Single-molecule localization software applied to photon counting imaging

Liisa M. Hirvonen; Tiffany Kilfeather; Klaus Suhling

doi:10.1364/AO.54.005074

Applied Optics
Vol. 54,
Issue 16,
pp. 5074-5082
(2015)
•https://doi.org/10.1364/AO.54.005074

Single-molecule localization software applied to photon counting imaging

Liisa M. Hirvonen, Tiffany Kilfeather, and Klaus Suhling

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Liisa M. Hirvonen, Tiffany Kilfeather, and Klaus Suhling, "Single-molecule localization software applied to photon counting imaging," Appl. Opt. 54, 5074-5082 (2015)

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- Imaging Systems
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About this Article
History
- Original Manuscript: March 17, 2015
- Revised Manuscript: April 30, 2015
- Manuscript Accepted: May 1, 2015
- Published: May 25, 2015
Virtual Issues
Virtual Journal for Biomedical Optics Vol. 10, Iss. 6

Abstract

Centroiding in photon counting imaging has traditionally been accomplished by a single-step, noniterative algorithm, often implemented in hardware. Single-molecule localization techniques in superresolution fluorescence microscopy are conceptually similar, but use more sophisticated iterative software-based fitting algorithms to localize the fluorophore. Here, we discuss common features and differences between single-molecule localization and photon counting imaging and investigate the suitability of single-molecule localization software for photon event localization. We find that single-molecule localization software packages designed for superresolution microscopy—QuickPALM, rapidSTORM, and ThunderSTORM—can work well when applied to photon counting imaging with a microchannel-plate-based intensified camera system: photon event recognition can be excellent, fixed pattern noise can be low, and the microchannel plate pores can easily be resolved.

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	Single-Photon	Single-Molecule
Event shape and size	Determined by image intensifier and camera system	Determined by microscope PSF
Resolution	Limited by MCP pore size	Limited by localization precision and photon count
Background	Flat, camera noise only, negligible thermal emission background from intensifier	Uneven, variable, contains out-of-focus light
Pulse height distribution	A saturated, peaked pulse height distribution discriminates photons from readout noise, straightforward setting of threshold	Not peaked, decreasing single-molecule intensity distribution, threshold-setting not straightforward
Other features	Photon timing	3D imaging (astigmatism or bi-plane)

Program	Original Application	Timing	Spot Finding Method	Centroiding Method	References
QuickPALM	Molecules	–	Högbom CLEAN [74]	Modified center-of-mass	[68,69]
rapidSTORM	Molecules	–	Threshold	MLE with Gaussian PSF	[70,71]
ThunderSTORM	Molecules	–	Several (see text)	Several (see text)	[72,73]
In-house program	Photons	$\sqrt$	Threshold + size	Nonfitting (several)	[22]

Method	Time	Photons	FPN (%)
QuickPALM	105	1,044,088	27.55
rapidSTORM	7	1,265,365	8.67
ThunderSTORM: C-R	30	1,102,714	9.18
ThunderSTORM: C-IG-LS	54	1,067,230	4.98
In-house: Hyp. cos	12	673,982	43.06
In-house: Gaussian + CoG	12	1,113,517	83.33

Approximate Centroiding	Subpixel Centroiding	Time (min)	Photons	FPN (%)
Centroid of connected components	Centroid of local neighborhood	7	286,845	72.47
Centroid of connected components	Radial Symmetry	7	275,271	8.26
Centroid of connected components	LS (Gaussian)	15	266,485	5.07
Centroid of connected components	LS (Integrated Gaussian)	14	266,367	5.34
Centroid of connected components	MLE (Gaussian)	26	268,915	7.66
Centroid of connected components	MLE (Integrated Gaussian)	32	268,176	6.93
Local Maximum	Centroid of local neighborhood	6	321,872	136.58
Local Maximum	Radial Symmetry	7	332,262	32.21
Local Maximum	LS (Gaussian)	11	321,767	10.77
Local Maximum	LS (Integrated Gaussian)	10	321,768	11.22
Local Maximum	MLE (Gaussian)	14	321,865	30.95
Local Maximum	MLE (Integrated Gaussian)	20	321,866	30.39
Nonmaximum suppression	Centroid of local neighborhood	6	319,642	138.69
Nonmaximum suppression	Radial Symmetry	6	319,638	29.01
Nonmaximum suppression	LS (Gaussian)	11	319,640	11.43
Nonmaximum suppression	LS (Integrated Gaussian)	11	319,640	11.15
Nonmaximum suppression	MLE (Gaussian)	14	319,642	30.86
Nonmaximum suppression	MLE (Integrated Gaussian)	21	319,642	29.65

	Single-Photon	Single-Molecule
Event shape and size	Determined by image intensifier and camera system	Determined by microscope PSF
Resolution	Limited by MCP pore size	Limited by localization precision and photon count
Background	Flat, camera noise only, negligible thermal emission background from intensifier	Uneven, variable, contains out-of-focus light
Pulse height distribution	A saturated, peaked pulse height distribution discriminates photons from readout noise, straightforward setting of threshold	Not peaked, decreasing single-molecule intensity distribution, threshold-setting not straightforward
Other features	Photon timing	3D imaging (astigmatism or bi-plane)

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Applied Optics