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Smart-aggregation imaging for single molecule localisation with SPAD cameras

View Article: PubMed Central - PubMed

ABSTRACT

Single molecule localisation microscopy (SMLM) has become an essential part of the super-resolution toolbox for probing cellular structure and function. The rapid evolution of these techniques has outstripped detector development and faster, more sensitive cameras are required to further improve localisation certainty. Single-photon avalanche photodiode (SPAD) array cameras offer single-photon sensitivity, very high frame rates and zero readout noise, making them a potentially ideal detector for ultra-fast imaging and SMLM experiments. However, performance traditionally falls behind that of emCCD and sCMOS devices due to lower photon detection efficiency. Here we demonstrate, both experimentally and through simulations, that the sensitivity of a binary SPAD camera in SMLM experiments can be improved significantly by aggregating only frames containing signal, and that this leads to smaller datasets and competitive performance with that of existing detectors. The simulations also indicate that with predicted future advances in SPAD camera technology, SPAD devices will outperform existing scientific cameras when capturing fast temporal dynamics.

No MeSH data available.


The smart-aggregation scheme.A sequence of bit-plane images are processed according to (i) filtering, (ii) molecule detection and positive photon events registration and (iii) summation of original bit planes according to aggregated photon arrival times.
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f2: The smart-aggregation scheme.A sequence of bit-plane images are processed according to (i) filtering, (ii) molecule detection and positive photon events registration and (iii) summation of original bit planes according to aggregated photon arrival times.

Mentions: The steps in the bit-plane smart aggregation algorithm are outlined in Fig. 2. The input to the system is the raw or unprocessed bit-plane images from the sensor. The first step is to apply a standard spatial Gaussian filtering, to enhance any molecule flashes16. The size (or width σ) of the Gaussian kernel is chosen so as to match the expected point spread function (PSF) of the molecule, readily estimated from the system optics. More specifically, σ is chosen to be one-third of the Airy disk radius, which is estimated from r = 0.61 λ/NA, where λ is wavelength and NA is the numerical aperture of the objective. The spatial filtering is followed by time averaging, carried out with a rolling “window”17, whose size corresponds to the shortest blink that can be reliably detected.


Smart-aggregation imaging for single molecule localisation with SPAD cameras
The smart-aggregation scheme.A sequence of bit-plane images are processed according to (i) filtering, (ii) molecule detection and positive photon events registration and (iii) summation of original bit planes according to aggregated photon arrival times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120354&req=5

f2: The smart-aggregation scheme.A sequence of bit-plane images are processed according to (i) filtering, (ii) molecule detection and positive photon events registration and (iii) summation of original bit planes according to aggregated photon arrival times.
Mentions: The steps in the bit-plane smart aggregation algorithm are outlined in Fig. 2. The input to the system is the raw or unprocessed bit-plane images from the sensor. The first step is to apply a standard spatial Gaussian filtering, to enhance any molecule flashes16. The size (or width σ) of the Gaussian kernel is chosen so as to match the expected point spread function (PSF) of the molecule, readily estimated from the system optics. More specifically, σ is chosen to be one-third of the Airy disk radius, which is estimated from r = 0.61 λ/NA, where λ is wavelength and NA is the numerical aperture of the objective. The spatial filtering is followed by time averaging, carried out with a rolling “window”17, whose size corresponds to the shortest blink that can be reliably detected.

View Article: PubMed Central - PubMed

ABSTRACT

Single molecule localisation microscopy (SMLM) has become an essential part of the super-resolution toolbox for probing cellular structure and function. The rapid evolution of these techniques has outstripped detector development and faster, more sensitive cameras are required to further improve localisation certainty. Single-photon avalanche photodiode (SPAD) array cameras offer single-photon sensitivity, very high frame rates and zero readout noise, making them a potentially ideal detector for ultra-fast imaging and SMLM experiments. However, performance traditionally falls behind that of emCCD and sCMOS devices due to lower photon detection efficiency. Here we demonstrate, both experimentally and through simulations, that the sensitivity of a binary SPAD camera in SMLM experiments can be improved significantly by aggregating only frames containing signal, and that this leads to smaller datasets and competitive performance with that of existing detectors. The simulations also indicate that with predicted future advances in SPAD camera technology, SPAD devices will outperform existing scientific cameras when capturing fast temporal dynamics.

No MeSH data available.