Confocal.nl: The beauty of simplicity
Image: Dr Erik Manders has pioneered high resolution, high sensitivity, yet simple confocal microscopy for life scientists.
In late 2015, confocal microscopy pioneer, Dr Erik Manders, and Nikon Instruments manager, Peter Drent, formed Confocal.nl, to bring high resolution, high sensitivity, yet simple confocal microscopy, to life scientists worldwide.
Across two decades, Manders had watched how the image quality and sensitivity of confocal microscopes had undoubtedly improved, but was dissatisfied with the set-up, complexity and cost of new instruments.
"I wanted to make an optics-only microscope that didn't use image processing," explains Manders. "I knew how biologists think - if there is a 'black box' between their sample and the images on their screen then they do not feel in control of the imaging."
"So this was why I developed an optics-only method," he says. "The image that you get on your screen is a direct image of your sample; there is nothing in between, only light."
COS7 cells: These fibroblast-like cells are immunostained for alpha-tubulin AlexaFluor647. This image is a stitching from 3x3 field of views, blended together according to their optimal path of structure. Single images were taken using the 2s configuration of the RCM in imaging software, NIS Elements. [Andreas Kurz/Wuerzburg University].
So-called Re-scan Confocal Microscopy (RCM) is based on standard confocal microscopy extended with an optical, re-scanning unit that projects the user's image directly onto a CCD camera.
Crucially, the method provides a lateral resolution of 170 nm at 488 nm excitation - some 40% better than a conventional confocal microscope - as well as improved sensitivity, alongside the sectioning capability of a standard confocal microscope (see 'A higher resolution' at end of article).
During imaging, excitation lasers are directed towards a first scanning unit, which scans the laser light across the sample. Emitted light is then directed and focused though the system pinhole, to a second re-scan unit. From here, the light is then directed to the camera chip and scanned, point by point, to produce an image of the object.
Crucially, the set of re-scanning mirrors move with a larger amplitude than the first set of scanning mirrors. This gives an extra magnification of the image on the camera chip and leads to a higher resolution image.
Manders first taste for microscope development came in the mid 1990s, when after his degree in experimental physics at the University of Amsterdam and as part of his PhD, also in Amsterdam, he developed multi-colour confocal microscopy.
Working with confocal microscopy pioneer, Professor Brakenhoff, he built a two-colour confocal microscope and used this to analyse DNA replication. As part of his research, he also developed a series of now well-used coefficients (the Manders’ coefficients) to characterise the degree of overlap between images and demonstrate correlation between biomolecule pairs.
Post-doctoral positions followed at the Universities of Stockholm and Oxford, in which Manders focused on building new systems for the 3D imaging of live cells. Then, come the late 1990s, and now well-versed in the issues surrounding fluorescence live cell imaging, he returned to Amsterdam University, with PhD student, Ron Hoebe, to develop a method to reduce photobleaching and phototoxicity, caused by excitation light.
Induced pluripotent stem cell-derived (iPSC) neuron: Blue, MAP2 and Red, LIMPII [Image by Jeroen Kole, VU University Medical Center/Confocal.nl, and sample prepared by Sonia Vasquez, VU University].
By 2007, Manders and colleagues had developed Spatially Controlled Illumination Microscopy (SCIM) - originally known as Controlled Light Exposure Microscopy - which at the time, could reduce photobleaching and phototoxicity during fluorescence live cell imaging by up to ten times.
And working with Peter Drent, then general manager at Nikon Europe, the pair set out to commercialise the method which culminated in the sale of commercial systems worldwide.
Come 2012, and following the development of SCIM, Manders was focused on taking super-resolution microscopy further. He had set-up a national programmes, ‘Nanoscopy’ and 'Ultra Sensitive Confocal Microscopy', in The Netherlands to improve the resolution and sensitivity of confocal microscopy, and importantly, had learnt about 'Image Scanning Microscopy' as pioneered by Jörg Enderlein, Georg August University, Germany.
Enderlein and colleague, Claus Müller, had combined confocal-laser scanning microscopy with fast wide-field CCD detection to double the lateral optical resolution in fluorescence imaging.
As Manders says: "I had read their paper and was inspired by this well as research I had seen at 'Focus on Microscopy'."
So, with his PhD student, Guilia de Luca, and technician Ronald Breedijk, he spent, as he says 'night and day' in the lab for about three months working evening shifts and starting again early in the morning.
"Then, somewhere in the middle of the night we saw our first image... so that was a real scientific kick," he says. "And at the time, my student and I couldn't understand why no-one had invented this super-simple method a long time ago."
From here, and with funding from the 'Ultra Sensitive Confocal Microscopy' programme, Nikon and US-based Coherent, Manders and his colleagues built a user-friendly prototype of the re-scan confocal microscope, which they demonstrated at conferences.
They also reconfigured their set-up to be compatible with standard imaging techniques including multi-colour and time-lapse imaging, as well as Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET), calcium imaging and more.
Indian Muntjac, deer skin fibroblast cell, Blue: DAPI, Green: Phalloidin-Alexa488, Red: Mitotracker CMXRos. [Jeroen Kole, VU University Medical Center/Confocal.nl]
By the end of 2015, Manders had joined forces with Peter Drent to set up Confocal.nl.
As he puts it: "Guilia and I realised that we weren't business people, we were inventors and this was our strength... so Peter left Nikon and we started our company with Peter as chief executive."
Financial investment from the University of Amsterdam's, UvA Ventures, followed and in early 2017, the first commercial RCM units were shipped to customers. Confocal.nl has since won more capital funds from Dutch venture capitalist, Value Creation Capital, and is now expanding operations.
Importantly, Confocal.nl's technology is available as a module, that can be added to the existing wide-field fluorescence microscope systems.
To this end, the company has been working with the likes of Nikon, Olympus, Leica and more, to ensure its modules are compatible with microscopes.
"We sell complete systems and modules but the upgrade market is important to us so users can upgrade existing systems at a relatively low cost," highlights Manders.
Units are manufactured by Dutch company, Hittech, with systems now set up in China, Taiwan, the US, UK, and of course Germany. For example, the Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich, recently integrated a RCM module to its super resolution STORM microscope.
So what now for Confocal.nl? Beyond plans for company growth, Manders is keen to extend the imaging depths of RCM.
"We think that re-scan microscopy could be a good alternative to two-photon microscopy, for deep tissue imaging, and this is a development that we are working on," he says. "Sales are rising and systems are spreading around the world," he adds. "The first publications from researchers using our instruments are now getting published, and these are all great steps forward."
A higher resolution
At 170 nm, the lateral resolution of RCM alone, doesn't match that of Airyscan nor Structured Illumination Microscopy. These software-based methods claim 120 nm lateral resolution.
But, as Manders highlights, when RCM is combined with deconvolution technology, a similar lateral resolution can be achieved. Therefore, he has been collaborating with SVI, The Netherlands, to produce a dedicated RCM-deconvolution module in the existing Huygens software. This combination of techniques stretches the resolution down to 130 nm.
"You have to deconvolve your images with software that is developed for image scanning microscopy methods, such as RCM, and with SVI we have has done just this," he points out. "We have produced very, very nice clear images with this software that I believe actually look better than those produced using structured illumination microscopy."
"I think this is the beauty of our method," he adds. "The user can say, 'I want my image with optics-only-RCM' or they can say, 'I need to go up a gear and switch the deconvolution-RCM on' and then they can reach 130 nm resolution."