Transgenic mouse models have been developed to recapitulate the complex effects of genes known to be involved in human breast cancer. These models can help to elucidate the mechanism of action of these genes during carcinogenesis, as well as their impact on normal mammary gland development. Imaging methods for mouse models of normal and cancerous mammary glands are in the developing stages and can help in the search for better ways to diagnosis human breast cancer earlier .
In order to study early phenotypic effects of gene over-expression or lack of expression on mammary gland development and cancer traditional methods require that tissue is harvested from the animal and subjected to histological techniques to detect morphologically aberrant growth. These invasive procedures preclude further examination of the effects of these genetic changes on the process of carcinogenesis. Later, once the tumor becomes palpable, the size of the developing tumor can be measured and followed in vivo over time to determine proliferative capacity. However, no information about initiation and progression can be gathered; only at the end of the experiment  can information about the morphology or gene expression profile of the developing tumor be obtained.
The implementation of in vivo imaging modalities to study normal mammary gland growth and disease progression has greatly improved the utility of these models, allowing the study of mammary differentiation or disease process, not simply the final effect . Biochemical and morphologic changes associated with early cancer change the optical properties of tissue, especially the absorption, scattering, and fluorescence, allowing the detection of these early carcinogenic effects with optical spectroscopy techniques . Imaging can allow researchers to, with minimal invasiveness, detect and follow abnormalities in ductal development during mammary differentiation in the same living animal. In cancer studies, imaging can detect undissected preneoplastic lesions and follow the behavior of these cancer cells, interactions with their stromal environment during the development of a tumor, angiogenesis, and metastatic disease. This can all be studied over time in the context of the cancer cells own physiological environment with an intact blood supply and interaction with surrounding tissues in 3-D and in real-time [1, 5, 6]. Imaging regimens can also be adapted to evaluate efficacy and response of a cancer to prevention and therapeutic interventions [7, 8] and to detect the presence of chemoresistance . Serial minimally invasive imaging of mice reduces the number of mice needed per experiment or in preclinical drug development since multiple time points can be observed in the same animal . The imaging modalities reflectance confocal microscopy (RCM), green fluorescent protein (GFP) imaging, and ultrasound imaging were utilized in this paper to image mammary glands and mammary tumors.
RCM provides real-time minimally invasive 3-D sectioning of in vivo (living) or ex vivo (newly biopsied) individual cells and tissues using variations in the optical properties of the natural backscattering of light from different cellular and subcellular structures without the use of labeling cells fluorescently or otherwise [10, 11]. Optical techniques such as RCM have demonstrated high sensitivity for detecting cancer in their natural environment without using ionizing radiation  and without time-consuming and potentially destructive fixation and staining, both of which may introduce artifacts and damage tissue . Tissue studied with RCM is treated with acetic acid, which induces DNA condensation providing increased reflectance to contrast nuclear versus cytoplasmic structure. We have shown that tissue treated with acetic acid can then be subjected to histological and immunohistological analyses without detrimental effects on the tissue , facilitating further study into signaling pathways which may be active in the imaged structure . RCM has been performed on biopsy specimens to assess tumor margins  and to identify precancerous lesions in human breast core needle biopsies , the cervix , and skin .
Fluorescent protein labeling and epi-illumination spectroscopy microscopy are very powerful tools to follow primary tumor growth and metastasis with fluorophores in vivo and in real time . Transgenic GFP optical imaging is one type of fluorescent protein label imaging and involves the detection of reporter transgene expression, namely a genetically encoded fluorescent protein, which is utilized to image cells within living tissue [3, 20]. The specimen, often exposed surgically, is illuminated with blue light (488 nm excitation wavelength) which is absorbed by green fluorescent protein, a protein originally from the jellyfish Aequorea Victoria . GFP then emits green light (509 nm peak shifted emission wavelength) which is collected by CCD cameras . GFP imaging can be used as a cell marker in both the living animal and in tissue culture and does not require a substrate for visualization .
GFP transfected tissue culture cells and GFP transgenic mice have been used to monitor real time tumor growth and for mechanistic studies [23, 24], evaluate the efficacy of therapy in a tumor xenograft model with metastasis , monitor specificity of in vivo gene therapy studies , mark and sort potential mammary stem cells , and examine mammary epithelial tumor cell behavior in metastasis . In addition to monitoring mammary gland development on the whole at the ductal morphology level as in our current study, GFP can be used to image single cells. This high resolution GFP imaging of cells in vivo has been combined in a dual labeling approach with red fluorescent protein (RFP) to monitor tumor-stroma interactions and drug response of cancer and stromal cells [29, 30].
Ultrasound imaging involves exposing tissues to high-frequency ultrasound waves (20–60 MHz in animals; 2–10 MHz in humans) by placing a transducer (which contains crystals that vibrate when exposed to small electrical currents and produce sound waves) on the skin and then detecting the ultrasound reflections from internal organs under investigation [6, 31, 32]. This non-invasive technique produces a dynamic real-time image of the tissue from which structural and functional information can be obtained because sound waves travel though soft tissue based on the acoustic impedance of each tissue, which is a function of the tissue density . When two tissues with different densities are next to each other, a mismatch in the acoustic impedance causes sound waves to be reflected relative to the degree of mismatch; a greater acoustic impedance mismatch leads to a greater reflected pressure magnitude or intensity and is seen as a brighter image .
Ultrasound is a rapid non-radiation method that has been used to detect cystic masses  and superficial tumors , differentiate between fibroadenomas and carcinomas in animal models , noninvasively track liver metastases growth and evaluate potential therapy in liver metastasis models , measure blood flow by Doppler [37, 38], guide biopsy of a palpable breast mass , and guide injections into target organs .
In the present study, we use RCM, GFP, and ultrasound to visualize mammary gland and mammary tumor characteristics in vivo. We show that RCM can be used to study mammary development in an ex vivo whole organ culture setting with good resolution using a lower concentration of acetic acid. We show that GFP expression can be used to visualize mammary gland ducts, mammary tumor, and tumor vasculature, to follow lobuloaveolar development in an ex vivo whole organ culture experiment, and can be used to follow development of transplanted mammary glands. We show that ultrasound imaging can be used to visualize normal mammary gland, hyperplastic areas of preneoplasia, to follow tumor progression and liver metastases, and can be used to distinguish between mammary tumor and enlarged lymph node. In conclusion, we show that these modalities are, individually and in combination, useful in studying normal and carcinogenic biological processes in the mouse mammary gland longitudinally and with minimal invasiveness.