Advanced Light Microscopy Unit

The Advanced Light Microscopy Unit (ALMU) of the CRG and UPF serves as a core facility for high-end light microscopy for PRBB researchers. A range of instruments with unique capabilities fully covers the spectrum of advanced imaging applications from thick tissue reconstruction to fast in-vivo imaging to the sensitive detection of very faint signals of single molecules. The staff of the facility provides advice in the initial experiment planning, training of the researchers on the instruments and assistance with the subsequent data analysis. It is the aim of the facility to provide a link for the biological questions of researchers to the full capabilities of advanced light microscopy at the organismic, cellular and molecular level. Methods available in the facility include super-resolution microscopy by stimulated emission depletion (STED) and localization based methods like Stochastic Optical Reconstruction Microscopy (STORM) and Ground State Depletion Imaging Microscopy (GSDIM), optical sectioning (single photon and multi-photon microscopy), spectral imaging, in-vivo time-lapse imaging, Total Internal Reflection Fluorescence (TIRF) Microscopy and methods for the study of molecular properties and interactions like Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Imaging Microscopy (FLIM), Fluorescence Resonance Energy Transfer (FRET) detection, Fluorescence Recovery after Photobleaching (FRAP) and confocal and widefield screening microscopy. Additionally, dedicated software packages for data visualization and analysis are available for 3D rendering, particle tracking and image analysis.

In 2018, the total booked microscope usage time of the unit reached more than 19000 hours in more than 6300 separate bookings. This corresponds to approximately eight hours of daily usage on the bookable main microscope systems plus many additional hours on equipment without mandatory booking and on special equipment. During the year, 130 users from 26 CRG groups and 60 users from 20 UPF-CEXS groups have used the unit. Additionally, the unit was used by 26 users from 12 groups of other institutes (EMBL Outstation Barcelona, IMIM) and one company located in the PRBB and for projects from external visitors (VHIO, IrsiCaixa). On average, 94 investigators use the unit every month.

During the first months of 2018 the unit finished moving into its new location in the basement of the PRBB building. Instead of one main room, three small microscope rooms and three separate offices in different parts of the building, all instruments are now centrally located in two adjacent microscope rooms (with the exception of one super-resolution microscope which remains in its separate location) and the staff is located in two adjacent offices right next to the image processing workstations. With the move into the new location, the unit has gained additional lab and visitor space and a microscope development room for special projects. ALMU is now located in the same space as the newly created Mesoscopy Imaging Unit of the EMBL Barcelona Outstation and shares the lab and office spaces with this unit. Due to the complementary imaging fields of mesoscopy and microscopy, a wide range of methods and instrumentation is thus available between the two units and synergies are aimed for.

The instrumentation of the unit was kept up to date by targeted upgrades of the existing equipment. A transmission light detector was added to a confocal microscope without it to offer the same imaging modalities on all systems. A piezo z-focusing stage was installed on the Dragonfly spinning disk fast confocal imaging system to increase the speed for z-stack generation and multiposition imaging. A mobile environmental control unit was acquired to meet the increasing demand for in-vivo imaging and to supplement the existing fully incubated microscopes. Similarly, a cooling device was developed for the screening microscope to accommodate temperature sensitive organisms.

The unit is currently developing transient darkstate imaging protocols for the fast quantitative assessment of fluorophore blinking and the optimization buffers for super-resolution imaging (see figure).

Timo Zimmermann is the Spanish representative for Biological Imaging in the Interim Board of the ESFRI project Euro-BioImaging. The microscopy unit participates in two highly evaluated Spanish node proposals for Euro-BioImaging, for super-resolution microscopy (with the Photonic Sciences Institute ICFO) and for in-vivo and intravital imaging (with the University of Barcelona and IRB Barcelona) which are participating in the interim operation phase of Euro-BioImaging which has started in May 2016.

All members of the staff are frequently participating as speakers and instructors in master’s courses from the CRG and UPF, as well as in conferences and microscopy courses both at the PRBB as well as at other institutions, nationally and internationally.

The unit participated in the execution of BIST Winter School on Microscopy and Imaging Sciences 2018 which took place from January to February for the BIST Master Course of Multidisciplinary Research in Experimental Sciences.