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

Colyer, Ryan A.; Scalia, Giuseppe; Villa, Federica; Guerrieri, Fabrizio; Tisa, Simone; Zappa, Franco; Cova, S.; Weiss, Shimon; Michalet, Xavier

doi:10.1117/12.874238

Cited by 32 publications

(32 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This can be addressed in the future by back-illuminated sensors and microlens arrays [69][70][71][72][73]. Also larger SPADs and thus a larger sensitive area per pixel become possible with newer microchip production processes that reduce the size of the electronics in each pixel, leaving more room for the SPAD [27,74]. These developments could give SPAD arrays the edge over other devices.…”

Section: Camera Comparisonmentioning

confidence: 99%

See 1 more Smart Citation

The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy

et al. 2013

View full text Add to dashboard Cite

Abstract:Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, transport, flow velocities and concentrations in an imaging mode. SPIM-FCS records correlation functions over a whole plane in a sample, which requires array detectors for recording the fluorescence signal. Several types of image sensors are suitable for FCS. They differ in properties such as effective area per pixel, quantum efficiency, noise level and read-out speed. Here we compare the performance of several low light array detectors based on three different technologies: (1) Single-photon avalanche diode (SPAD) arrays, (2) passive-pixel electron multiplying charge coupled device (EMCCD) and (3) active-pixel scientific-grade complementary metal oxide semiconductor cameras (sCMOS). We discuss the influence of the detector characteristics on the effective FCS observation volume, and demonstrate that light sheet based SPIM-FCS provides absolute diffusion coefficients. This is verified by parallel measurements with confocal FCS, single particle tracking (SPT), and the determination of concentration gradients in space and time. While EMCCD cameras have a temporal resolution in the millisecond range, sCMOS cameras and SPAD arrays can extend the time resolution of SPIM-FCS down to 10 μs or lower. References and links1. K. M. Berland, P. T. So, and E. Gratton, "Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment," Biophy. J. 68, 694-701 (1995). 2. P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, "Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one-and two-photon excitation," Biophy. J. 77, 2251-2265 (1999). 3. R. Brock, G. Vàmosi, G. Vereb, and T. M. Jovin, "Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy," Proc. Natl. Acad. Sci. U.S.A. 96, 10123-10128 (1999 0-1 (2010). 53. Z. Petrásek, P. Schwille, and Z. Petrášek, "Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy," Biophy.

show abstract

Section: Camera Comparisonmentioning

confidence: 99%

“…Multi-spot FCS approaches range from using stopped spinning disks [14] to spatial light modulators [19,26,27]. However, all of these approaches allow the measurement of only a limited number of widely separated spots, and thus cannot provide a contiguous image of a sample.…”

Section: Introductionmentioning

confidence: 99%

The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy

et al. 2013

View full text Add to dashboard Cite

show abstract

“…k d 6k s, where k is a constant much larger than 1, and the diameter of the excitation spot d is that of the Airy spot. In practice, larger separation may be needed to account for imperfect (nondiffraction-limited) PSF-generating excitation away from the PSF centre, an effect that will be compounded by the presence of many neighbouring spots [143] …”

Section: (A) Sample Excitation Parallelizationmentioning

confidence: 99%

“…The array function is controlled by an FPGA board containing 32 megabytes (MB) of memory and a universal serial bus (USB) communication module for data transfer to Owing to the lower sensitivity and larger dark count rate of this detector (greater than 1 kHz per SPAD, with some SPADs reaching greater than 100 kHz) [158], detection of single-molecule bursts turned out to be impossible [143]. Moreover, although we could generate up to 32 Â 32 excitation spots in the sample using a 40Â oil immersion objective lens (Plan-Neofluar 40Â, Zeiss, NA ¼ 1.3), the out-of-focus light generated by this large number of spots resulted in significant background signal in FCS measurement, leading to very low amplitude ACF curves.…”

Section: (Iii) 32 â 32 Pixel Cmos Spad Cameramentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

View full text Add to dashboard Cite

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

show abstract

“…In recent years, the availability of fast cameras has triggered the development of different spacially resolved modalities for FCS: these modalities are based on single-spot confocal microscopy [20], multi-spot confocal microscopy [21,22], line-confocal microscopy [23,24], spinning-disk microscopy [25], total internal reflection microscopy [26] and selective plane illumination microscopy [27,28]. All these techniques map the diffusion coefficients and other dynamic properties by calculating an ACF for each of potentially many pixels in software.…”

Section: Introductionmentioning

confidence: 99%

FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series

Buchholz¹,

Krieger²,

Mocsár³

et al. 2012

Opt. Express

View full text Add to dashboard Cite

Abstract:With the evolving technology in CMOS integration, new classes of 2D-imaging detectors have recently become available. In particular, single photon avalanche diode (SPAD) arrays allow detection of single photons at high acquisition rates (≥ 100 kfps), which is about two orders of magnitude higher than with currently available cameras. Here we demonstrate the use of a SPAD array for imaging fluorescence correlation spectroscopy (imFCS), a tool to create 2D maps of the dynamics of fluorescent molecules inside living cells. Time-dependent fluorescence fluctuations, due to fluorophores entering and leaving the observed pixels, are evaluated by means of autocorrelation analysis. The multi-τ correlation algorithm is an appropriate choice, as it does not rely on the full data set to be held in memory. Thus, this algorithm can be efficiently implemented in custom logic. We describe a new implementation for massively parallel multi-τ correlation hardware. Our current implementation can calculate 1024 correlation functions at a resolution of 10 μs in real-time and therefore correlate real-time image streams from high speed single photon cameras with thousands of pixels.

show abstract

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

Cited by 32 publications

References 17 publications

The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy

The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy

Development of new photon-counting detectors for single-molecule fluorescence microscopy

FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series

Contact Info

Product

Resources

About