The Bioimaging unit is part of i3S and INEB (an Institution with a wide experience and international recognition in the biomaterials and biomedical imaging fields), and it is open to all academic researchers and industry on a fee-for-service basis. Our aim is to advance in the development, improvement, integration and use of bioimaging solutions through research, technology development, training and education with focus on the fields of Biomaterials, Nano- and Regenerative Medicine. We operate on the basis of core projects and programs at the bioimaging/biomaterials and/or regeneration interface. Resources available include imaging at the molecular level, surface topography and force spectroscopy (atomic force microscopy); non-destructive chemical imaging (confocal Raman); high resolution multidimensional imaging for cells, tissues, biomaterials, live cell and high-throughput imaging (imaging flow cytometry and laser scanning confocal microscopy); in vivo anatomical and functional imaging for small animals (micro-ultrasounds and microCT (to be acquired soon)); and qualified staff for helping users from the experimental design and data acquisition to analysis.

The Bioimaging unit was created in 2010 following a successful application to structural funds of the ON2 program (2007-2013 North Regional Operational Program; SAIECT-IEC/2/2010). The Platform daily activities are supported by our technical staff and its governance assured by the Scientific and Management Committees, composed by a panel of excellent researchers from the biomedical and bioengineering fields.

In order to improve our assistance we look forward to receive your input (b.image@ineb.up.pt).

The collection of resources available allow for the imaging/characterization from molecules to organisms (small experimentation animals). Technologies available and example applications include:

• Raman spectroscopy (RS), which allows for chemical characterization of unlabelled sample. Providing information, in a non-destructive manner, of the structure and chemistry of not only materials but also biological samples with a resolution down to the optical diffraction limit (~ 200 nm). RS provides information about molecular vibrations useful for sample identification (fingerprint) and quantitation. With our confocal Raman microscope we can also obtain chemical images of the samples (mapping), which are useful to analysed distributions. Example applications include: chemical characterization of biological and synthetic samples; compound distribution within a sample; disease detection; drug design; etc 

• Atomic force microscopy (AFM) is a scanning probe microscopy technique that allows for the image acquisition down to the nanometre resolution. With this technology, it is possible to acquire 3D topographic images and surface roughness, at the nanometric scale, as well as adhesion force determination using Force Spectroscopy. Our AFM system is coupled to an inverted fluorescence microscope allowing the precise analysis of fluorescently labelled samples. Example applications include: characterization of surface properties, including the measurement of adhesion and friction forces, quantification of viscoelasticity and stiffness; imaging the topography of the sample; by force spectroscopy, interaction forces and binding strength can also be measured; etc.

• Optical microscopy provides multidimensional functional and structural information of a sample in a non-invasive manner. With our light and confocal microscopes applications include: multimodal imaging of biological samples; acquisition of series of overlapping tiled images acquired over a defined area; live cell imaging; FRAP; and FRET amongst others.

• Imaging flow cytometry constitutes a powerful combination between flow cytometry and microscopy, associating statistical strength and imaging with high speed acquisition. Example applications of the Imagestream include: localization and colocalization analysis; cell cycle and mitosis; morphological changes; and spot count amongst others.

• Critical point drying is used not only for dehydrating biological tissues, but also any other material that requires dehydration prior to examination in the Scanning Electron Microscope to better preserve the sample’s ultrastructure. This preservation is obtained considering that during the critical point temperature of a certain fluid, there is no apparent difference between the liquid and gas state, reducing the surface tension to zero. The best suitable inert fluid, whose critical point is just above ambient and does not cause temperature damage to the sample, is CO2.

• Microultrasounds is a real time imaging technology based on the transmission and the reception of ultrasound waves, which is capable of generating high resolution images for true longitudinal studies in animals. We can obtain in vivo anatomical, functional, physiological and molecular data simultaneously, in real-time and with a resolution down to 30 µm, allowing for the visualization of vasculature. Example applications of the Vevo 2100 include: cardiovascular phenotyping; image-guided cardiac injection; pregnancy research; tumour volume quantification; and bladder imaging amongst others.

• Microcomputed tomography is a nondestructive and noninvasive technique requiring no sample preparation that uses low dose X-ray imaging and computed tomography to generate 3D images of samples showing morphology and internal microstructure with resolution down to the micrometric level. Frequently used for imaging bone tissue, this equipment allows also for the image acquisition of body fat and lungs, with the use of contrast agents is potential use expand to the imaging of soft tissues and the vasculature. The stand-alone micro-CT is capable of delivering high quality images at an X-ray dose low enough to enable longitudinal true micro-CT preclinical studies, allowing the monitoring of disease progression throughout the complete study, and, as a result, a reduction of the number of animals used per experiment. Example applications include: characterization of bone morphometrics; tumour and blood vessel imaging; cardiovascular phenotyping; imaging of body fat and lungs; etc.

More information on the systems configuration can be checked in the corresponding Resources area.

Equipment

 

Our equipment includes:

 

Atomic force microscope with Inverted Fluorescence Microscope/ Coupling Stage

The equipment has two multipurpose scanners for AFM with 10 and 100 µm2, and performs in the following modes: Contact, Non-Contact, and Intermittent-contact (Tapping™ mode and Magnetic Acoustic Mode - MAC™). The MAC Mode™ is generally recognized as the AFM “gentle” mode, and consists in a magnetic cantilever directly controlled by a magnetic field. It integrates the oscillating mode (Acoustic AC) and the phase imaging (digital) as well. This mode is indicated for measurements in fluids, being characterized by low contact forces applied in extremely delicate samples, such as: proteins, DNA, RNA, cells and other biological structures. The AFM is coupled to an inverted fluorescence microscope that has light sources for both transmitted light and epi-fluorescence (allowing for the visualization of fluorochromes such as DAPI and eGFP).
The available equipment is capable of performing measurements both in air and in liquid (a specific liquid cell is available).

 

Inverted Fluorescence Microscope, Axiovert 200M (Zeiss)

Inverted fluorescence microscopes allow for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway.
Our system is equipped with a motorized stage and chamber for temperature and CO2 control. The Axiovert 200M is equipped also with the modules: Timelapse, Z-Stack, Mark&Find, MosaiX, AutoMeasure and AutoMeasure Plus.
Configuration: fluorescence filters for: 365, 470, 565 and 640 nm; objectives: 5X air, 10X air, 20X air, LD, 40X air, LD, 40X oil, 63X oil, 63X LD, 100X oil; adapters for 35 mm in diameter plates and adapter for well plates.

 

Confocal Raman Microscope, LabRAM HR800 UV, Horiba Jobin-Yvon

Our system allows for spectra collection in 2D. Thanks to the confocal setup, detailed images and analysis can be obtained and fluorescence interference can be reduced.
Configuration: lasers: 325, 514, 633 and 785 nm; objectives: 10X, 50X and 100X detector: CCD air cooled; spectrograph with gratings: 600, 1800 and 2400 l/mm and software for data acquisition and analysis: LabSpec 5.

 

Critical point dryer (CPD 7501, Polaron Range)

Our critical point dryer (CPD 7501, Polaron Range) is used for not only dehydrating biological tissues, but also any other material that requires dehydrating, prior to examination in the Scanning Electron Microscope to better preserve the sample’s ultrastructure. This preservation is obtained considering that during the critical point temperature of a certain fluid, there is no apparent difference between the liquid and gas state, reducing the surface tension to zero. The best suitable inert fluid, whose critical point is just above ambient and does not cause temperature damage to the sample, is CO2.

 

Imaging Flow cytometer ImageStreamX

This equipment combines the speed, fluorescence sensitivity and statistical strength of a flow cytometer with a high resolution microscopy system, giving a picture of every event. The high versatility of the analysis software allows for multiple data analysis. Our system allows the acquisition of six different channels: one for brightfield images, one for sidescatter images and four additional for different fluorochromes in use.
The ImageStreamX is equipped with a 488 and a 788 nm lasers. Objective: 40X. The table of suggested fluorochromes for being used in our ImageStreamX can be seen here. In addition, a workstation for data analysis is available.

 

In vivo microCT (Bruker SkyScan1276)

X-ray micro-computed tomography (microCT) is a nondestructive and noninvasive technique requiring no sample preparation that uses low dose X-ray imaging and computed tomography to generate 3D images of samples showing morphology and internal microstructure with resolution down to the micrometric level. Micro-CT opens a window on the micro-scale 3D structure of a wide range of samples relevant to the life sciences and bioengineering fields. Frequently used in imaging bone tissue, this equipment allow also for the image acquisition of body fat and lungs, with the use of contrast agents is potential use expand to the imaging of soft tissues and the vasculature. The stand-alone micro-CT, capable of delivering high quality images at an X-ray dose low enough to enable longitudinal true micro-CT preclinical studies, allowing the monitoring of disease progression throughout the complete study, and, as a result, a reduction of the number of animals used per experiment.

 

In vivo micro-ultrasounds imaging, Vevo 2100 (230 V)

Our system allows working in M-mode, PW Doppler Mode and Color Doppler Mode. Image analysis for physiological trace including ECG, respiration waveform and body temperature.
The Vevo2100 is equipped with a MS400: 38Hz Microscan transducer with a broadband frequency: 18 MHz – 38MHz. Capable of working in M-mode, PW Doppler Mode and Color Doppler Mode. Image station plus imaging station extension with injection mount. Also the software package can be used for B-mode (2D) image capture and analysis, Cineloop image review, capture and display with integrated physiolical trace including ECG, respiration waveform and body temperature.

 

Spectral Confocal Laser Scanning Microscope Leica TCS-SP5 AOBS

Our Leica TCS SP5 inverted confocal microscope provides a full range of scan speeds at the highest resolution. With three detectors and working in the AOBS (Acousto Optical Bream Splitter) mode, we can acquire bright, noise-free images with minimal photo damage at high speed.
Our system allows: 3D imaging, colocalization, excitation fingerprinting, FRET, FRAP and FLIP, photo-switching and photo-activation. Live cell imaging experiments can also be performed (controlled temperature and CO2).

Configuration: lasers available: 405, 488, 561, 594 and 633 nm; objectives: 10X dry, 20X immersion, 40X and 63X oil and 63X glicerol; adapters for 35 mm in diameter plates, well plates and 25 mm in diameter coverslips; 3 detectors: 1 Hybrid detector and 2 PMTs; motorized stage. In addition, a workstation for the analysis of the data is available.

Software

Software developed at INEB

MIQuant

The MIQuant is an open source software developed with the objective of automating the infarct extension estimation in animal models (mice). In the specific operation of the MIQuant software the user is asked to provide input seed points for the left ventricle’s lumen and the healthy heart tissue areas, sequentially.

 

Cell File Analyzer

Cell File Analyzer is an automatic tool to detect cell files, the main structure in plant roots. The obtained cell file detection enables the automatic extraction of important data on the plant development stage and of characteristics for individual cells, reducing in more than 90% the time required for the analysis.

 

LCFs - Local Convergence Filters

LCFs allow the detection of cell nuclei and joint detection of cytoplasm without the use of image segmentation. LCFs are local interest point detectors based on the direction of the image gradient and not magnitude, making them robust to image contrast variations.

 

Equipment specific software

Leica LAS AF Lite

LAS AF Lite is the free version software from the Leica LAS AF developed by Leica Microsystems CMS GmbH. This software allows you to read and export -lif files generated by the SP5 Leica Confocal. You can downloaded a free version from here.

 

IDEAS

IDEAS software integrates imaging and population statistics for the data obtained in the ImageStream. With this software populations can be defined by discriminating them with different features and statistically analyzed. Analysis scheme ideas can be saved and used as a template for batch processing future experiments.
You can download the software at the AMNIS website.

 

LabSpec 5

LabSpec 5 software is a fully configured data acquisition and analysis software designed for HORIBA Scientific’s range of Raman spectrometers and microscopes. It enables a full range of experiment types including single spectrum acquisition, multidimensional spectral array acquisition (including time profiles and XY maps), and video image capture. Many commonly used data analysis and processing routines are also available, including peak labelling and fitting, smoothing, noise reduction, baseline subtraction, linear and non-linear filtering and direct classical least squares modelling.

 

Vevo Software

This software from VisualSonics facilitates the real time visualization and measurement of anatomy and the quantification of physiology in adult, neo-natal and (in utero) embryonic mice. Data and measurements can be exported and summarized to standard formats for offline statistical analysis and calculation as well as use in presentations reports and publications.

Image analysis software

FIJI/ImageJ (fiji.sc)

ImageJ is an open source software for image processing and analysis developed at the National Institutes of Health. It can read images in different formats including TIFF, PNG, GIF, JPEG, BMP, DICOM, FITS, as well as raw formats. It supports standard image processing and also allows the creation of plugins for new analysis.
FIJI is just ImageJ. FIJI includes a large amount of plugins from ImageJ focused on assisting research in life sciences. You can download the software from here.

 

Open Microscopy Environment - OME

OMERO is a client-server software for visualization, management and analysis of biological microscope images.

 

Octave

GNU Octave is a high-level interpreted language, primarily intended for numerical computations. It provides capabilities for the numerical solution of linear and nonlinear problems, and for performing other numerical experiments. It also provides extensive graphics capabilities for data visualization and manipulation. The Octave language is quite similar to MATLAB so that most programs are easily portable.

 

MATLAB

MATLAB® is a high-level language and interactive environment for numerical computation, visualization, and programming. Using MATLAB, you can analyze data, develop algorithms, and create models and applications. MATLAB can be used for a wide range of applications, including signal processing and communications, image and video processing, control systems, test and measurement, computational finance, and computational biology.

 

Gimp2 

Gimp2 is a powerful image editor that it is freely available.

 

R

This open-source software constitutes a powerful environment for statistical and graphics analysis.

We are open to all academic researchers and industrial clients on a fee-for-service basis. Although data acquisition can be ensured by our technicians, we encourage our users to undergo the corresponding training period to become independent users.

New users should fulfill the Experimental Design Form and send it to the following email: b.image@ineb.up.pt. This form will be evaluated by the Technician in charge of the respective equipment and the Scientific Coordinator of the Center that will define the necessary training scheme for the user. This form will help you to know if your experiment can be undertaken in the equipment and will help us to direct your training. Additionally if you have doubts we can discuss with you the specific experiment. To download the form click here. When a new user has been trained and validated as a qualified user, he/she will be allowed to book the equipment online.

Our staff also organizes different hands on workshops related to the technologies available and participates in the organization of Scientific Conferences.

Past Training Actions

Workshop in Atomic Force Microscopy coupled to Inverted Fluorescence Microscopy | November 15th - 17th 2017

Atomic Force Microscopy (AFM) has revealed as a powerful tool to study human pathology, in fields ranging from cancer, cardiovascular and blood diseases, since it is suitable to perform studies on different cell/ tissue types, at physiological conditions. The main principle is based in the interatomic forces stablished between a probe tip and the sample causing the cantilever to deflect as the samples’ surface topography changes. A laser light reflected from the back of the cantilever measures the deflection of the cantilever. In this lab session the participants will be introduced to the AFM/IFM techniques, having the opportunity to learn about the determination of morphometric parameters to the characterization of biomechanical properties of cells/ tissues. As well, students will learn how to use different software for the analysis of the data arising from these studies. Applets software will be used to analyze the Thermal tune data to obtain the force constant of the cantilevers and to analyze AFM force distance curves, to calculate the mechanical properties of the samples.

 

Upcoming Training Actions

High Throughput Screening and Image Analysis for BioSciences | June 18th - 22nd, 2018

Fundamentally practical, this course aims at introducing the participants in experimental design, image acquisition, image and data analysis for high throughput experiments. Essential skills when generating massive amount of imaged-based data in the biosciences field. Lectures will be given by specialist in the field and participants will learn how to use state of the art technology for high throughput (HT) experimentation and open-source software for image and data analysis. Attendants will get acquainted with use of automated liquid handling equipment and high content (HC) imagers such as IN Cell Analyzer (HCS microscope) and ImageStream (imaging flow cytometer). Interaction with HTS/HCS specialists will be fostered due to the restricted amount of participants.

The Bioimaging is a node of the PPBI-Portuguese Platform of BioImaging, an open-access research infrastructure integrated in the Roadmap of National Research Infrastructures of Strategic Relevance.

The b.IMAGE is a node of the Portuguese Platform of Bioimage (PPBI), which constitutes a functional platform dedicated to promote the technical integration and centralized management of shared resources in bioimaging in the Country.