Fluorescence localization and quantification
Fluorescence is emitted when a fluorophore interacts with an incident photon (excitation). Absorption of the photon causes an electron in the fluorophore to rise from its ground state to a higher energy level. Then, the electron reverts to its original level, releasing a photon (fluorescence emission) whose wavelength depends upon the amount of energy that is released during reversion. A given fluorophore may emit at single or multiple wavelengths (creating an emission spectrum), as electrons drop from various orbitals to their ground states. The emission spectrum is constant for each species of fluorophore. Imaging finds many uses in fluorescence. As examples, consider the following:

	An imaging system tuned to a specific emission spectrum can be used to localize a fluorophore. For example, cells expressing green fluorescent protein can be imaged and counted.
	Changes in the fluorophore molecule (such as binding of fura-2 to Ca++) will lead to alterations in the emission spectrum. An imaging system can be used to measure these spectral changes, as an indication of changes in the environment of the fluorophore.
	By measuring the intensity of fluorescence, an imaging system can estimate the concentration of a fluorescently tagged molecule. A common example of this is in the use of fluorescent microarrays for gene expression analyses.

Localization: monochrome and multispectral fluorescence imaging
In the simplest case (monochrome fluorescence imaging), a single fluorophore is used to mark a single molecular species. For example, glial fibrillary acidic protein (GFAP) labeled with fluorescein isothiocyanate (FITC) can be used to visualize regions of repair following CNS trauma. Similarly, a specific chromosomal DNA location can be shown by fluorescence in situ hybridization.
Multispectral fluorescence imaging demonstrates multiple molecular species in the same image. Each discrete fluorescent tag is visualized as a different color. For example, we might show Cy3 (green) and Cy5 (red), with the regions of overlap shown as mixtures of colors (e.g. red and green overlap shown as yellow). MCID® and AIS handle multispectral fluorescence in two ways.

Best quality
Each fluorophore is visualized independently, under optimal conditions. For example, discrete images of FITC and rhodamine fluorescence are created. The Image Fusion function then combines the two images into a single color image that shows inter-relationships among the tagged tissue components (Figure). This method yields the best image quality, for three reasons.

	High resolution, very sensitive cooled cameras can be used.
	The fluorescence optics (e.g. excitation and emission filters) may be optimally tuned for each wavelength.
	You have flexible control over the contribution of each discrete image to the final fused image.

Figure: Three discrete monochrome images, are represented in their original colors (rhodamine on mictochondria, fluorescein on GFAP, nuclei stained with DAPI. Each image was acquired (10-bit camera) with exposure and filters optimal for that stain. At bottom right, the images are fused into a single color image. Click on the image for a larger version.

Most convenient
Multiple fluorophores are visualized simultaneously. In this case, we use optics that provide simultaneous multispectral excitation and discrete emission wavelengths for each fluorophore. A color camera is used to image the multicolored specimen. As standard color cameras are not sufficiently sensitive to visualize fluorescence emission, an integrating color camera is used.
Figure: The same specimen as above, but captured with a 3-CCD color camera, integrating for about 1/2 sec. Note that image quality is not quite as good as with discrete color acquisition. Click on the image for a much larger version.

Quantification: changes in fluorophore environment
Changes in pH, binding of the fluorophore to specific ions, and many other environmental factors can lead to an alteration in the emission spectrum of a fluorophore. Measurements of such changes were traditionally performed in cuvettes. However, various methods have been developed that allow imaging systems to be perform similar measurements at the cellular and subcellular levels. MCID includes dedicated functions for the quantification of changes in fluorophore environment.
Figure: Time course of intracellular Ca++ concentration following administration of bradykinin. Note that the cell bodies show a monotonic response, while the processes tend to cycle. Click on the image for a much larger version.

Variety of measurement functions
Typical fluorescence measurements include area and proportional area, number of fluorescent targets, and fluorescence intensity. The spatial measurements are quite straightforward, and are performed more or less well by most image analyzers. In contrast, intensity measurements can be rather tricky because fluorescence fades, and good calibration standards are difficult to create. MCID's proven competence in quantitative intensity measurement lets you concentrate on the specimens, not on the weaknesses of the measurement instrument.

Support for both intensified and integrating cameras
Standard video cameras are not well suited to fluorescence applications, and a specialized low-light camera is usually necessary. We have a state-of-the-art intensified camera, and a broad variety of integrating cameras available for use with MCID.

Intensified CCD cameras
Intensified CCDs (ICCDs) consist of a video camera mated to an image intensifier. The intensifier amplifies incident illumination by an adjustable factor. ICCDs are fast, in that they take a short time image relatively dim specimens. Their main drawbacks are grainy images at high amplification, poor rendition of contrast in fine details, and a severely limited intrascene dynamic range. That is, ICCDs cannot see both bright and dim material within one image (typical dynamic range of about 40:1). ICCDs are best suited to dynamic fluorescence imaging, where their ability to provide images quickly is a critical advantage. For most purposes, we recommend a GEN IV intensifier, which exhibits much better image quality than other variants. We have various ICCD cameras available, but we recommend the Roper Instruments video ICCD with GEN IV intensifier, integrating CCD camera, and control unit. This is about as sensitive as a single-stage ICCD gets, and has the added benefit of being very flexible. For extremely dim specimens, multistage intensifiers are available, and are often used in photon counting applications. In our opinion, the trials of working with a multistage ICCD are significant, and we prefer to use our Black Ice cryogenic integrating cameras when ultimate sensitivity is required.  

Integrating cameras
Integrating cameras are like film. They accumulate incident illumination over time. In general, integrating cameras provide better image quality and broader dynamic range than intensified cameras. MCID and AIS support a variety of integrating cameras.
Integrating video cameras are low in cost and suitable for moderately bright specimens such as many immunolabeled cells. For bright specimens, the camera does not need to be cooled. For dimmer specimens, chilled (above 0 degrees C) or cooled (below 0 degrees C) integrating video cameras are still cost-effective. However, do not expect any video camera to function with demanding specimens. Integrating video technology sacrifices sensitivity and dynamic range (limited to 8-10 bits) in exchange for low cost.
The next step above video is a family of moderately priced integrating cameras (e.g. the Roper Sensys or Hamamatsu 4742), which use high resolution CCDs that can be operated in integration mode. Typically, these cameras are chilled to above-zero temperatures, and make fine images with fluorescent specimens.

For more difficult specimens, we use scientific-grade, cooled cameras. The exact definition of a "scientific grade" camera varies but, generally, these devices use full-coverage CCDs, high precision digitizers (>12 bits), and deep cooling. The most advanced of these cameras use special, high-sensitivity CCDs and cryogenic cooling (cool below -100 C). Our Black Ice camera incorporates every technical advantage that we know of, to yield performance that is absolutely state-of-the-art. Unfortunately, Black Ice technology is costly, but there are many scientific-grade cameras that are reasonably priced and yield excellent performance. Contact us for details.

The imaging system
A single video frame (made in 1/30 sec) from an intensified camera will be very grainy. The quality of the low-light image is improved by real-time averaging. Therefore, ICCD cameras may be interfaced to any imaging system capable of rapid frame averaging. It is useful if the imaging system can also construct ratios and perform fluorescence background subtraction in real time.
Integrating cameras can present more of a challenge to the imaging system. Efficient use of an integrating camera presents the following requirements:

	Integrated camera and software :Although MCID/AIS can use images from any camera (by importing TIFF files), it is convenient if the image analysis software also controls the various exposure and data transfer parameters of the camera. Doing image acquisition within dedicated camera software and image analysis in a separate package is very tedious.
	Accept high precision data: The imaging system must accept and calibrate to data at high bit densities (integrating cameras supply data at 8 - 16 bits).
	Fast interface: The imaging system should include a fast interface to the integrating camera. The best cameras come with a dedicated connection (e.g. RS422) to the imaging system interface board, or with their own interface card. Acquiring images via a SCSI or other slow connection is cheaper and easier for the manufacturer to implement, but really degrades imaging throughput.

MCID includes fast and efficient control of integrating cameras, and can be calibrated to high bit densities.

Single excitation, single emission procedures are much simpler than ratiometry. All that is necessary is that we acquire images at timed intervals, and then measure fluorescence intensity values from those images. Changes in fluor location escence intensity or fluor location (e.g. internalization of a receptor labeled with GFP) can be tracked. Changes in intensity are generally qualitative. That is, we can state that a change in fluorescence emission occurs, but we cannot quantify the change in terms of ionic concentrations.
An example of a single emission procedure is use of the Ca++ indicator fluo-3. It is excited at 503-506 nm, in the visible portion of the spectrum. Fluo-3 has a weaker affinity for Ca++ (KD about 400 nm) than do fura-2 or indo-1, permitting measurement of lower Ca++ concentrations. It also exhibits very marked changes in fluorescence intensity (about 4 decades) with Ca++ binding. Compare this with the tenfold change in fluorescence intensity exhibited by fura-2. MCID's single emission option is similar in use to fura-2 imaging, though there is only one excitation wavelength. As filter wheel changes are not required, rather short inter-image intervals are possible.

MCID includes an equation editor, that lets you create output images by specifying mathematical operations between input images. Using the editor, you can create your own ratiometric methods.