advanced imaging laboratory
Zeiss LSM 410 laser scanning microscope
A major problem with the light microscope is the inherently shallow depth of field that goes along with high magnification. This becomes a serious problem with thick tissue and commonly results in images made of a combination of sharp information and blurred or out of focus information. Confocal microscopes are designed to reject the out of focus light while keeping the in focus light, resulting in a sharp optical section.
There are a number of techniques for achieving this, some using spinning disks with many pinholes. The laser scanning confocal microscope uses a laser beam that is scanned back and forth across the selected area of the specimen to excite fluorescence emission. The resulting emitted light is imaged onto a photo multiplier (PMT) via a very precisely aligned pinhole. The microscope stage can then be stepped upward under computer control and a new "optical section" acquired. If this is repeated a number of times the result is a "stack" of images from which 3-d reconstructions of the inner structures of the tissue can be made.
Lasers (an acronym for Light Amplification by Stimulated Emission of Radiation) are devices that contain an optical cavity with mirrors at each end that are filled with lasable material. This might be a gas such as argon or a crystal such as ruby, the atoms of which can be excited to a semi stable level by light or electric discharge. The light emitted by each atom as it drops back to its ground state stimulates other atoms and the intensity increases as light bounces back and forth between the mirrors. The mirror at one end of the laser is slightly transparent (1% to 2%) and when the intensity becomes sufficient an intense beam of highly collimated light emerges.
Much contemporary biological research involves the examination of material that has been labeled or stained with specific fluorochromes. These dyes absorb higher energy, short wavelength light and emit lower energy, longer wavelength light. The Zeiss LSM 410 invert is a modern incarnation of the confocal microscope. The Advanced Imaging Laboratory's LSM 410 is built around a Zeiss Axiovert inverted microscope and is equipped with an Argon/Krypton laser that emits light at 488, 568 and 647 nanometers. The instrument's optics allow two fluorescence channels to be imaged simultaneously.
Our confocal microscope is normally set up for material stained with fluorescein isothiocyanate (FITC), propidium iodide (PI), Texas Red (TR) and rhodamine (TRITC). TRITC filter settings, FITC filter settings and dual label filter settings are schematic drawings of the instrument showing the correct filter settings for some of these labels. There is also a complete schematic of all the filters and dichroic beam splitters on our confocal microscope. The instrument is equipped with a video camera that feeds onto a frame buffer, Nomarski DIC and has Achroplan 10x na .25, Plan Apo 20x na .60, Plan Apo 40x na 1.0 oil iris and Plan Apo 63x na 1.4 oil objectives. You might want to read a bit about laser safety before using the instrument.