The Laser Scanning Confocal Microscope (LSCM, or confocal) is a specialized type of light microscope. A laser beam is scanned across the sample to form the image, which is stored on a computer as a digital file. The key advantage of the confocal microscope over a conventional “wide-field” light microscope is that it images only a narrow slice of the sample, or in other words it has a narrow depth of field (as small as 0.4mm). Only information from the plane of focus is detected. This allows the operator to take a single image (or optical section”) from deep within the sample. The confocal can also be instructed to acquire a series of images from several discrete focus levels (Figure 1). The resulting data sets allow the investigator to view their sample in 3-D and manipulate and measure structures in those 3 dimensions.
In addition to its narrow depth of field the confocal has several other advantages. Under optimal conditions the LSCM can resolve smaller detail than a conventional light microscope (~100 nm). Collection of the image is rapid and it can be previewed before saving. Confocal imaging also avoids the difficulties associated with photographic imaging such as developing and printing delays and chemical waste generation.
The confocal microscope is usually operated in epi-fluorescence mode although it can also be used in reflected and transmitted light mode. Most confocals are equipped with one or more lasers that can excite most common fluorescent probes excited by visible light (e.g. FTIC, Rhodamines, Cyanine dyes.) UV lasers are available (to excite probes such as DAPI or Fura-2) but are quite expensive and have safety and phototoxicity issues. Visible light excited fluorescent dye analogs are available for most UV dye applications. Sample preparation is identical to that for conventional epi-fluorescence with heavier staining sometimes being an advantage. Thick and thin, living and fixed biological specimens, as well as materials samples can be examined. The reflected mode is used to image surfaces or reflective stains within samples. Samples of this type include geological materials, semi-conductors, optical storage disks and tissues or cells labeled with colloidal gold or silver stains. The transmitted mode functions the same as that of a conventional light microscope with samples able to be viewed by bright field, darkfield, phase contrast or differential interference contrast. Some confocal manufacturers support full color transmitted light imaging.
All three of these modes are computer controlled and take advantage of the digital image processing capabilities of the confocal computer. Images may be annotated on screen with labels, arrows, scale bars and measurements. The images may be printed on color laser or dye-sublimation printers or to 35mm film. Files can be archived on Zip, Jaz, CD-R or CD-RW disk or may be transferred to other computers via the Internet.
THEORY
In a confocal microscope (Figure 2) the fluorescent label in the sample is excited using a laser instead of a mercury or xenon lamp (as in a conventional epi-fluorescent microscope.) This laser beam is focused to a point and scanned across the sample, point-by-point, line-by-line. At each point along the scan the emitted fluorescence light is collected by the objective lens, passed through a aperture and then detected by a photomultiplier tube (PMT.) A pixel of the appropriate brightness is then stored in memory and displayed on the monitor. A typical image consists of an array of 512 by 512 pixels giving an image size of 256k bytes. The key to the confocal effect is the pinhole, or iris. This adjustable aperture prevents the out of focus light from being detected by the PMT thereby causing the system to image only a thin slice of the specimen, i.e. “an optical section.” Closing this iris gives a thinner “optical slice” and better resolution while opening it gives a thicker slice and a brighter image.
CONFOCAL DESIGNS
Most modern confocal microscopes follow one of two designs; “laser spot scanning” or “spinning disk”. In a laser spot scanning system (Figure 3) one or two galvanometer-controlled mirrors scan a focused spot of light across the sample. The fluorescent light emitted from each spot passes through the pinhole (if from the plane of focus) and is detected by the PMT. Depending on the resolution setting (which can be 2048 by 2048 pixels or greater) the scan times can range from fractions of a second to minutes. Up to 5 different fluorescent probes can be detected at one time. This is the most common type of confocal sold and is the most light efficient. It produces the highest resolution images and is the most flexible in imaging modes.
The spinning disk confocal employs one or two rotating disks with regularly spaced holes (Figure 4). As light (from a mercury lamp or a laser) is projected on to the disk the holes trace concentric arcs of illuminating light across the sample. When the fluorescent or reflected light returns through the disk only light from the plane of focus makes it past the holes. The disk(s) in effect act as a high-speed light chopper. Because of this the spinning disk confocal works well in reflected, white-light mode. They are commonly used in the semi-conductor industry for wafer inspection and in dental and material research. The real-time images can be viewed by eye and are recorded by a digital or analog camera for storage. The spinning disk confocal can scan much more quickly (as fast as 360 frames per second) than a spot scanner but has less resolution and is not as well suited for fluorescence.
Figure 4
A recent advance in spinning disk confocal microscopy replaces the holes with microlenses. This greatly increases the sensitivity of the system and improves its ability to image fluorescently labeled samples.
In his original patent Minsky describes a transmitted light, stage scanning confocal microscope (figure 5). Light shown through a pinhole (A) is focused by the condenser to a point on the sample (D). Light from the plane of focus is collected by the objective lens(O) and passes through the confocal pinhole (B) to be detected by the PMT (P). As shown by the arrows in Figure 5 the sample had to be scanned around the point of light produced by the condenser lens. This required long scan times and could result in distorted images if the sample was not rigid. There were also safety and alignment problems so this design was not widely pursued. Points A, D and B correspond to “conjugate focal planes” hence the term “confocal.”
Figure 5
A confocal of a very different design has been recently gaining popularity. A “digital deconvolution” (Figure 6) system takes images from a conventional wide-field fluorescence microscope and digitally removes the out of focus light. The computer has a map, or “point spread function” of how light passes through a particular microscope. The software uses this map to determine what light is out of focus in origin and removes it through various algorithms. Digital deconvolution systems can be inexpensive and retrofitted to existing fluorescent microscopes. The images are prone to artifact however and are not as reliable as spot scanned images. Deconvolution is often performed on tradition confocal images to further narrow the “slice.”
COMMONLY USED FLUORESCENT PROBES
In general, allmost any fluorescent probe that is excited by visible light can be imaged on a confocal. UV probes (such as DAPI, Hoechst, Fura-2) cannot be used. However, there are visible light dye options for most traditional UV excited probes.
FLUORESCENT PROBES FOR CONJUGATION
to antibodies, biotin, strepavidin, etc,Green Fluorescent
FITC, Alexa 488, Cy2
Red Fluorescent
Rhodamines, Cy3, Texas Red, Alexa 568
Far Red Fluorescence
Cy5, Phycoerythrins
DNA/RNA, NUCLEUS
Green Fluorescing
YO-YO, Sytox green, Actimomycin
Red Fluorescing
Propidium lodide, Ethidium Bromide
Far Red Fluorescing
TO-TO3, TOPRO
VIABILITY DYES
Live Cells
Calcein AM, CFDA
Dead Cells
Propidium lodide
Apoptosis labels
ORGANELLE PROBES
Mitochondria
Mito Tracker
Mito Fluor
Rhodamine-123
DiOC6
MEMBRANE MARKERS
DiA, Dil, DiO
FM 4-64
Fluorescent Lectins
ELECTROLYTE/ION CONCENTRATION
Ca++
Fluo-3, Calcium Green, Calcium Crimson
Mg++
Mag-Fura Red, Magnesium Green
pH
SNAFL, SNARF
FLUORESCENT PROTEINS
GFP
EGFP
CFP
YFP
RFP
