Fluorescence microscopy is a very powerful technique widely used in biomedical research. Though eminently useful for most applications, the technique suffers from serious limitations, notably hazed images and phototoxicity. The haziness arises from interference originating from structures above and below the plane of focus and phototoxicity derives from non-productive excitation of the fluorophore. A high numerical aperture objective lens provides an in focus image of a thin plane within the specimen. However, the information from this optical section is often obscured by out of focus interference from fluorescent structures above and below the plane of focus. Confocal imaging and computer deconvolution are two techniques that have been developed to combat this problem. Typically a confocal imaging system consists of a laser scanning microscope with a pinhole aperture in the image plane of the objective. The confocal aperture only passes light that emanates from the point being illuminated. Unwanted fluorescence that originates away from this point is blocked. Computer deconvolution techniques use computer algorithms using the knowledge of the point spread function of the imaging system and information from a sequence of adjacent focal planes to remove out of focus components from a given focal plane.

Although both confocal imaging and computer deconvolution can be effective in virtually eliminating out of focus interference. These techniques do nothing to alleviate the other major problem, phototoxicity. When a fluorophore is excited, there is a probability that instead of decaying to a singlet state and emitting a fluorescence photon, intra-system crossing will occur to a triplet state. These relatively long lived states are very reactive and can damage living cells and bleach the fluorophore in both living and fixed cells. One of the most significant damage mechanisms is the generation of highly reactive singlet oxygens from triplet states (1). When a specimen is being observed in a fluorescence microscope, fluorophore is excited throughout the bulk of the sample, even though only one focal plane is being observed at any time. Most of the phototoxic load, therefore, comes from regions away form the thin focal plane being observed. This problem can be circumvented to some extent by soaking fixed specimens in antioxidants. However, this approach is not practical for living material and bleaching can reach very high rates and phototoxicity can become very extensive in living samples.

The technique of multi-photon excitation provides an elegant solution to the problem of unwanted out-of-focus excitation in which the excitation is confined only to the optical section being observed (2). The sample is illuminated with light of a wavelength which is approximately twice (or three times) the wavelength of the absorption peak of the fluorophore in use. In the case of fluorescein, which has an absorption peak at approximately 500 nm, 1,000 nm excitation could be used. Essentially no excitation of the fluorophore will occur at the 1,000 nm wavelength because it is so far removed from the peak excitation wavelength of the fluorophore. In addition, no bleaching will occur, nor will phototoxic products be generated in the bulk of the sample. A high peak power pulsed laser source with a peak power of more than two kilowatts is used in pulses that are shorter than a picosecond so that the peak mean power levels are moderate and do not damage the specimen. With this source, the photon density at the point of focus will be sufficiently high for significant numbers of two and three photon events to occur. In a laser scanning microscope, the point of focus will describe a raster point as it scans and only at this focal point is the photon density high enough during the brief pulse for two or more photons to be absorbed simultaneously by the fluorophore. The absorption of two photons of long wavelength is equivalent to the absorption of one photon of half the wavelength, resulting in fluorescence excitation. Thus, in a 2-photon fluorescence microscope, one has the ideal probe in which fluorescence excitation is confined to the focal point under observation. Out of focus interference is eliminated simply because it is never generated.

Multi-photon excitation fluorescence microscopy has important advantages over other imaging modes of microscopy, particularly for the study of live cells and/or for thick tissues (3).

1. The lack of fluorophore excitation in regions away from the focal plane has the consequence that the bulk fluorophore bleaching and generation of toxic by-products is reduced to minimum levels during imaging.
2. Multi-photon images are less prone to degradation by light scattering. This is because the longer wavelengths used for excitation suffer less scattering from microscopic refractive index differences within the sample, allowing much greater penetration of the tissues. In addition, as all the resolution is defined by the geometry of the excitation beam, the fluorescence emission is unaffected by light scattering. The insensitivity of multi-photon imaging to light scattering is particularly advantageous for the study of living specimens as live tissue is highly light scattering due to the presence of many refractive index interfaces.
3. Due to the additive effects of 1 and 2 above, images to can obtained from deeper within a sample using multi-photon imaging than with any other type of light microscopy (3).
4. Because wavelengths of different colors of light originating from one point source focus at different focal planes, in standard multi-probe confocal microscopy registration of multiple probes along the Z axis is problematic. Because multi-photon microscopy excites at one discreet diffraction limited point and because emitted light is collected without being deconvolved to different focal planes, true registration in the Z axis is achieved.

The common scientific need of the major users is the ability to obtain confocal optical sections from deep within intact tissue, often of multiple probes such as Dapi, GFP and Rhodamine, with minimum bleaching and, in the case of live material, minimum phototoxicity. A comparison of our laser scanning confocal microscope, the MRC-600, with a multi-photon microscope, the MRC-1024MP, was done using live anesthetized rats containing primary breast tumors composed of tumor cells expressing green fluorescent protein (GFP). The GFP in the tumor cells is an excellent cytoplasmic volume marker which allows the entire cell outline to be defined (4). The video supplied with the application contains two sequences each showing a series of optical sections obtained by steping at 4 um intervals through a primary tumor in a live animal. The first series was obtained with the conventional MRC-600 and the second series was obtained with the MRC1024MP in multiphoton mode. The comparison of these two z-series demonstrates the superiority of the multi-photon microscope in penetrating deep within the live tissue to generate high resolution confocal images. The greater penetrating power of the multi-photon microscope is even more impressive when one considers that the high quality image set shown was taken inside of the primary tumor and not at the surface, ie. the deepest section is approximately 150 um into the tissue. The deepest penetration possible when generating high quality images with the conventional confocal was about 40 um.

References for Justification

1. Widengren & Rigler (1996). Mechanisms of photo bleaching investigated by fluorescence correlation spectroscopy. Bioimaging, 4; 149-157.

2. Denk, Strickler & Webb (1990). Two photon laser scanning fluorescence microscopy. Science, 248; 73-76.

3. Centonze & White (1998). Multi-photon excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J. 75(4):2015-24.  2015-2024.  PMID: 9746543; UI: 98427250

4. Farina, K.L., Wyckoff, J.B., Rivera, J., Lee, H., Segall, J.E., Condeelis, J.S., Jones, J.G. (1998) Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Research 58:2528-2532.