What is fluorescence intensity contrast in tumor imaging?

To date, there have been two major steps toward implementing fluorescence intensity contrast in tumor imaging. They can be summarized as:

(i) whole body optical imaging, employing stereomicroscopy (fluorescent and bioluminescent reporters) and transgene expression in mouse models, and (ii) Intravital imaging, employing optical sectioning approaches and single cell visualization up to a few hundred microns depth in tissues.

The major emphasis of these two approaches has been to visualize a tumor cell in intact animals. These approaches had not been previously employed in quantifying the tumor burden in three-dimensions or in reconstructing the primary tumor in its early stage. This limitation was partly due to the bottleneck inherent in intensity-based approaches. Mammalian tissue is a turbid medium that can be characterized by an isotropic scattering coefficient and an absorption coefficient. Scattering in tissue occurs due to the index of refraction mismatch between fluid and cellular organelles. Absorption is primarily affected by the presence of chromophores such as hemoglobin. In vivo imaging systems detect the diffuse emission on the surface of the animal and therefore the optical properties of the tissue affect the detected signal and the minimum level of detection.

The two major impediments in small animal imaging modalities that employ fluorescence intensity contrast are (i) inability to provide depth information and (ii) ubiquitous autofluorescence background that reduces the signal-to-noise ratio. Together, these two factors decrease signal-to-noise ratio in the final images.


What is the principle of two-photon excitation?

Fluorescence microscopy involves visualization and quantitative imaging of microscopic objects (~0.3 microns and above). Fluorescence imaging techniques are optimal for biological cells (unlike electron microscopy), since live cell processes can be monitored noninvasively, in their native conditions. Conventional fluorescence imaging methods (1-photon excitation) employ excitation/emission schemes for various fluorophores in visible range (400-800nm). Although these methods work very well for monolayers of cells and thin tissue sections (<10 microns), they have fundamental limitations in thick tissue imaging and/or in vivo animal imaging. The critical limitation arises from the poor penetrability of visible wavelengths (owing to significant absorption by the aqueous tissue environment) thereby limiting the depth from which useful information can be obtained. In 2-photon excitation, a high-power pulsed laser and near-infrared excitation wavelengths (700-1000nm) are used to excite fluorophores. In a narrow window of optimal conditions, this configuration combines the energy of “two near-infrared photons” to excite the fluorophore, resulting in fluorescence emission akin to the 1-photon excitation. The immediate advantage is that near-infrared photons (longer wavelengths) can penetrate deeper than visible wavelengths (blue or green alone); useful information can therefore be obtained from even deeper tissues and from live animals (at least up to 500-600 microns as compared to <100microns in 1-photon excitation).


What are the advantages of multiphoton excitation in addressing depth penetration problem in thick tissues?

Wide-field fluorescence microscopy and laser scanning confocal microscopy methods have been successfully employed to understand a variety of phenomena in fixed or living cells in the UV to far red optical window. Tissue absorption and scattering make these fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of microns. In this context, multiphoton excitation methods have proven more suitable in providing clear images even from deeper tissue layers, owing to their near infrared excitation wavelengths (700-1000 nm) and hence reduced absorption of the excitation light.

Intravital imaging in tissues has been very successful in providing information from deeper layers (depth ~ a few hundreds of microns), although the emphasis has been on “visualization” rather than on “quantification” of the fluorescent signals. It is therefore possible to achieve high-resolution images from deeper layers by sophisticated optics. Even though multiphoton excitation provides a partial solution for penetrating deeper layers of the tissue, complications arise as one progresses from imaging cells to tissues to whole animals—which include enormous autofluorescence background in tissues, and poor signal from regions of interest.

Endogenous autofluorescence background has a fairly wide emission band, encompassing the wavelength range 450–700 nm, and is convolved in every pixel of the image with varying amplitudes. Use of NIR probes can reduce the autofluorescence background only minimally.


What contributes to redox metabolism in tumors?

A variety of intracellular molecules such as NAD(P)H, flavins, glutathione, and thioredoxin contribute to the overall redox status of mammalian cells. In particular, the reducing species have an impact on how tumor tissues respond to oxidative stress induced by a particular chemotherapeutic agent or radiation modality. Thus any noninvasive means of monitoring the tumor redox status can supplement routine clinical oncology procedures. The feasibility of real-time redox mapping and endogenous collagen architecture in living tissues utilizing multiphoton excitation methods leads to more interesting avenues of interrogating healthy and disease phenotypes in a variety of animal models.