detectoo blind-time bach pixel) whi

wing frame. Th with a conve

24 pixels [14]. he array chip, wg resolution of

nnels providindes (SPADs) of fluorescence

r exploiting ann. One digital arge mechanismD and the quents the destruce bias voltage ovide a digital called dark-coMOS technolo

means of a 32m CMOS technsummed up anxel comprises The pitch betwtire array and a

eal for confocactive area relat

ay. The inset shenching Circuit

xcess-bias, whixels and afterpor is gated-off

between consecile photon couhe maximum enient 100MHzEven higher fr

with an absoluf 10ns. In this p

ng high sensitivare ideal cane microscopy

n operation mopulse is produ

ms. In fact, upoenching circuitrtion of the devagain to the opulse signal o

unt [10]. ogy and single-

2 x 32 SPAD nology. The arrnd the digital a 20 µm-diam

ween pixels is a scheme of a l microscopy ative to the spo

hows a schema[8], an 8 bit cou

ile Dark-Counpulsing probabf). All pixels ocutive frames. unters are reseframe-rate canz system clock

frame rates canute maximum opaper we repor

vity together wdidates to reawith SPAD ar

ode also calleduced for every on photon detery stops the av

vice, while the operating levelonly when a p

-chip monolith

array developeray operates inoutput is there

meter SPAD, a V100µm and thesingle pixel. T

and spectroscopot spacing intr

atic of the pixeunter and a latch

t Rate (DCR) bility is about 3operate in para

In fact at eacet and readoutn be set by tk we achieved

n be easily achiof 100,000,000rt measuremen

with high ach high rrays has

d Geiger-detected ction, an valanche recharge . SPADs

photon is

ic arrays

ed in the n photon-efore the Variable-e overall

Thanks to py, since rinsically

el h

at room 3%, with allel in a ch frame t can be the user, d a free-ieved by

0 frame/s nts on an

Proc. of SPIE Vol. 7905 790503-4

This arrasingle-photonsmall active

2.1 Data tra

In order

compact boainterface prowith LabViewtool for mult

Figuand

The paramafter each desub-array regaccumulatingin order to afrom the FPGpossible to parray or withimage displaemphasizingperformed acompleteness

The secophoton counfunction (ACorder to extradiffusion coewith high Dinformation. noisy pixel wtool avoids can adjustable

ay detector fulfn level), very area [2].

ansmission an

to readout the ard featuring aovides fast comw, in order to cti-channel data

ure 4. Screenshosetup alignment

meters used to etection, duringgion-of-interesg counts (in facachieve higher GA to the PC plot in real-timh time traces, anay the average the desired s

and both actus of the inform

ond LabView pnts in time-depCF) [16,17]. It aact the parameefficient and th

DCR (higher thMoreover, sin

would negativecomputing the e threshold leve

fills the main rhigh acquisitio

nd postprocess

array chip, wa Xilinx Spartammunication frconfigure the aanalysis and p

ot of the two cust program; on the

configure the g which the detst so that data ct in many appframe rate) an

at a maximumme the counts o

nd to focus on e DCR can bsignal during ual and expecmation retrievedprogram, creatependent wavefalso enables enters of interesthe dye concenhan about 20 nce in the calibely affect the reACF and perfel.

requirements oon speed (i.e.

sing

e developed a an-3 FPGA, 32rom FPGA to array settings aplotting. Figure

stom tools devele right the multi-

array chip are tector is gated- readout can o

plication with lnd the duration

m transfer rate oof each pixel by

and compare tbe automaticallalignment. In

cted durations d by and from ted for post-proform traces wintering diffusiot ( and ) [ntration, a featu

kcps) could hbration procedesults of the en

forming the cal

of HT-FCS: lareither high fra

compact detec2 MB 16-bit wthe remote PC

and download te 4 shows a scr

loped with LabV-channel data an

the integration-off in order toobtain only thow photon raten of the overaof 22 MByte/sy means of intthe photon-couly subtracted order to veriof each mea

the FPGA. ocessing of theith adjustable on models for f[3]. Since a calure has been dhave too low

dure some averntire array. Thelibration proce

rge number of ame-rates or v

ction module ewide SDRAM C [15]. We devthe photon coureenshot of the

VIEW: on the lenalysis program.

n time (durationo reduce afterpuhe desired pixee, such as FCSall measuremens (USB 2) and tensity maps, tunting time depfor each pixefy the correctasurement are

e acquired dattime binning, fitting of all chlibration procedeveloped to f

of a signal-torage operationerefore, a darkedure on those

pixels, high severy short integ

employing an and an on-boa

veloped also a unts from the F

two tools.

ft the SPAD arr

n of each framulsing probabilels, the numbe, it is preferablnt. Photon coustored for pos

to see frames apendence of indel, which is cot data transmise compared in

ta, makes it poand to compu

hannels indepenss is required i

fully automate o-noise ratio t

ns among all pk count measur

pixels that sho

ensitivity (dowgration time-sl

Opal Kelly XEard PLL. The Uuser friendly i

FPGA, and also

ray configuration

me), the dead timlity), the addreer of bits-per-ple not to read a

unt data is dowst-processing. Iacquired by thedividual pixelsonvenient for ssion, crosschn order to ve

ossible to repreute the autocondently or comin order to comthe processing

to provide meixels are made

re is performedow a DCR hig

wn to the lots) and

EM3010 USB 2.0 interface

o another

n

me (time ess of the pixel for all 8 bits, wnloaded It is also e overall s. During

visually ecks are

erify the

esent the orrelation mbined in mpute the g. Pixels eaningful e, a very d and the gher than

Proc. of SPIE Vol. 7905 790503-5

With these two developed programs HT-FCS data acquisition and analysis is an automated, straight-forward, and user-friendly process.

3. RESULTS

3.1 Validation of the calibration process

To evaluate the capability of our system for performing an appropriate calibration we used 100nm fluorescent beads in H2O. Figure 5 shows ACFs for the beads, where the data are acquired simultaneously from 64 different channels (using an 8x8 sub-array of the SPAD Array). The comparison between row curves and calibrated curves shows that the calibration process correctly adjusts the curves so that every channel is yielding a similar measurement.

Figure 5. Left: Raw ACF curves of 100nm beads in H2O acquired from 8x8 channel. Right: Calibrated ACF curves, where the channel dependence is removed and all the curves collapse to show the diffusion behavior of the sample.

After this calibration it is possible to merge the curves obtaining an ACF curve 64 time faster than using one single channel. Therefore we increased the throughput by 64 times. 3.2 Single molecule measurements To evaluate the capability of our system for performing single molecule measurements with single fluorophores, we performed a number of measurements of Cyanine 3B (Cy3B) under various conditions. Figure 6 shows raw and calibrated ACFs for Cy3B 5nM in 200mM NaCl buffer with 40% w/w of sucrose. The data were acquired simultaneously from 64 different channels. Though the correlation amplitude, dampened by the background, is a little lower then with beads, after calibration and fitting the curves still give reliable diffusion times and concentrations.

Proc. of SPIE Vol. 7905 790503-6

F

Increasinoptical crossarray. To solvthe spot sepain identifyingof view, and

The feasib

array, using CA highly

position, spaphoton-counmolecule spacquisition afocus on impHT-FCS and

This work wWe thank Drstamped and

[1] Tho

1), J[2] X. M

Mill"Hig760

[3] R. AFCS201

[4] R.A"Hig

Figure 10. Exam

ng the number o-talk interferesve this problemaration in the sag the optimal ewill be the sub

bility of singleCy3B at low coflexible spot

acing, and angting SPAD arr

pectroscopy thand analysis of proving the illud other high-thr

was supported ir. Ted Laurencebinned data, r

ompson, N.L., J.R. Lakowicz,Michalet, R. A.lauda, I. Rech,gh-throughput 8, 2010.

A. Colyer, G. SS using an LCO0.

A. Colyer, G. Scgh-throughput

mple ACFs for 5n

of excitation sps with the abilitm the geometryample plane inexcitation geombject of future w

4. D

e molecule HToncentration. Ngeneration app

gle, and it is vray has been emhroughput. Nef the substantiaumination controughput single

in part by NIHe and Dr. Michespectively.

"Fluorescence , Plenum Press.Colyer,, G. Sc D. Resnati, S.single-molecu

Scalia, I. Rech, OS spatial light

calia, T. Kim, Imultispot sing

nM Cy3B in 40%

pots also causety to trivially s

y of the excitatin order to avoidmetry for good work.

DISCUSSION

T-FCS has beenNew measuremproach using avery convenienmployed for theew user-friendal quantity of dtrast of the exce-molecule spe

5. ACKNO

H grants R01 Ghael Culbertson

REF

Correlation Sps, New York, (1calia, T. Kim, M. Marangoni, Aule fluorescence

A. Gulinatti, Mt modulator an

I. Rech, D. Resgle-molecule sp

% sucrose before

es an increase inscale to higher ion pattern mud cross contribusignal strength

N AND CON

n demonstratedments with largan LCOS has nt for the come first time in H

dly LabVIEW data generated citation geometectroscopy tech

OWLEDGM

GM084327 andn for providing

FERENCES

pectroscopy", i1991). M. Levi, D. AhA. Gulinatti, Me spectroscopy

M. Ghioni, S. Cnd an 8x1 SPAD

snati, S. Maranpectroscopy," P

e (left) and after

n the backgrounumbers of ch

ust be changed, utions from othh while also fit

NCLUSIONS

d with a sub-aer sub-arrays abeen develop

mplicated task HT-FCS, whic

programs hawith multi-chatry when scalinhniques can ma

MENTS

d R01 EB00635g C code to imp

in (Topics in Fl

haroni, A. Chen. Ghioni, S. Ti

y using parallel

Cova, S. Weiss,D array," Biom

ngoni, M. GhioProceedings of

(right) calibratio

und due to out-hannels, such as

such as by sigher spots. This tting all 32x32

S

array (8x8 pixeare in progress.ped. It allows

of multi-pixeh has led to a 6

ave been deveannel FCS. Futng to larger nuake use of the f

53, and by NSplement multit

luorescence Sp

ng, F. Guerriersa, F. Zappa, Sl detection," Pr

, X. Michalet, medical Optics E

oni, S. Cova, S.f SPIE, 2010. 7

on.

-of-focus light.s using the full

gnificantly incrintroduces chaspots within th

els) of a 32x3. rapid changes

el alignment. A64X increase ineloped for auture developmmbers of pixelfull 32x32 SPA

SF grant DBI-0tau algorithms

pectroscopy, Vo

ri, G. Arisaka, JS. Cova, S. Weroceeding SPIE

"High-througExpress 1(5):1

. Weiss, X. Mic7571: p. 75710G

This l 32x32 reasing allenges he field

2 SPAD

s to spot A 32x32 n single-utomated ents will ls so that AD array

0552099. for time

olume

J. iss,

E, Vol

hput 408-31,

chalet G.

Proc. of SPIE Vol. 7905 790503-7

[5] I. Rech, D.R., S. Marangoni, M. Ghioni, S. Cova, "Compact-eight channel photon counting module with monolithic array detector," Proc. of SPIE 6771(13), 1-9 (2007).

[6] H.T. Dai, Y.J. Liu, X. Wang, J. Liu, "Characteristics of LCoS Phase-only spatial light modulator and its applications," Optics Communications, 2004. 238: p. 269-276.

[7] X.W. Wang, H. Dai, and K. Xu, "Tunable reflective lens array based on liquid crystal on silicon," Optics Express, 2005. 13(2): p. 352-357.

[8] S. Marangoni, I. Rech, M. Ghioni, P. MacCagnani, M. Chiari, M. Cretich, et al., "A 6 × 8 photon-counting array detector system for fast and sensitive analysis of protein microarrays," Sensors and Actuators, B: Chemical,2010 149(2), 420-426.

[9] E. P. Dupont, E. Labonne, C. Vandevyver, U. Lehmann, E. Charbon, M.A. Gijs, "Monolithic Silicon Chip for Immunofluorescence Detection on Single Magnetic Beads," Anal. Chem. 2010, 82 (1), 49–52.

[10] F. Zappa, S. Tisa, A. Tosi, S. Cova, "Principles and features of Single Photon Avalanche Diode Arrays," Sensors & Actuactors. A: Physical, Vol. 140, Issue 1, 103-112 (2007).

[11] F. Zappa, A. Tosi, A. Dalla Mora, F. Guerrieri, and S. Tisa “Single-photon avalanche diode arrays and CMOS microelectronics for counting, timing, and imaging quantum events,” Proc. of SPIE Vol. 7608, 2010.

[12] E. Charbon, “Single-photon Imaging in CMOS,” Proc. of SPIE Vol. 7780, 2010). [13] S.Tisa, F. Guerrieri, F. Zappa, “Variable-Load Quenching Circuit for Single-Photon Avalanche Diodes”, Optic

Express, Vol. 16 Issue 3, pp.2232-2244, January 2008. [14] Guerrieri F, Tisa S, Tosi A, Zappa F, “Two-Dimensional SPAD-Imaging Camera for Photon Counting”, IEEE

Photonic Journal, October 2010. [15] www.opalkelly.com/products/xem3010 (User Manual) [16] T.A. Laurence, S.F., T. Huser, "Fast, flexible algorithm for calculating photon correlations," Optics Letters

31(6), 829-831 (2006). [17] M.J. Culbertson, D.L.B., "A distributed algorithm for multi-tau autocorrelation," Review of Scientific

Instruments 78(4), 044102-1-6 (2007).

Proc. of SPIE Vol. 7905 790503-8