magnification, number of axes, processing method). To optimize the values for these settings, the software SQUIRREL
(Super-resolution Quantitative Image Rating and Reporting of
Error Locations) can be used [35]. SQUIRREL estimates a
resolution scaling function (RSF), which converts the pointspread function (PSF) of the super-resolved image to the PSF
of the diffraction-limited image. The RSF can also be manually
entered by the user as a Gaussian function, calculated from the
PSF of the diffraction-limited image and the PSF of the superresolution image [36]. SQUIRREL applies the RSF to the
SRRF image to generate a theoretical diffraction-limited
image. An experimental diffraction-limited image is generated
by averaging all the raw images in a SRRF burst. The theoretical diffraction-limited image is quantitatively compared to the
experimentally obtained one by calculating the absolute difference, the Pearson Correlation Coefficient, and the root-meansquare error [35]. Since the experimental and the theoretical
diffraction-limited images are generated from the same raw
data, any differences can be attributed to artifacts caused by
the SRRF reconstruction. Thus, it is possible to vary both
image acquisition and SRRF reconstruction parameters and
use the results of SQUIRREL to determine the parameter
values that optimize image reconstruction (Fig. 5d).
Acknowledgements
We thank Katheryn Rothenberg for assistance with Fig. 3, and
Christopher McFaul and Eric The ´veneau for comments of the
manuscript. G.S. is supported in part by an Ontario Graduate
Scholarship. Work in our lab is funded by grants to R.F.G. from
the Natural Sciences and Engineering Research Council of Canada
(418438-13), the Canada Foundation for Innovation (30279), the
Ontario Ministry of Economic Development and Innovation
(ER14-10-170), the Ted Rogers Centre for Heart Research
TBEP Seed Program, and the Canadian Institutes of Health
Research (156279). RFG is the Tier II Canada Research Chair in
Quantitative Cell Biology and Morphogenesis.
References
1. Yang X, Dormann D, Munsterberg AE, Weijer
CJ (2002) Cell movement patterns during gastrulation in the chick are controlled by positive
and negative chemotaxis mediated by FGF4
and FGF8. Dev Cell 3:425–437
2. Wyngaarden LA, Vogeli KM, Ciruna BG,
Wells M, Hadjantonakis A-K, Hopyan S
(2010) Oriented cell motility and division
underlie early limb bud morphogenesis. Development 137:2551–2558. https://doi.org/10.
1242/dev.046987
3. Gaggioli C, Hooper S, Hidalgo-Carcedo C,
Grosse R, Marshall JF, Harrington K, Sahai E
(2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for
Live Imaging in Drosophila Embryos
221
(Super-resolution Quantitative Image Rating and Reporting of
Error Locations) can be used [35]. SQUIRREL estimates a
resolution scaling function (RSF), which converts the pointspread function (PSF) of the super-resolved image to the PSF
of the diffraction-limited image. The RSF can also be manually
entered by the user as a Gaussian function, calculated from the
PSF of the diffraction-limited image and the PSF of the superresolution image [36]. SQUIRREL applies the RSF to the
SRRF image to generate a theoretical diffraction-limited
image. An experimental diffraction-limited image is generated
by averaging all the raw images in a SRRF burst. The theoretical diffraction-limited image is quantitatively compared to the
experimentally obtained one by calculating the absolute difference, the Pearson Correlation Coefficient, and the root-meansquare error [35]. Since the experimental and the theoretical
diffraction-limited images are generated from the same raw
data, any differences can be attributed to artifacts caused by
the SRRF reconstruction. Thus, it is possible to vary both
image acquisition and SRRF reconstruction parameters and
use the results of SQUIRREL to determine the parameter
values that optimize image reconstruction (Fig. 5d).
Acknowledgements
We thank Katheryn Rothenberg for assistance with Fig. 3, and
Christopher McFaul and Eric The ´veneau for comments of the
manuscript. G.S. is supported in part by an Ontario Graduate
Scholarship. Work in our lab is funded by grants to R.F.G. from
the Natural Sciences and Engineering Research Council of Canada
(418438-13), the Canada Foundation for Innovation (30279), the
Ontario Ministry of Economic Development and Innovation
(ER14-10-170), the Ted Rogers Centre for Heart Research
TBEP Seed Program, and the Canadian Institutes of Health
Research (156279). RFG is the Tier II Canada Research Chair in
Quantitative Cell Biology and Morphogenesis.
References
1. Yang X, Dormann D, Munsterberg AE, Weijer
CJ (2002) Cell movement patterns during gastrulation in the chick are controlled by positive
and negative chemotaxis mediated by FGF4
and FGF8. Dev Cell 3:425–437
2. Wyngaarden LA, Vogeli KM, Ciruna BG,
Wells M, Hadjantonakis A-K, Hopyan S
(2010) Oriented cell motility and division
underlie early limb bud morphogenesis. Development 137:2551–2558. https://doi.org/10.
1242/dev.046987
3. Gaggioli C, Hooper S, Hidalgo-Carcedo C,
Grosse R, Marshall JF, Harrington K, Sahai E
(2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for
Live Imaging in Drosophila Embryos
221