Areas of activity

The activity of the Microscopy and Molecular Imaging Unit is focused on epifluorescence microscopy and confocal laser microscopy, which allows to analyze the localization of molecules of interest using immunofluorescence techniques and fluorescent fusion proteins such as CFP, GFP, YFP, mRFP, mCherry, among others, both in fixed and living cells, which makes it possible to develop studies on dynamic cellular processes, among others. Furthermore, another activity of the Unit is laser capture microdissection technique, which facilitates the isolation of specific cell populations in heterogeneous tissue sections or individual cells for subsequent genomic and proteomic study.

Epifluorescence Microscopy

The light source in epifluorescence microscopy is a lamp with light containing different wavelengths. A filter is used to gain the adequate excitation wavelengths, depending on the fluorochrome of the specimen, so that the reflected light or emitted fluorescence light after passing through an emission filter system, reaches the detector or camera, then obtaining a digital image in the computer.

The Microscopy and Molecular Imaging Unit of GENYO is provided with the following epifluorescence microscopy equipment : Nikon Eclipse 50i, Nikon Eclipse 80i, Nikon Eclipse 90i with spectral camera and motorized plate, Nikon Eclipse TE2000-U, and Zeiss Axio Imager A.1, providing images acquisition with transmitted-light techniques (bright field, phase contrast, and DIC), and fluorescence images acquisition from fixed specimens with one or several fluorochromes (single or multiple labeling, respectively).

 

Confocal Laser Microscopy

In contrast to epifluorescence microscopy, confocal laser microscopy allows to obtain high-resolution images, as well as sharpness, and contrast of only one focal plane of the specimen when light from planes out of focus is eliminated. Confocal laser microscope captured images of the specimen that reflect fluorescent light; in that case, the light source is a laser line, which excites the specimen point by point and moves along the focal plane by a laser scanning system.


 

Fluorescence from the focal plane of the specimen is detected by a photomultiplier tube, and converted into electrical signal, which is digitalized and converted into a signal for display into a video monitor, resulting in an exceptional quality image. Confocal laser microscope allows the study of specimens marked with several fluorochromes, by the capture, without overlap, of the signals coming from the fluorochromes through the tailor-made detection of the emission spectra of each fluorochrome; Then, multiple images are generated, one for every fluorochrome detected, which can be finally overlapped in one image.

One of the main advantages of confocal microscopy is the possibility of changing the focal plane position, and capturing images at different Z-axis heights of the specimen. Then, obtaining a set of optical sections to which it is possible to apply reconstruction techniques able to provide visualization of 3D structure of the specimen.

GENYO is equipped with the last generation technology of confocal laser microscopy such as the LSM 710 (Zeiss) confocal laser microscope, equipped with an incubation chamber with temperature and CO2 control to perform assays with living specimens.

Laser Capture Microdissection allows the isolation of regions of interest from cryo frozen tissue sections or FFPE tissues, cell populations or even specific individual cells from fixed or living specimens marked with immunohistochemical, immunofluorescence methods, or techniques expressing molecules of interest fused to fluorescent proteins in order to perform a later genomic or proteomic study. The impossibility of isolating pure groups of cells was a major constraint in the study of a specific type of cell. However, the development and evolution of microdissection techniques resulted in laser capture microdissection system. It is an efficient, accurate and safe from environmental contamination technique, addressed to investigators interested in separating cell populations or specific unique cells for the later molecular analysis. The Microscopy and Molecular Imaging Unit of GENYO is equipped with the Zeiss PALM Microbeam IV Laser capture microdissection system. The Zeiss Axio Observer Z.1 inverted microscope is available for tissue or cellular sections observation providing excellent quality images. Thanks to the PALMRobo software, tissue or cellular regions of interest can be easily and rapidly localized on the screen. The drawing tools of the program allow toselect cellular groups or unique cells. The system’s UV laser (355 nm) is used to both cut and catapult cellular groups or unique cells into the adhesive cap of the collection tube to be studied. The “Cap Check” system of the program is used to verify the catapulted sample in the cap, which provide a higher reliability of the system.

Laser Capture Microdissection

Laser Capture Microdissection allows the isolation of regions of interest from cryo frozen tissue sections or FFPE tissues, cell populations or even specific individual cells from fixed or living specimens marked with immunohistochemical, immunofluorescence methods, or techniques expressing molecules of interest fused to fluorescent proteins in order to perform a later genomic or proteomic study.

The impossibility of isolating pure groups of cells was a major constraint in the study of a specific type of cell. However, the development and evolution of microdissection techniques resulted in laser capture microdissection system. It is an efficient, accurate and safe from environmental contamination technique, addressed to investigators interested in separating cell populations or specific unique cells for the later molecular analysis.

The Microscopy and Molecular Imaging Unit of GENYO is equipped with the Zeiss PALM Microbeam IV Laser capture microdissection system. The Zeiss Axio Observer Z.1 inverted microscope is available for tissue or cellular sections observation providing excellent quality images. Thanks to the PALMRobo software, tissue or cellular regions of interest can be easily and rapidly localized on the screen. The drawing tools of the program allow toselect cellular groups or unique cells. The system’s UV laser (355 nm) is used to both cut and catapult cellular groups or unique cells into the adhesive cap of the collection tube to be studied. The “Cap Check” system of the program is used to verify the catapulted sample in the cap, which provide a higher reliability of the system.