Software & Techniques

Epifluorescence Microscopy

Fluorescence microscopy is a rapidly evolving technology, and the Laboratory of Cancer Biology and Genetics Microscopy Core Facility has likewise evolved to maintain state-of-the art services for the scientists in the Center for Cancer Research of the National Cancer Institute. Our facility houses multiple systems that can be used to analyze cell structure, protein expression, and cell dynamics using immunofluorescence. These include inverted epifluorescence microscopes, a confocal microscope, and slide scanning microscopes.

Although fluorescent markers have been used extensively to study biological systems for decades, technological advances in fluorescent probes have tremendously broadened their spectrum of applications. Fluorescent proteins now include reporters of transcriptional regulation, targeted indicators of organelles and other subcellular structures as well as fusion proteins that can be used to follow the dynamics of living cells. As fluorescent proteins and their uses have evolved, the microscopes and cameras used to detect them have also been improving. The LCBG Core Facility has both a Zeiss AxioObserver and Nikon Ti2 inverted epifluorescent microscopeds equipped with scientific CMOS cameras and LED light sources that provide high resolution fluorescence and DIC optics along with up-to-date image acquisition and analysis capability.

Confocal Microscopy

Confocal microscopy is a powerful optical imaging technique that can increase both horizontal and vertical resolution of an image by eliminating out-of-focus light in specimens thicker than the focal plane. This type of microscopy is ideal for localization of organelles or molecules in a sample or reconstructing 3-dimensional images of a specimen. In confocal microscopy the focal point in the specimen is conjugate to the pinhole (i.e. they are confocal). Consequently, only the light coming from the focal point can pass through the pinhole and be detected by the photomultiplier behind it. Any light above or below the focal plane is excluded by the confocal pinhole resulting in a well-defined image, virtually free from scattered light. This unique feature makes confocal microscopy an ideal technique for 3-dimensional analysis of specimens. High resolution optical sections can be generated and then combined into a 3D projection image that is in focus at all levels. This type of microscopy allows the generation of superior, high-resolution images, particularly in the case of thick specimens such as tissue sections or embryos. In addition, decreased light scatter leads to increased resolution of fine structures such as microtubules, stress fibers, focal adhesions, mitochondria, and centrosomes.

The LGCP Core Facility has a Zeiss LSM 780 laser scanning confocal microscope. The sensitivity of LSM 780 is outstanding. The GaAsP detector achieves 45 percent quantum efficiency compared to 25 percent typically by conventional PMT detectors. This results in accurate details and contrast-rich images of specimens. The system’s illumination and detection design allow you to simultaneously acquire up to ten dyes. You excite any common fluorophore with seven different lasers, detecting the signals with the 32 channel GaAsP detector.

Slide Scanning

One of the problems scientists face with using immunofluorescence is the short lifespan of the specimens, relative to immunohistochemistry. Finding the time to image the data before it is lost is always a challenge. Users of the LCBG Core Facility are able to digitize their specimens and create high-quality virtual slides with the Zeiss AxioScan.Z1 slide scanner. The AxiScan is highly automated and simple to operate, and capable of imaging in both brightfield, and in multiple parallel fluorescence channels. The virtual slides are able to be stored long term in an online database.