2.1.

Cell Culture

T47D cells were obtained from the American Type Culture Collection. NMuMG cells were a kind gift from Dr. Caroline Alexander (University of Wisconsin). EGFP-Vinculin was a kind gift from Dr. Anna Huttenlocher (University of Wisconsin). For GFP-RBD, the Rho binding domain (RBD) of Rhotetkin was excised from GFP-RBD (a generous gift of Dr. Bill Bement, University of Wisconsin), subcloned into pEGFP-C1 (Clontech), and stably expressed in T47D breast carcinoma cells. T47D human breast cells were maintained in RPMI supplemented with 10% fetal bovine serum and insulin. NMuMG mouse mammary cells were maintained in DMEM supplemented with insulin and 10% fetal bovine serum. Both cells lines were cultured at $37°\mathrm{C}$ with 5% ${\mathrm{CO}}_2$ .

Cells were cultured and imaged under standard 2-D conditions or within 3-D collagen matrices. For 3-D culture, cells were cultured within a 1.0 to $4.0\mathrm{mg}∕\mathrm{mL}$ type-I collagen gel (rat-tail collagen solution, BD Biosciences) neutralized with $100\mathrm{mM}$ HEPES in $2\times$ PBS. Following gel polymerization, gels were soaked in cell-specific media (described earlier) or serum-free media containing BSA and maintained at $37°\mathrm{C}$ with 10% ${\mathrm{CO}}_2$ until imaged as described in the text.

2.2.

Mammary Tumors

All animal experiments were approved by the institutional animal use and care committee and meet NIH guidelines for animal welfare. To generate mammary tumors, polyoma middle-T mice⁴¹ were employed. For MPLSM imaging of live, unfixed, intact (not sectioned), nonstained PyVT tumors, tumors were harvested and live tissue maintained in buffered media at $37°\mathrm{C}$ . All tissues were imaged immediately following tissue harvest.

2.3.

Instrumentation

For all imaging, two different custom multiphoton systems that are part of the University of Wisconsin Laboratory for Optical and Computational Instrumentation (LOCI, www.loci.wisc.edu) were utilized.^{19, 40, 42} The first system is an MPLSM workstation constructed around a Nikon Eclipse TE300 that facilitates multiphoton excitation (MPE), SHG, and FLIM. All SHG imaging was performed on this microscope and was detected from the backscattered SHG signal.³⁸ A 5-W mode-locked Ti:Sapphire laser (Spectra-Physics-Millennium/Tsunami) excitation (laser field) source producing around 100-fs pulse widths was tuned to 780 to $900\mathrm{nm}$ . The beam was focused onto the sample with a Nikon CFI Plan Fluor $20\times$ multi-immersion objective (NA of 0.75 and WD of 0.33 with water), a Nikon CFI Plan Fluor $40\times$ oil immersion lens $(\mathrm{NA}=1.3)$ , or a Nikon CFI Plan Apo $60\times$ water-immersion lens $(\mathrm{NA}=1.2)$ . Endogenous fluorescence and SHG signals were isolated with 464-nm (cut-on) long-pass and 445-nm narrow bandpass filters, while GFP signals were isolated with a 480 to $550\mathrm{nm}$ (bandpass) filter (all filters: TFI Technologies, Greenfield, Massachusetts). Intensity and FLIM data were collected by a H7422 $\mathrm{GaAsP}$ photon counting photomultiplier tube (PMT) (Hamamatsu) connected to a time-correlated single photon counting (TCSPC) system (SPC-730, Becker & Hickl).

The second microscope has been recently described in detail^{19, 29, 40} and allows generation of multiphoton excitation intensity images in conjunction with FLIM and SLIM. In short, the system is built around an inverted microscope (TE 2000, Nikon, Melville, New York) with source illumination from a Ti:Sapphire mode-locking laser (Coherent Mira, Coherent, Santa Clara, California), with a tuning range of $\sim 700$ to $1000\mathrm{nm}$ . FLIM images were acquired with an electronic system for recording fast light signals by time-correlated single photon counting (SPC-830, Becker & Hickl). The system has several detectors, including a 16-channel combined spectral lifetime detector (utilizes a Hamamatsu PML-16 PMT), detection range 350 to $720\mathrm{nm}$ , and an H7422P $\mathrm{GaAsP}$ photon counting PMT (Hamamatsu) for intensity and lifetime imaging. The same lenses and filters were used as on the first microscope system. Both FLIM and SLIM data were collected over $60\mathrm{s}$ , and the pixel frame size for the MPE/SHG images is $1024\times 1024$ , while the FLIM and SLIM images are $256\times 256$ .

Acquisition for both MPLSM systems was performed with WiscScan,⁴³ a LOCI-developed software acquisition package that can control both the MPLSM and the lifetime collection. Image analysis was performed with ImageJ⁴⁴ and VisBio⁴⁵ software. Fluorescent lifetime analysis was carried out with SPCImage (Becker & Hickl), which can fit fluorescent decay data to an exponential function [Eq. 1], for one, two, or three terms, and sum individual photon counts for each pixel to construct a contrast image. The incomplete model approach in SPCImage was used to compensate for instances where the fluorescence decay is slow compared to the time window defined by the repetition rate of the laser system. SLIM analysis was performed with SPCImage (Becker & Hickl) and SLIM Plotter (described in detail in Sec. 3).

3.1.

Nonlinear Optical Imaging: MPE, SHG, FLIM, and SLIM

Increased understanding of tumor-stromal interactions is a critical aspect of the study of breast tumor formation and progression, since stromal-epithelial interactions play a critical role in both tumorigenesis and metastasis. ^{14, 26, 46, 47, 48, 49} Since optical imaging modalities allow deep, noninvasive imaging of epithelial and stromal components of live breast tissue and tumors, they provide a valuable set of tools to better understand the tumor-stromal interaction. As seen in Fig. 1 , multimodal multiphoton excitation (MPE)/SHG imaging offers a clear view of intact live mammary tumor tissue. Not only can epithelial and stromal cells be clearly seen, but their interaction with the stromal collagen matrix can also be readily imaged. By taking advantage of the fact that SHG signals are exactly half of the excitation wavelength, while fluorophores under MPE excitation obey the fundamental physical relationship of energy loss after excitation (Stokes shift), the MPE and SHG signals can be separated by filtering the emission signal. In Fig. 1, live mammary tumor tissue was excited at $890\mathrm{nm}$ to produce endogenous fluorescence from tumor and tumor-associated cells and SHG from collagen. (In our hands, with our biological systems, this wavelength has provided the strongest SHG signal for collagen.) To separate the emission signals, a 464-nm (cut-on) long-pass filter was used to isolate the cellular fluorescence (MPE signal), while SHG was isolated with a 445-nm narrow bandpass filter. By performing this filtering scheme, the tumor cell’s interaction with collagen can be studied. Consequently, the use of combined MPE/SHG has the ability to identify and differentiate features that are either not obtainable or not easily obtained with more traditional fluorescence microscopy techniques. For instance, using this approach, we previously defined three tumor-associated collagen signatures (TACS; Ref. 14) in mammary tumors by imaging stromal collagen density and organization within and around tumors of varying stages. These TACS are diagnostic, allowing identification of early neoplastic regions as well as signatures that identify invading metastatic cells.¹⁴ TACS-1 characterizes the presence of increased SHG signal intensity due to increased locally dense collagen within the globally increased collagen concentration surrounding tumors, serving as a reliable hallmark for locating small neoplastic regions. TACS-2 classifies straightened collagen fibers stretched around the tumor, constraining the tumor volume and indicating that the tumor has substantially expanded and is straining the space of its microenvironment. TACS-3 is the identification and characterization of radially aligned (distributed at approximately $90\mathrm{deg}$ relative to the tumor boundary) collagen fibers that facilitate local invasion, and support evidence presented by Condeelis and colleagues that metastasizing tumor cells migrate along collagen fibers.^{3, 26}

Fig. 1

MPE/SHG imaging of live tumor tissue. Combined (a) and separated (b) MPE/SHG images acquired following ${\lambda }_{ex}=890\mathrm{nm}$ of live mammary tumor tissue. Combined MPE/SHG clearly demonstrates the ability to image deep into live tissue and obtain meaningful information from endogenous signals. In (b), emission signals were isolated with a $464\text{-}\mathrm{nm}$ (cut-on) long-pass filter for MPE (pseudocolored red) and a $445\text{-}\mathrm{nm}$ narrow bandpass filter SHG (pseudocolored green). This approach permits identification of epithelial tumor cells, tumor-associated stromal cells, and the collagen matrix. As such, combined MPE/SHG imaging of intact tumors can provide relevant information about the cell-matrix interaction and how matrix density and organization influence cell behavior. Bar = $25\mu \mathrm{m}$ .

Multiphoton FLIM provides an additional data dimension—allowing the measurement of the fluorescence lifetimes of endogenous fluorophores within normal and tumor cells while simultaneously identifying collagen in and around the tumor by exploiting the fact that the collagen signal is not fluorescent and as such has a theoretical lifetime of zero. Figure 2 demonstrates the utility of multiphoton FLIM in identifying and measuring lifetimes of the relevant metabolic components NADH and FAD, as well as identifying collagen. To characterize and present lifetime information, the data was fit with a multiexponential model:

Fig. 2

FLIM imaging of live mammary tumor tissue. Tumors were imaged at ${\lambda }_{ex}=780\mathrm{nm}$ [(a) and (b)] and ${\lambda }_{ex}=890\mathrm{nm}$ [(c) and (d)] to produce endogenous fluorescence from NADH and FAD, respectively. Intensity [(a) and (c)] and color-mapped lifetime [(b) and (d)] images are shown. Color mapping represents the weighted average of the mean lifetime $\left({\tau }_m\right)$ (Eq. 2) following fitting with a two-term exponential model [Eq. 1]. Note that emission from collagen (d) maps to the yellow end of the spectrum, indicating SHG.

In addition to the ability to image live tissues, studies in more reductionist in vitro systems that reconstruct aspects of the cell’s microenvironment, such as reconstituted 3-D matrices, can provide valuable information regarding cell phenotype and signaling events. Figure 3 demonstrates the ability of mammary epithelial to recapitulate ductal structure when cultured under the appropriate conditions within a collagen matrix, supporting the concept of this system as a valuable method to study breast epithelia. Although transmitted light [Fig. 3a; Video 1 ] provides information regarding the morphology of the cells and allows visualization of the ductal structure, little information can be obtained regarding the cellular interaction with the microenvironment. By exciting signals from endogenous sources, insight into this interaction can be gained. Combining MPE excitation of endogenous fluorophores with SHG imaging, a more complete view of the structure and its interactions can be obtained [Fig. 3b; Video 2 ]. Furthermore, multiphoton FLIM can be utilized as described earlier to acquire lifetime data that can identify the metabolic state of the cell or isolate and characterize fluorophores present with and around the cells. This is of even greater utility when the desired goal is to image both exogenous and endogenous signals simultaneously, such as is the case for protein localization (Fig. 4 ) or FRET experiments within 3-D collagen environments. As seen in Fig. 4, combined MPE/SHG imaging allows a clear view of the cell-matrix interaction in 3-D. GFP emissions from GFP-Vinculin were separated using a 480- to 550-nm (bandpass) filter, while SHG was isolated with a 445-nm narrow bandpass filter. Three-dimensional matrix adhesion to the collagen matrix can clearly be seen (see Fig. 4, inset) allowing a reconstruction of cell morphology, collagen organization, and the cell-matrix interface. However, in more complex studies, such as FLIM, the presence of a strong SHG signal (or other endogenous or exogenous signal) in close proximity to the fluorophore can be very problematic. Moreover, while FRET measured with FLIM can be superior to intensity-based FRET measurements due to the fact that FLIM is independent of fluorophore concentration, FLIM is susceptible to multiple signal contamination issues in data analysis, particularly when more than two lifetime components are potentially present. This can be overcome by employing spectral lifetime imaging, as discussed earlier. By separating the spectral signal of interest from contaminating background²⁸ or other fluorophores of interest, the lifetime of the fluorophore of interest, can be cleanly obtained (Fig. 5 ). However, in order to efficiently analyze the large multidimensional data sets from SLIM, a computational infrastructure needs to be developed. As a means to effectively visualize and exploit the information from spectral lifetime data, we have developed a novel computational package.

Fig. 3

Imaging mammary ductal structure in reconstituted 3-D matrices. Nontransformed NMuMG mammary cells were cultured within a 3-D collagen matrix $(3.0\mathrm{mg}∕\mathrm{mL})$ for 7 days to demonstrate the ability of combined MPE/SHG/FLIM to study cellular differentiation and the extracellular microenvironment. The recapitulated ductal structure can be seen with transmitted light (a), but use of combined MPE/SHG (b) and/or multiphoton FLIM (c) provides information regarding the collagen matrix and the physiologic state of the cells. In (c), color represents the weighted average following a two-term exponential fit as described in Fig. 2. Scale bar= $25\mu \mathrm{m}$ ; color bar 0.08 to $1.0\mathrm{ns}$ (red to blue).

Fig. 4

Imaging 3-D matrix adhesions with MPE/SHG. Combined MPE/SHG imaging at ${\lambda }_{ex}=890\mathrm{nm}$ of GFP-Vinculin expressing NMuMG cells within a 3-D collagen matrix $(4.0\mathrm{mg}∕\mathrm{mL})$ for 7 days allows a clear view of collagen matrix organization, vinculin localization, and the cell-matrix interaction. Emissions signals from GFP were isolated with a 480 to $550\text{-}\mathrm{nm}$ (bandpass) filter (pseudocolored red) and a $445\text{-}\mathrm{nm}$ narrow bandpass filter SHG (pseudocolored green). Three-dimensional matrix adhesion to the collagen matrix can clearly be seen (inset). Bar= $25\mu \mathrm{m}$ .

Fig. 5

SLIM to optimize exogenous and endogenous signals. (a) MPE/SHG intensity image of seven T47D breast cells (each designated by a star), composing a developing ductal tubule within a 3-D collagen matrix $(1.3\mathrm{mg}∕\mathrm{mL})$ after 5 days in culture. Each cell expresses various levels of EGFP–Rhotekin binding domain (GFP-RBD) and therefore has different intensity in the MPE image. (b) An FLIM image demonstrates collagen fibers in juxtaposition to localized GFP-RBD. Arrows demonstrate regions of localized RBD, suggesting local Rho GTPase activation. Note that Rho is activated at “stress” points where the forming tubule contacts collagen fibers, which is biologically quite interesting but can make data analysis and interpretation difficult. Therefore, the spectral channels for GFP [see the SLIM Plotter output in (d)] were selectively reimaged, producing a new SLIM image, and were color mapped with a narrower lifetime scale (c). This allows elimination of the collagen signal when desired and shows additional localization of GFP-RBD. Hence, spectral lifetime imaging has the ability to facilitate separation of signals of interest for analysis or elimination when the large data set can be efficiently managed and visualized (see Sec. 3.2). Bar= $20\mu \mathrm{m}$ .

Video 1

Movie of a transmitted light $z$ -stack $\left(200\mu \mathrm{m}\right)$ , showing mammary ductal structure with a reconstituted 3-D matrix (QuickTime, $2.7\mathrm{MB}$ ). 2.

Video 2

Pseudocolored movie of a combined MPE/SHG $z$ -stack $\left(200\mu \mathrm{m}\right)$ , showing ductal structure and the surrounding collagen microenvironment (QuickTime, $4.49\mathrm{MB}$ ). 1.

3.2.

Combined Spectral Lifetime Visualization: SLIM Plotter

Slim Plotter is a lightweight application for the visualization of combined spectral lifetime data (Fig. 6 ). The main purpose of the program is to allow exploration of regions of data collected with an SLIM system, but the software also provides some features to assist hardware engineers in proper calibration of the acquisition hardware.

Fig. 6

SLIM Plotter Visualization window. Example of the SLIM Plotter Visualization window. Data used for this example is from a mammary tumor, and the image shown in the 2-D viewer is a single spectral channel.

The program is written in Java, which enables cross-platform deployment. Launchers are provided for Microsoft Windows (EXE file), Mac OS X (application bundle), and ${}^{*}\mathrm{nix}$ (shell script). It uses the VisAD Java visualization toolkit for displaying data (http://www.ssec.wisc.edu/ $\sim$ billh/visad.html), which in turn uses Java3D (https://java3d.dev.java.net/). Slim Plotter requires Java version 1.4 and Java3D 1.3—these versions come bundled with Mac OS X, so Slim Plotter should work out of the box on Apple computers, while Windows and Linux users must set up the Java runtime environment and Java3D if they do not already have them installed. Although memory requirements vary depending on the size of the data set, at least $512\mathrm{MB}$ of system RAM is strongly recommended. See the Slim Plotter website (http://www.loci.wisc.edu/ome/slim.html) for download and detailed installation instructions.