Microscopy on the edge: pushing CrestOptics technology to the limit at the European Institute of Oncology in Milan

The world of research at European Institute of Oncology

The IEO (European Institute of Oncology) is one of the world’s most prestigious hospitals and the fastest growing comprehensive cancer centre in Europe. This institute integrates the various areas related to the fight against cancer such as prevention, diagnosis, treatment, training and education, basic and translational research. At IEO, 300 scientists aim at discovering the molecular mechanisms involved in the development of cancer with a complete integration between clinical and research activities to translate scientific results into therapy, as quickly as possible. Basic and translational research takes place at the Department of Experimental Oncology (DEO) that is located on the campus in Via Adamello 16 in Milan, Italy.

Imaging Unit at European Institute of Oncology, Milan (Italy)

The Imaging Unit supports the IEO scientific groups, providing state-of-the-art optical imaging platforms and developing on-demand specific imaging and image analysis protocols. It provides its experienced assistance in users’ training to grant direct access to the microscopes, in assisting both microscopy beginners in setting up their experiments and the experienced researchers in pilot experiments or advanced imaging techniques. The available systems cover different microscopy techniques including wide-field and confocal, total internal reflection (TIRF), stochastic optical reconstruction microscopy (STORM), high content and time-lapse microscopy and the Unit services include the microscopes’ data back-up and storage and the imaging reagents management. “Our Imaging Unit has been recently equipped with the CrestOptics X-Light V3 Spinning Disk confocal and DeepSIM X-Light module mounted on a Nikon Ti2 inverted microscope. This piece of equipment filled an existing gap in our instrument fleet,” said Dr. Simona Rodighiero, Unit Coordinator of the Imaging Unit, European Institute of Oncology (IEO).

Imaging Development Unit at European Institute of Oncology, Milan (Italy)

The Imaging Development Unit at the IEO in Milan has been created to develop high-end imaging applications requiring super resolution, high content microscopy and computer-aided image acquisition. It works on selected projects to create tools of general usefulness for cancer research collaborating with research groups. “The Imaging Development Unit does not directly perform single experiments on demand but trains researchers actively participating in the development process. Once completed the setup, the personnel of the Imaging Unit help us in disseminating the usage of the produced tools to make them available for the entire Department,” said Dr. Mario Romolo Faretta, Unit Coordinator of the Imaging Development Unit, European Institute of Oncology (IEO). “We push any technology in our lab to its limit and make the most out of the imaging instrumentation for our project’s development. This cutting-edge and challenging attitude we adopt in IEO paves the way to unprecedented opportunities of technological and scientific discoveries,” Dr. Mario Faretta added.

Imaging-based spatial proteomics at Imaging Development Unit, IEO

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section, has raised considerable interest for in situ characterization of neoplastic, reactive, inflammatory, and normal cells; the major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities [1]. “We have validated a method based on four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied,” Dr. Mario Faretta said. “The images are digitally registered and the autofluorescence is subtracted. The procedure employs an open-source algorithm to automatically realign the x, y, z axis coordinates of the tissue section acquisition from consecutive immunostaining cycles, that involve removing and replacing the very same slide on the scanner or on the microscope  stage. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue.” Dr. Mario Faretta added. “The pipeline we created allows the collection of several informative data at different optical resolutions employing a high level of automation and a coupling between image acquisition and analysis”.

“For this target application the CrestOptics X-Light V3 Spinning Disk Confocal system coupled with the Nikon Ti2 Eclipse inverted microscope outperforms the other immunofluorescence slide scanners we have in our Imaging Unit. When it comes to running tile scan for mosaic images, the micro-lenses block in the X-Light V3 illuminator delivers unparalleled illumination uniformity across the Nikon Ti2 Eclipse 25 mm FOV. The resulting stitched image is high-quality even with a small percentage overlap between consecutive tiles,” Dr. Mario Faretta stated. “Furthermore, a remarkable aspect to mention is the extremely fast acquisition speed of the X-Light V3 Spinning Disk Confocal which comes with good optical sectioning and sensitivity for important markers,” Dr. Mario Faretta added.

Figure 1. Expansion microscopy of human colorectal carcinoma (HCT) cells. HCT cells were labelled with DAPI and indirect immunofluorescence for Pol2S5P (shown in green) and ZC3H4 (shown in red), expanded of a factor of ~ 6 and acquired with the CrestOptics X-Light V3 Spinning Disk Confocal. A single plane is shown. Scale bar: 20 µm.

A multi-modal approach with the combination of Spinning Disk Confocal and Structured Illumination Microscopy

In situ multiplexing analysis and in situ spatial -omics require a continuous interplay among different technologies during all the phases of the experimental procedures [2]. “New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity,” Dr. Simona Rodighiero said. “The combination of CrestOptics and Nikon technologies allows a ‘Multi-Modal Microscopy’ approach through the seamless switching between wide-field, spinning disk confocal and SIM super-resolution imaging modalities. This setup makes us embrace the concept of analysis-driven acquisition that means choosing the right image collection conditions for the biological task addressed by the current experimental question.,” Dr. Mario Faretta added. “In the X-Light V3 to act as a master is the fast acquisition of large and homogeneously illuminated 25 mm FOVs. The DeepSIM X-Light, conversely, helps us untangle nanometric structures, like cell foci, beyond the light diffraction limit of 200 nm. Featuring three different SIM 2D lattice patterns, the DeepSIM X-Light has proven to be highly compatible on a whole bunch of samples available in our labs, from cell monolayers to 3D cell cultures and tissue sections.” Dr. Simona Rodighiero stated. Although fluorescence scanners provide an easy-to-use solution to automate image acquisition, their major limitation is the use of dry, low-mag objectives.

The consequent exclusion of high Numerical Aperture objectives precludes the investigation of diffraction-limited details and lowers the sensitivity [2]. The analysis of high spatially resolved details in pathology samples is a growing demand that is stimulating new imaging approaches. Even if the developed amplification techniques greatly enhance signals in spatial -omics from single molecules making it detectable even with medium sensitivity and resolution, high-resolution optical microscopy provides a signal-to-noise ratio that reduces error in the correct detection, localization and assignment of the low expressed markers [2]. “In our multimodal approach, the CrestOptics DeepSIM X-Light technology can be applied in spatially-confined target regions, where augmented resolution is required. With the DeepSIM X-Light, the single cell segmentation performances are greatly, thus providing the classical analysis performed by pathologists with twice the spatial resolution of a confocal system”, Dr. Mario Faretta concluded.

Figure 2. Fluorescence colocalization analysis in single cells. DAPI (blue), BrU incorporation (red) and Intronic Cyclin B1 RNA smRNA FISH (green) are acquired with the CrestOptics DeepSIM X-Light. Signal colocalization shows the efficient detection of the nascent transcript on MCF10A cells.

Technical Overview: the combo setup with X-Light V3 Spinning Disk Confocal and DeepSIM X-Light module

The high-end system adopted at the European Institute of Oncology (IEO) consists of a X-Light V3 Spinning Disk Confocal (CrestOptics) and a DeepSIM X-light (CrestOptics) mounted on the left camera port of a Nikon Eclipse Ti2 inverted microscope with 25 mm FOV. The back-illuminated Kinetix sCMOS camera (Teledyne Photometrics) is used as a detector. This camera covers the large 25mm FOV of Nikon microscope with 96% Quantum Efficiency and low 0.7 e Read Noise for enhanced sensitivity to detect dim signals. With its 6.5 um x 6.5 um Pixel area, sharp images are acquired with DeepSIM X-Light module. Taking advantage of an 8-bit readout mode, the Kinetix sCMOS delivers a tremendous 498 frames per second (fps) in full frame for fastest acquisitions with the X-Light V3 Spinning Disk Confocal. Confocality is achieved by means of a motorized spinning disk with 50 µm pinhole diameter. Illumination consists of a Celesta-7 (Lumencor) multimode high-power laser source for higher flexibility in fluorophores choice when performing multiplex staining. The system is also equipped with microfluidic platform (Elveflow) for expanding the range of available experiments employing liquid handling automation.

CrestOptics & Nikon Europe B.V.: a long-lasting collaboration in Life Sciences Microscopy Market

Alberto Zorloni

Lombardia Research Market Sales Manager, Nikon Italy, Branch of Nikon Europe B.V.

The fruitful collaboration between Nikon Instruments and CrestOptics has generated many successful stories across Italian Life Sciences Microscopy market. Nikon has been the direct interface with the IEO staff, acting as a bridge with the system manufacturer, CrestOptics, and adding an extra-value in terms of scientific and technical support for the end user. Promoting the interaction between CrestOptics and IEO will have a positive impact in the development of next-generation technologies.

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