Ultra high-throughput single molecule spectroscopy with a ... · Ultra high-throughput single...
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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.
* [email protected], [email protected], 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
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Proc. of SPIE Vol. 7905 790503-3
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Proc. of SPIE Vol. 7905 790503-4
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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
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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
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