Advanced Light Microscopy Facility

1. LeicaSP5/STED

This microscope is an inverted fully motorized confocal microscope from Leica (SP5) combined with a super resolution module (Super Resolution Light Microscope).

Figure 1: Leica TCS SP5 II confocal microscope, Inverted research microscope Leica DMI6000 CS Life Imaging Services chamber, temperature and CO2-air unit STED CW module

The SP5 is configured with

• Five (5) detectors: three PMT’s and two highly sensitive Avalanche Photodiodes (APD) for very low signal imaging.
• Two (2) scanners: a classical line scanner and a resonance scanner for faster imaging.
• Six (6) laser lines: 405 nm, 458 nm, 488 nm, 514 nm, 561 nm, and 640 nm are available for excitation.
• Six (6) objective lenses: APO CS 10.0x0.40 DRY UV, APO CS 20.0x0.70 IMM UV, APO CS 40.0x1.25 OIL UV, APO CS 63.0x1.20 WATER UV, APO CS 63.0x1.40 OIL UV and APO CS 100.0x1.40 OIL UV

Figure 2: Example of triple immunofluorescence confocal laser scanning microscopy, Scale bar, 10μm (Fotsis and Murphy Group-Unpublished Data)

Note that the 405 nm laser is not present because its slot is occupied by the CW laser. DAPI should be replaced by DRAQ5 whenever a nuclear staining is needed.

In addition to the basic applications, with SP5 we can perform additional microscopy applications. Software wizards for FRAP (Fluorescence Recovery After Photobleaching) and FRET (Fluorescence Resonance Energy Transfer) are available.

FRAP (Fluorescence Recovery After Photobleaching)

Fluorescence recovery after photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. The resulting information can be used to determine kinetic properties like the diffusion coefficient, mobile fraction and transport rate of the fluorescently labeled molecules.

FRAP in TCS SP5

Figure 3: Principle of FRAP

http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

Figure 4: FRAP data collected from transfected cells after photobleaching a single heterochromatic focus per nucleus (circles). Enlargements of the bleached regions are shown in insets. The data obtained in 50-100 independent experiments with each cell type used have been averaged (I/I₀) is the relative fluorescence intensity; (t) is the time elapsed. Scale bar, 5μm (Georgatos Lab)

FRET (Fluorescence Resonance Energy Transfer)

Fluorescence Resonance Energy Transfer (FRET) is a technique, which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution. An excited fluorophore, called the donor, transfers its excited state energy to a light absorbing molecule which is called the acceptor. This transfer of energy is non-radiative. Sensitized Emission is one established method for the evaluation of FRET efficiencies. It can be applied to live cells as well as to fixed samples.

FRET in TCS SP5

Figure 5: Principle of FRET

https://cam.facilities.northwestern.edu/588-2/fluorescence-resonance-energy-transfer/

Figure 6: FRET experiment between CFP-Protein A and YFP-Protein B in EEA1-positive vesicle in endothelial cells (Fotsis and Murphy group)

Super Resolution Light Microscope

The Leica TCS SP5 gated STED CW is a super-resolution confocal microscope. Stimulated Emission Depletion (STED) is a special illumination technique allowing the resolution of sub-cellular details below 80 nm. For STED imaging, a Continuous Wave (CW) laser enables the use of conventional dyes such as Alexa 488, FITC and Oregon Green and established fluorescent proteins such as YFP. Suppression of laser light and improvement of the resolution in the STED mode is achieved by time gating available for the two APDs. STED depletion is done with a 592 nm laser. For excitation in the STED mode the 458 nm, 488 nm, 515 nm can be chosen. This STED setup is ideally suited for one or two color STED with conventional “blue” and “green” dyes including TFP, YFP, and Alexa 488. In addition, our STED is a high-end confocal microscope suitable for most confocal applications.

2-color STED CW Sample Preparation (Leica)

Figure 7: Principle of STED

http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/stedconcept/indexflash.html

CLSM	STED

Figure 8: Confocal Laser Scanning Microscopy vs. STED Microscopy (Fotsis and Murphy Group)

Figure 9: Comparison between Confocal Microscopy (upper panel) and STED Microscopy (lower panel) (Kyrkou et al., 2013)

2. TIRF Microscope

Total internal reflection fluorescence microscopy permits high signal-to-noise imaging of fluorescently-labeled molecules at surfaces and interfaces. By adjusting the angle of the illuminating light beyond the critical angle at an interface between media of two different refractive indices (i.e. achieving total internal reflection), a shallow evanescent wave is generated into the medium of lower refractive index that can excite fluorescent molecules that lie within 100 nm of the interface. This results in high signal-to-noise images, due to the lack of signal beyond the 100 nm depth of illumination. This is ideal for studying signal molecule fluorescence, cell-substrate interactions and membrane dynamics [Video (MPG format) - See  TIRF video from Basagiannis et al., 2016 - http://www.jbc.org/content/291/32/16892/suppl/DC1].

Figure 10: Principle of TIRF http://jcs.biologists.org/content/joces/123/21/3621/F2.large.jpg

The facility houses Leica AF 7000 TIRF system equipped with a 100x/1.45 NA Plan Apochromat TIRF objective lens (in addition to 10x, 20x, 40x, and 60x lenses), DIC & phase optics, UV, FITC, TRITC, a low-profile stage incubator (to facilitate live cell imaging) and a CCD camera. The system integrates four wavelengths; 405nm, 488nm, 561nm, and 635nm with fast AOTF control.

Figure 11: Leica AF 7000 TIRF system-Leica DMI 6000B inverted microscope-Pecan Chamber and temperature CO2-air control unit

3. IncuCyte ZOOM System 

The IncuCyte ZOOM® system is a live-cell imaging and analysis platform that enables automated quantification of cell behavior over time (from hours to weeks) by automatically gathering and analyzing images around the clock. The system provides insight into active biological processes in real-time which is not possible using single-point and end-point measurements. The system resides within the controlled environment of a standard cell incubator. All imaging is completely non-invasive and non-perturbing to cell health. The system can process multiple plates, flasks and dishes in parallel and does not depend on shuttling plates into and out of the incubator.

The purchase of the incucyte zoom system has been financed by the project “BioImaging-GR: The Greek Research Infrastructure for the Imaging and Monitoring of Fundamental Biological Processes” (MIS 5002755) which is implemented under the “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

IncuCyte® ZOOM System - Essen BioScience Brochure

Figure 12: The IncuCyte ZOOM System

Figure 13: Example of live cell imaging snapshots using IncuCyte® ZOOM System - Essen BioScience (Fotsis and Murphy group)

Video 1 (AVI format) - Proliferation assay using IncuCyte Zoom System (Fotsis and Murphy group)

Video 2 (AVI format) - Migration assay using IncuCyte Zoom System (Fotsis and Murphy group)

If you are not an independent user yet or think you need help for a new application, please call or write to Sofia Bellou to help you.
Mail Address: This email address is being protected from spambots. You need JavaScript enabled to view it.
Tel.: +302651007816, +306932047088

References

1. Christogianni A at al., Heterochromatin remodeling in embryonic stem cells proceeds through stochastic de-stabilization of regional steady-states. Biochim Biophys Acta. 2017
2. Kyrkou A et al., The RhoD to centrosomal duplication. Small GTPases. 2013 Apr-Jun;4(2):116-22.
3. Basagiannis D et al., VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation through macropinocytosis. J Cell Sci. 2016 Nov 1;129(21):4091-4104.
4. https://www.leica-microsystems.com
5. https://www.dzne.de/en/research/core-facilities/light-microscope-facility/
6. https://anatomy.vcu.edu/microscopy/microscopy2/tirf/index.html
7. http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html)
8. https://cam.facilities.northwestern.edu/588-2/fluorescence-resonance-energy-transfer/
9. http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/stedconcept/indexflash.html
10. http://jcs.biologists.org/content/joces/123/21/3621/F2.large.jpg
11. https://www.essenbioscience.com