The view of a tumor as an aberrant tissue whose growth and differentiation is sustained by cancer stem cells is now largely accepted for a wide variety of cancer diseases. Cancer stem cells, as their physiological counterparts, possess the ability to control their fate by maintaining themselves in a quiescent and undifferentiated state or self-renewing to geometrically expand their number. The first situation leads to an asymmetric division which gives origin to two well different daughter cells. The first progeny is devoted to expansion by multiple cellular division and differentiation to generate the whole heterogeneous cell population composing the tissue (or the tumor). The second one does not enter the cell cycle and rests as a reservoir able to self renew when requested. A typical stimulus involving the stem cell compartment expansion is, for example, the response to injury, e.g. repopulation of a damaged bone marrow in case of hematopoietic stem cells. In this second case, stem cells divide in a perfectly symmetric manner giving rise to two new cells with identical stem cell fate and proliferation potential.

The comprehension of stem cell biology and its molecular basis is now acquiring paramount importance in cancer research. However the low representativeness of the stem cell compartment, e.g. in the order of 1 out 1000 in the hematopoietic system, makes population averaging techniques, as the vast majority of molecular biology assays (just think to Western Blots) are inapplicable in most cases. The need to look at a single, possibly living, cell makes fluorescence microscopy and confocal microscopy invaluable allies in the study of stem cells.

The need to replace standard cell culture with more representative assays in terms of biological complexity led to the development of three dimensional cell culture techniques to produce in vitro biological prototypes of the different tissues and organs. In the field of breast physiology and pathology the ability to reconstitute in vitro the mammary gland is measured by the formation of mammospheres (figure 1).

Purified putative stem cells are plated at very low density on specific substrates and their growth observed by living cell microscopy. In a physiological situation, asymmetric division prevails with only one of the two daughter cells prosecuting with multiple rounds of divisions [1]. The transformed stem cell, as in the case of cells derived from the ErbB2 transgenic mice which express the activated ErbB2 oncogene in the mammary epithelium, abrogates asymmetric divisions preferring to give rise to multiple actively proliferating cells with self-renewal potential.
Asymmetric mitosis possesses a specific molecular signature characterized by a polarized three dimensional redistribution of certain segregating determinants, proteins that are differentially partitioned between the daughter cells and contributing to determine their stem cell fate. The optical sectioning ability of a confocal microscope consequently becomes fundamental to reconstruct the volumetric localization of a polarity marker as shown in figure 2. The extremely different signal distribution of the segregating determinant Numb in normal mammary (panel A) and cancer (panel B) stem cells confirmed that asymmetric division is lost upon transformation.

The tight link existing between stem cells fate and the physiological environment surrounding them (the stem cell niche), calls for imaging cells in living organisms or in fixed thick tissue slices maintaining the native histological architecture. As a consequence, two-photon microscopy is now massively entering the field. Nonlinear fluorescence excitation contribution could be also extremely relevant in in vitro studies. Mature mammospheres dimension can exceed 100 microns in thickness representing a relevant challenge for conventional confocal imaging.

Besides increased penetration depth, use of infrared pulsed sources for imaging provides superb performances in functional microscopy assays. Simultaneous absorption of two low energy quanta opens the possibility to excite fluorescence in the UV range, even at relevant depths, making visible and quantifiable the spatial distribution of a wide variety of endogeneously expressed metabolic products as NADH, a relevant readout of the glycolysis activity of the cell (figure 3). The ability to browse through the spectrum of emitted light by spectral analysis allows to simultaneously quantify fluorescence from different molecular sources excited in the same light range as reported in figure 3 where NADH and flavin contribution were identified according to their photophysical properties.

Nonlinear microscopy can consequently represent a revolution in the field of stem cell biology revisiting the role of the microscope from a "simple" tool for purely structural analysis to a relevant and unique assay for the in vivo functional characterization of cells according to their self-renewal potential.