PPBI-Portuguese Platform of BioImaging

The Advanced Fluorescence Microscopy Facility at the Institute for Bioengineering and Biosciences (iBB) is specialized in quantitative imaging to describe the organization, dynamics and molecular interactions in both in vitro and cellular systems. The facility stands out in the application of cutting-edge fluorescence microscopy techniques (including at the single-molecule level) and in the development of quantitative modeling. We have a well-established expertise in:

- Förster Resonance Energy Transfer (FRET) Microscopy;
- Two-photon microscopy;
- Fluorescence recovery after photobleaching (FRAP);
- Fluorescence lifetime imaging (FLIM, time-domain) and FLIM-FRET;
- Fluorescence correlation spectroscopy (FCS);
- Fluorescence cross-correlation spectroscopy (FCCS);
- Fluorescence anisotropy imaging microscopy (FAIM);
- Single-molecule FRET;

The team attained international recognition in molecular and cell biophysics. Remarkably, we implemented for the first time FAIM and single-molecule FRET in Portugal. The unit also provides scientific support in project development, technical training, and guidance in image acquisition/ analysis for iBB researchers and also academic visitors or companies. The facility is a node of the PPBI-Portuguese Platform of BioImaging, part of the National Roadmap of Research Infrastructures (funded by FCT).


Microscope #1: Leica SP5 Multiphoton/Confocal Fluorescence Microscope for distinct applications:
• colocalization of fluorescent molecules and 3D sectioning/reconstruction;
• spectral imaging;
• two-photon microscopy;
• functional imaging (FRET, FRAP, FLIM, FAIM, FCS and FCCS);
It is equipped with:
• CW lasers: Ar ion laser (458, 465, 488, 496 and 514 nm) and HeNe (633 nm);
• Pulsed laser: Ti:Saphire (710-990 nm, 100 fs, 80 MHz);
• Objectives: HCX PL APO 10x/0.4 Dry CS; HC PL FLUOTAR 50x/0.8 Dry; and  HCX PL APO 63x/1.20 W (collar correction);
• Microinjection: combination of InjectMan® 4 and Femtoject® 4i from Eppendorf;
• Autocorrelator and two APDs for single-molecule FCS and FCCS;
• TCSPC module coupled to a DCC-100 PMT or two hybrid detectors;

Microscope #2: Olympus IXplore IX83 Widefield Microscope equipped with:
• Digital Hamamatsu CMOS camera (C11440-36U);
• Imaging Workstation (HP Z2 SFF G4);
• cellSens dimension software;
• Filter cubes: (1) Exciter filter: 340-390 nm, beam splitter: 410 nm, barrier filter: 420 nm; (2) Exciter filter: 470-495 nm, beam splitter: 505 nm, barrier filter: 510 nm; (3) Exciter filter: 530-550 nm, beam splitter: 570 nm, barrier filter: 575-625 nm;
• Objectives: UPLXAPO10X and UPLXAPO60XO;

Microscope #3: Home-built confocal microscope for single-molecule FRET:
It is based on an inverted microscope (Olympus IX-73) containing a high numerical aperture objective (60x/1.2NA water immersion objective) and 2 APDs detectors (SPCM-AQR-14 from Excelitas). It integrates a Sapphire LP 488 diode laser (Coherent), 50 or 100-μm diameter optical fibers to achieve confocal aperture; and a Correlator Flex03LQ-12.

Image analysis softwares: MATLAB, ImageJ and Imaris


Ana M. Melo - Senior Researcher and Facility Manager
Ana Coutinho - Senior Researcher
Fábio Fernandes - Senior Researcher
Manuel Prieto - Senior Researcher
Mario Berberan-Santos - Senior Researcher
Sandra Pinto - Senior Researcher


Please contact Ana M. Melo