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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.

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(a) Schematic representation of the output of a binary SPAD imager. Each pixel registers a 0 (no photon count, white) or 1 (photon count, grey) in time. (b) Representation of the operational frame capture of an emCCD and bit-plane imager for an example, single blinking molecule. The top graph represents the molecule intensity trace in time, the middle graph the collected intensity from a fixed frame rate emCCD. The grey regions represent data readout sectors. The bottom graph shows the difference between fixed frame capture and smart-aggregated frame capture for a SPAD CMOS camera. (c) Simulated point spread functions from the molecule emitter in each time frame as imaged in the emCCD.
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f1: (a) Schematic representation of the output of a binary SPAD imager. Each pixel registers a 0 (no photon count, white) or 1 (photon count, grey) in time. (b) Representation of the operational frame capture of an emCCD and bit-plane imager for an example, single blinking molecule. The top graph represents the molecule intensity trace in time, the middle graph the collected intensity from a fixed frame rate emCCD. The grey regions represent data readout sectors. The bottom graph shows the difference between fixed frame capture and smart-aggregated frame capture for a SPAD CMOS camera. (c) Simulated point spread functions from the molecule emitter in each time frame as imaged in the emCCD.

Mentions: The Quanta Image Sensor (QIS) concept11 projects the recent image sensor developments of read noise reduction, decreasing pixel size (and diminishing full well) to an imaging array of single photon photodetectors with a binary response. The binary states of either 0 (no photon detected) or 1 (at least one photon detected) provide limited information, therefore these are summed in space and/or time to form a spatio-temporally oversampled greyscale image frame. A binary SPAD camera is an example of a QIS, its raw output consisting of spatial information of binary bits or “bit-planes”, as illustrated in Fig. 1a, with each pixel producing a time-domain sequence of 1’s and 0’s.


Smart-aggregation imaging for single molecule localisation with SPAD cameras
(a) Schematic representation of the output of a binary SPAD imager. Each pixel registers a 0 (no photon count, white) or 1 (photon count, grey) in time. (b) Representation of the operational frame capture of an emCCD and bit-plane imager for an example, single blinking molecule. The top graph represents the molecule intensity trace in time, the middle graph the collected intensity from a fixed frame rate emCCD. The grey regions represent data readout sectors. The bottom graph shows the difference between fixed frame capture and smart-aggregated frame capture for a SPAD CMOS camera. (c) Simulated point spread functions from the molecule emitter in each time frame as imaged in the emCCD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: (a) Schematic representation of the output of a binary SPAD imager. Each pixel registers a 0 (no photon count, white) or 1 (photon count, grey) in time. (b) Representation of the operational frame capture of an emCCD and bit-plane imager for an example, single blinking molecule. The top graph represents the molecule intensity trace in time, the middle graph the collected intensity from a fixed frame rate emCCD. The grey regions represent data readout sectors. The bottom graph shows the difference between fixed frame capture and smart-aggregated frame capture for a SPAD CMOS camera. (c) Simulated point spread functions from the molecule emitter in each time frame as imaged in the emCCD.
Mentions: The Quanta Image Sensor (QIS) concept11 projects the recent image sensor developments of read noise reduction, decreasing pixel size (and diminishing full well) to an imaging array of single photon photodetectors with a binary response. The binary states of either 0 (no photon detected) or 1 (at least one photon detected) provide limited information, therefore these are summed in space and/or time to form a spatio-temporally oversampled greyscale image frame. A binary SPAD camera is an example of a QIS, its raw output consisting of spatial information of binary bits or “bit-planes”, as illustrated in Fig. 1a, with each pixel producing a time-domain sequence of 1’s and 0’s.

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.


Related in: MedlinePlus