Olympus FLUOVIEW FV1000 Confocal Laser Scanning Microscope Although "confocal laser scanning microscopy" is not exactly a household word to most people, it's a very important tool for the researchers who are working to save lives and advance the cause of medical science. And thanks to an innovative new Olympus scanning technology called SIM Scan, these researchers can now obtain a realtime, three-dimensional view of the processes at work inside living cells. Cell Structure (Click to enlarge) hen the human genome was first decoded, it represented a scientific breakthrough of such magnitude that the whole world took note. Articles on the subject appeared in the popular press, and there was talk that a whole range of miracle cures was just around the corner. In truth, decoding the human genome was just the first step in a long process, and scientists are only now undertaking the painstaking task of using genetic information to determine how cells work at the most basic level. To carry out this work, researchers need to understand the molecular processes that occur inside living cells. And that's where confocal laser scanning microscopy comes into play. But what is laser scanning microscopy, anyway? Most of us are familiar with microscopes, of course - those things we used to look at leaf structure and microorganisms in biology class. Properly speaking, this type of microscope is known as a compound microscope. There are digital microscopes, which are connected to personal computers so that specimen samples can be observed on a computer monitor. There are also many other types of microscope, each designed to handle a specific task. The confocal laser scanning microscope is one such specialized type, and to understand how it works requires a basic knowledge of the phenomenon known as "fluorescence." Olympus CX31 Compound Microscope Olympus Mic-D Digital Microscope The Principle of Fluorescence (Click to enlarge) A Primer in Fluorescence Most laser microscopes utilize the principle of fluorescence, which allows only a particular area of the specimen - the area that fluoresces- to be observed. From a research standpoint, this offers three distinct advantages: 1) It is possible to observe only certain structural elements, protein compounds or other macromolecules within a cell. 2) By using multiple fluorescent dyes, it is possible to simultaneously observe multiple structural elements, compounds, and macromolecules. 3) Fluorescence allows extraneous background and foreground elements to be suppressed, resulting in a much clearer picture of the area being studied. Since the specimens under observation generally do not fluoresce on their own, they need a little help. One method that is used is to dye the areas that the researcher wants to observe with a fluorescent dye. The dyed sample is then mounted in a laser scanning microscope, and a beam of laser light is directed at the sample. When light strikes the sample, the fluorescent dye molecules in the sample absorb light of a particular wavelength, which increases the energy of the molecules and causes them to release some of this energy as light of a slightly longer wavelength. This, in brief, is the process of fluorescence. Diagram of Confocal Laser Scanning Microscope (Click to enlarge) Confocal Laser Scanning Microscopy: A "Slice of Life" Let's now consider the advantages of confocal laser scanning microscopy. There are three major advantages: 1) It is possible to obtain highly detailed images with a very shallow depth of field. 2) Because the depth of field is extremely shallow, multiple images taken of different layers of the specimen can be combined to create a detailed three-dimensional image. 3) Both the interior and exterior of a cell can be observed. Shallow Depth of Field (background out of focus) Deep Depth of Field (background in focus) The key to understanding these advantages is in the words, "confocal" and "scanning." Let's look at the "confocal" aspect first. With any type of lens assembly there is a focal point at which the subject is precisely in focus. When people talk about "depth of field," they are talking about the degree to which things behind and in front of this point are in focus . When you are taking a picture with a camera, these things "behind and in front of" your main subject are often desirable, because such background and foreground elements can add interest and a sense of perspective to your photo. But in the world of microscopy, especially when trying to observe fluorescence within a living cell, such elements are just a distraction. Although the laser beam used to excite the fluorescent dye molecules can be aimed at a particular spot on the specimen, any fluorescent dye molecules located in front of or behind that spot will also fluoresce. This creates a haze of out-of-focus background and foreground fluorescence that obscures the primary point of observation. And that's where "confocal" technology comes in. In confocal microscopy, a pinhole mask/screen is interposed between th>?e viewing (upper) lens and the imaging device at a point that precisely corresponds to the focal point of the objective (lower) lens. (In optical terms, the point at which the pinhole screen/mask is interposed is called the "conjugal focal point," thus the name, "confocal.") This shuts out virtually all of the light rays from out-of-focus areas of the image, and allows only the light rays from the focal point to pass through, providing a clear image of the specimen at the precise point of focus.