Autofocus software

We developed an Arduino based controller for shifting the focus lock position of reflection based autofocus systems. It was developed just with internal rough and ready hacked-solution use in mind, but since it might be useful to other groups, I’ve put it up on our lab GitHub. Documentation is admittedly brief, but I’m happy to answer queries.

In a tribute to certain open-microscopy pioneers, we have named this tool FocusMcFocussy.

BioRxiv preprint: Super-resolution microscopy fight club!

Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

Daniel SageThanh-An PhamHazen BabcockTomas LukesThomas PengoRamraj VelmuruganAlex HerbertAnurag AgarwalSilvia ColabreseAnn WheelerAnna ArchettiBernd RiegerRaimund OberGuy M. HagenJean-Baptiste SibaritaJonas RiesRicardo HenriquesMichael UnserSeamus Holden


With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

Introduction to microscopy – online resources

I realised we were lacking a list of good microscopy 101 resources for new Holden lab arrivals. Hopefully this list should be useful to others too. Thanks to the lovely sci-twitter community for pointing to or donating many of these resources.

Outstanding, comprehensive iBiology course on Microscopy (look at this first) :

For quick overviews:

More detailed microscopy lecture series:

Microscope manufacturer tutorials:

Other commercial resources:

Key textbooks

  • The fluorescence textbook: “Principles of Fluorescence Spectroscopy”by Lakowicz (on the group server)
  • Excellent single molecule protocols (a little dated but still very useful) “Single-Molecule Techniques: A Laboratory Manual“, Ed. TJ Ha

BioRxiv preprint on Caulobacter cresentus size control

We submitted a new preprint to bioRxiv, read it here:

Ambroise Lambert, a post-doc in the lab of Suliana Manley, EPFL, led a really exciting peace of work where we showed that Caulobacter cresentus cells use modulation of their constriction rate as a hitherto unappreciated means of cell size control. Ie, one of the ways in which Caulobacter cells control their size is by controlling how fast they divide.

How a motile cytoskeleton drives bacterial cell division

We just solved a 25 year old mystery: the basic mechanistic principle of bacterial cell division. We found that a motile bacterial cytoskeleton drives the motion of enzymes which build a partition wall across the middle of the cell. In the longer term, this may help us find new antibiotics to target this process.

Animation credit TU Delft / Scixel.

The story has just been published in Science, here.

Newcastle’s press office wrote a great pop science article about the discovery, here.

A big thanks to Calum Jukes, a graduate student in my lab, for all his hard work on this. And to our wonderful collaborators in Harvard, Indiana and Delft on this multi-lab team-up, it’s been a pleasure!


Alexandre W. Bisson Filho, Yen-Pang Hsu, Georgia R. Squyres, Erkin Kuru, Fabai Wu, Calum Jukes, Yingjie Sun, Cees Dekker, Seamus Holden, Michael S. VanNieuwenhze, Yves V. Brun, and Ethan C. Garner, Treadmilling by FtsZ Filaments Drives Peptidoglycan Synthesis and Bacterial Cell Division. Science. 355 (2017) 739-742. doi:10.1126/science.aak9973.



Localization microscopy challenge 2016 launched

Here’s one for all my fellow super-res geeks:

We would like to announce the second edition of Single Molecule Localization Microscopy Challenge at the upcoming SMLMS 2016.

After the success of the first challenge in 2013, we would like to continue the benchmarking of SMLM software, and to extend the challenge to key unaddressed areas of the field. This challenge will focus on two aspects of localisation microscopy:

– 3D, which despite being one of the most exciting applications of SMLM from day one has seen significantly less algorithm development than 2D

– Ultra High-density 2D intended for live cell applications. The 2D UHD challenge is specifically intended to allow the direct competition of both “localization” methods and image-based methods which do not necessarily resolve individual molecules, such as SOFI, sparse deconvolution and 3B.

We will also be updating the 2013 challenge with additional structures and conditions.

In order to tailor the challenge to the needs of the community, we will open a community consultation of the SMLM challenge 2016 draft specification. You are kindly invited to join the discussion. The consultation will take place via the SMLM challenge discussion group which will also serve as a discussion platform during the rest of the competition:!forum/lm-challenge-discuss

The draft specification is here:

The challenge is open to all individuals or teams, academic or corporate, that developed or are currently developing super-resolution software tools and algorithms. We are also open to submissions by expert users of a software.Your participation is important. It will allow the research community to better understand the current state of localization algorithms and software, and to discuss future directions of the field. The challenge is also an excellent platform for exchanging ideas, sharing your experience, and making your software more well-known to the users.

SMLMS 2016 will be the fifth edition of the Single Molecule Localization Microscopy Symposium. It is becoming one of the major event in the field, providing a fruitful platform for engineers and practitioners to interact and collaborate. The conference will be held on 28-30 August 2016 in Lausanne, Switzerland, incorporating a special session for the SMLM challenge:

Looking forward for your input,

From the organisation committee,

Daniel Sage
Thomas Pengo
Junhong Min
Hagai Kirshner
Bernd Rieger
Ricardo Henriques
Nils Gustafsson
Hazen Babcock
Guy Hagen
Martin Ovesny
Tomas Lukes
Seamus Holden

Publication: Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging

Carlini L*, Holden S*, Douglass K*, Manley S. Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging. PLoS ONE 2015.
*Equal author contribution

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.



Finally… 5 months post-arrival, a website!

In the Holden lab, we study biophysics in bacteria, especially bacterial cell division, using super-resolution microscopy. We are based at the Centre for Bacterial Cell Biology at the University of Newcastle.

Currently we are microscope building and getting the hang of our new favourite model organism, Bacillus subtilis (pictured in the header). Watch this space for new developments.