- MICROSCOPIA CONFOCAL
The National Museum of Natural Sciences has recently purchased a Leica TCS SPE microscope equipped with brightfield, interferencial, fluorescency and confocal. We also provide a new version of the software METAMORPH to analyze images (alternative software is Huygens, Volocity, Amira, Imaris, etc.).
Confocal microscopy provides several advantages against the traditional microscopy, among them images with less out of focus. Briefly, the light falling on the sample comes from a laser that in this microscope can be of three wavelengths: 488 nm, 532 nm y 635 nm. This light excites the natural or artificial (by adding fluorochromes to the sample, e.g., Eosin) fluorescence, and the emitted fluorescence is received by the objective of the microscope, which allows the visualization and the study of the sample.
Due to the physical nature of the light falling on the sample and the mechanism receiving the fluorescence, the confocal microscope produces optical sections conceptually equivalent to histologic sections, with the advantage that these sections are registered and therefore ordered. These optical sections are later treated with one of the visualization programs as mentioned before to obtain a #D reconstruction of the object, or for any other application. The confocal equipment contains a program for image acquisition and basic treatment called Leica LAS AF. This software allows controlling of all the parameters of image capture. There is a free version of this software called ‘lite’ (download the software Leica LAS AF lite).
THIS SOFTWARE ALLOWS TO PROCESS IN OUR COMPUTER ALREADY EXISITNG- LEICA LAS AF LITE (compressed file program for PC)
The confocal microscope TCS SPE is one of the most basic in the market, presented by Leica as ‘easy to use and first results are quickly achieved even by confocal novices”, and therefore, this is the most common model offered by the majority of laboratories to their users 24/7. To facilitate the comprehension of the use of this machine I include here a four page tutorial from the University of Iowa to handle it.TUTORIAL TO ACQUIRE CONFOCAL IMAGES – TCS SPE QUICK START (English)
The software METAMORPH is more flexible and has more options than LAS AF, but it requires a longer learning period. Most functions available in our version (different versions of METAMORPH have different modules for different applications) are detailed in the section ‘help’ included in the software. In the following link you can download the tutorial (ppt file) from the Centro de Investigaciones del Cáncer, CSIC, which can provide an introduction tour version, although is not exactly the same.
There is a detailed manual to go into the TCS SPE system in depth:
TCS SPE MANUAL DE USO (Spanish version)
TCS SPE USER MANUAL (English version)
In Arthropods it is common to use autofluorescency, and when this is not enough, a fluorochrome like Eosin gives usually a good result. For other organisms it is needed to consult the existing literature (very easy: Zoological Record, ‘confocal’ + ‘group of organism of interest’)
The microscope accepts a range of sizes of only a few millimeters, but is an excellent tool for all organisms and structures traditionally studied with the optical microscope like insect genitalia.
Other aspectsIMAGEJ is a software freely available, widely used and relatively easy to use with applications relatively sophisticated. This software was developed with economical support from the National Institute of Health, USA, has a PC and a MAC version, and is still today being updated due to several ‘plugins’ (modules that perform special applications) developed by individual researchers that can be added to the software by downloading them and inserting them in the folder called ‘plugins’ to be recognized by the program. These plugins can be easily found in the search tool in Google, searching for ‘imagej’ and ‘plugin’ + ‘the function we are interested in’. It is very likely that the function we need has already been developed by someone, and is already available for public use. The last version of IMAGEJ can be downloaded at the following address: http://rsbweb.nih.gov/ij/
For all those interested in knowing in more detail the microscopic technology, there library at the Natural Science Museum has available the third edition of the Handbook of Biological Confocal Microscopy (Pawley, 2006).
Antonio G Valdecasas (author)