21 Non-invasive single-photon and multi-photon imaging of stem cells and cancer cells in mouse models

: The use of ﬂuorescent protein for in vivo imaging began a revolution in cell biology. In particular, non-invasive imaging of ﬂuorescent-protein expressing cancer cells, stem cells, immune cells and other cell types has enabled longitudinal studies on cancer spread or stem cell differentiation in real time. Fluorescent proteins come in many colors, enabling color-coded imaging of multiple cell types in vivo. Practical applications include evaluation of all types of agents on cancer and metastasis or stem cells in real time. The combination of ﬂuorescent proteins with multiphoton imaging techniques represents a powerful tool for the visualization and tracking of single cancer cells and stem-cell behavior in their natural niche. Multiphoton tomography(MPT)iscapableofhigh-qualityrecordingofﬂuorescenceandsecond-harmonic generation (SHG) images at submicron resolution, yielding greater structural details than possible with single-photon imaging technology. It also enables optical 3D biopsies without surgery. Most importantly, MPT has been adapted for in vivo imaging. Cell dynamics and subcellular effects of drugs can be investigated in living animals. Auto-ﬂuorescence/SHG imaging of the extracellular-matrix proteins elastin and collagen is highly complementary to other protein labeling methods, such as green ﬂuorescent proteins (GFP). This chapter discusses aspects of tomographic linear and nonlinear in-vivo optical imaging.

can be used for the color-coding of cancer cells growing in vivo and enable the distinction of host cells from cancer cells with single-cell or even subcellular resolution.Visualization of many aspects of cancer initiation and progression in vivo are possible with fluorescent proteins [1].
We first reported non-invasive, real-time, imaging of GFP-expressing tumors growing and metastasizing in live mice in 2000 [2].The simple whole-body optical imaging system was external and non-invasive.It enabled unprecedented continuous visual monitoring of tumor progression within intact animals.Whole-body non-invasive optical images showed metastatic growth in the brain, liver, and bone in real time enabling quantitative measurement of tumor growth in each of these organs.Either a trans-illuminated epifluorescence microscope or a fluorescence lightbox and thermoelectrically cooled color charge-coupled device (CCD) camera was used for imaging.The depth to which metastasis and micrometastasis could be imaged depended on their size [2].This was a totally unexpected discovery and was reminiscent of Roentgen's first use of X-rays to obtain images, but in the case of fluorescent proteins, only harmless blue light was needed.
We have also described a system for rapidly and non-invasively visualizing transgene expression with GFP in major organs of intact live mice.GFP linked to an adenovirus was expressed in the cells of brain, liver, pancreas, prostate, and bone upon injection, and its fluorescence was visualized non-invasively.For lowmagnification images, animals were illuminated in a fluorescence lightbox and directly viewed with a CCD camera.Higher-magnification images are made with the camera focused through an epi-fluorescence dissecting microscope.Within 5-8 h after adenoviral GFP injection, the fluorescence of the expressed GFP in brain and liver became visible non-invasively and whole-body images were recorded at video rates.The GFP fluorescence continued to increase for at least 12 h and remained detectable in liver for up to 4 months.The system's rapidity of image acquisition made it capable of real-time recording.The method requires only that the expressed gene or promoter be fused or operatively linked to GFP [3].
GFP-expressing Lewis lung carcinoma cells were injected into the subcutaneous site of the footpad of nude mice which is relatively transparent, with comparatively few resident blood vessels [4], allowing quantitative non-invasive imaging of tumor angiogenesis in the intact animal.Capillary density increased linearly over a 10-day period as determined by non-invasive imaging.A GFP-expressing human breast cancer MDA-MB-435 was orthotopically transplanted to the mouse fat pad, blood vessel density increased linearly over a 20-week period, shown by non-invasive imaging [4].
In 2003, we described the use of non-invasive imaging of tumors in mice to monitor drug response.RFP-expressing pancreatic cancer tumor fragments were surgically transplanted onto the nude mouse pancreas [5].The model reliably simulated the aggresive course of human pancreatic cancer.The anti-metastatic efficacy of drugs was followed non-invasively in real time by imaging the RFP fluorescence.Gemcitabine could delay tumor growth and metastasis but not cure the tumor, which matches clinical experience [5].
In 2004, dual-color fluorescent cancer cells with one color in the nucleus and the other in the cytoplasm were developed.RFP was expressed in the cytoplasm of cancer cells, and GFP linked to histone H2B was expressed in the nucleus.These cells enabled real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro [6].RFP was expressed in the cytoplasm of cancer cells and GFP linked to histone H2B was expressed in the nucleus.Nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes could be imaged.The cell cycle position of individual living cells was determined by the nuclear-cytoplasmic ratio and nuclear morphology.Apoptosis was observed by nuclear size changes and progressive nuclear fragmentation.Dual-color mitotic cells were visualized non-invasively by whole-body imaging after injection in the mouse ear [6].
Non-invasive imaging of RFP-expressing cancer cells growing in the lung, was enabled by spectral separation [7], using the Maestro In-Vivo Imaging System with a liquid tunable filter [7] (Fig. 21.1 and 21.2).

Fig. 21.1:
The Olympus IV100 microscope is a scanning laser microscope with a 488 nm argon laser was used and stick objectives (as small as 1.3 mm) which deliver very high resolution images [13].A three-color sub-cellular, non-invasive imaging model was developed consisting of GFP-expressing mice transplanted with the dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm [8].The Olympus IV100 Laser Scanning Microscope, with ultranarrow microscope objectives ("stick objectives"), was used for imaging the two-color cancer cells interacting with the GFP-expressing stromal cells (Fig. 21.3).Cellular dynamics were non-invasively imaged, including mitotic and apoptotic cancer cells, tumor vasculature, and tumor blood flow.In this model, drug response of both cancer and stromal cells in the intact live animal was also non-invasively imaged in real time [8] (Fig. 21.3).
RFP-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to transgenic GFP nude mice.Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by non-invasive whole-body imaging [9].
GFP-expressing human cancer cell lines, including HCT-116-GFP colon cancer and MDA-MB-435-GFP breast cancer were orthotopically transplanted to transgenic RFP nude mice.Dual-color non-invasive fluorescence imaging also enabled visualization of human tumor-host interaction [10] (Fig. 21.4).GFP-or RFP-expressing human pancreatic cancer cells were implanted into the bright blue fluorescent pancreas of the cyan fluorescent protein (CFP) nude mouse.The bright blue fluorescence of the pancreas in the CFP mouse was an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host [11].
Patient-derived orthotopic xenograft (PDOX) models of pancreatic cancer were passaged through transgenic nude mice expressing GFP and RFP [12].The patient tumors acquired brightly fluorescent stroma from the transgenic host mice, which was stably associated with the tumors through multiple passages.The tumors, with very bright GFP and RFP stroma, were then orthotopically passaged to nontransgenic nude mice.It was possible to image the brightly-fluorescent tumors non-invasively as they progressed [12].

Advantages of multiphoton imaging of stem cells and cancer cells in live mice
The imaging systems described so far are based on one-photon protein fluorescence excitation.In 2011, we applied multiphoton tomography (MPT) to image live transgenic mice.Our group demonstrated that imaging of specific fluorescence protein ex-pressing cancer and stem cells in live mouse models can be combined with autofluorescence as well as second harmonic generation (SHG) imaging using MPT.Unlike fluorescence, SHG is a coherent process involving only virtual energy transitions and does not induce photobleaching or phototoxicity [15].SHG is a largely frequencyindependent second-order nonlinear optical process in which two photons at the same frequency generate new photons with twice the energy (therefore twice the frequency) and half the wavelength of the initial photons [16].A variety of proteins, such as extracellular matrix collagen fibrils, tubulin, as well as the muscle-myosin lattices of muscle cells generate SHG [17].The intrinsic signal permits SHG microscopy of those structures non-invasively with no need for labeling.SHG signals can be collected efficiently and selectively using narrow bandwidth emission filters with minimal contribution from overlapping of any other fluorescence signals [15].Since SHG is frequency independent, a variety of excitation wavelengths can be chosen to easily separate SHG targets from any other fluorescence signals.
MPT typically employs excitation light in the near infrared (NIR).Fluorophores that have excitation spectra in the 300-600 nm range are excited with wavelengths of 600-1200 nm through absorption of two infrared photons simultaneously.In such a two-photon quantum event, each photon carries approximately half the energy necessary to excite the molecule than that needed for one photon excitation [18] and thus reduces photothermal and photodamage effects.Nevertheless, the simultaneous absorption of two photons requires a high flux of excitation photons which can be effected with femtosecond lasers.Due to the low scattering and absorption of NIR photons, MPT is capable of high-quality acquisition of images in 3D at higher penetration depths than possible with single-photon imaging technology, such as confocal microscopy.Dynamics of specific cells and responses to external agents in the cells' natural environment can be investigated in living small animals with sub-cellular resolution.

Real-time imaging of stem cells and their dynamics with subcellular resolution
Our group first applied high-resolution 3D MPT in order to non-invasively visualize hair follicle-associated pluripotent (HAP) stem cells in their natural environment.HAP stem cells have been found to express nestin, reside in the bulge area and the dermal papilla and have the capacity to develop various tissues of follicular and nonfollicular origin, such as nerves, blood vessels, and smooth muscle [19][20][21][22].MPT was employed to localize these stem cells in unperturbed niches in nestin-driven GFP transgenic athymic nu/nu nude and normal mice [23].The multiphoton tomograph MPTflex™ (JenLab GmbH, Jena, Germany) equipped with a sealed turn-key tunable 80 MHz titanium:sapphire femtosecond laser (710-920 nm, in situ laser pulse width 250 fs) and with an articulated arm suitable for animal imaging was used (Fig. 21.5 (a) and (b)).
The optical unit consists of an active optical power attenuator to regulate the in situ power of the laser corresponding to tissue depth, an active beam stabilization device, a safety unit and a flexible articulated mirror arm with a compact scan head.The scan head consists of a fast galvoscanning device and a piezodriven z-scanner to generate 3D scans, high NA focusing optics (NA 1.3) and a dual-photon detector unit for the simultaneous recording of a variety of fluorescence and SHG signals.The acquisition time for one optical section is typically 2 seconds.The overall field-of-view of the optical system covers 350 × 350 µm 2 .Autofluorescence of unlabeled cells from the skin tissue are also visible (images from [23,24]).
Multiphoton imaging provided the possibility of visualization of specific fluorescence protein nestin-GFP expressing stem cells within their intact microenvironment.Surrounding non-stem cells were visualized without exogenous labels based on twophoton excitation of intrinsic fluorophores such as nicotinamide adenine dinucleotide hydrogen (NADH), flavins and melanin in order not to cause any physiological imbalance on stem cells.In addition, SHG images were recorded simultaneously from collagen in the extracellular matrix.These imaging conditions were completely noninvasive and in particular, suitable for in vivo long-term tracking of intratissue stem cells without any significant effect on cell viability [23].
High-resolution multiphoton tomography allowed visualization of nestin-GFP expressing stem cells in their native niche at 930 nm excitation.Autofluorescence and SHG have been recorded simultaneously at 790 nm.3D optical sectioning without surgical intervention revealed cell morphology, cell size, and stem cell distribution in the tissue (Fig. 21.5 (c)) [23].Nestin-GFP expressing stem cells often occurred in clusters comprising varying numbers of cells per bulge.Long-term time-lapse imaging allowed detection of stem cell dynamics (Fig. 21.5 (d)) [23][24][25].Stem cells migrating from the hair-follicle bulge have been detected inside the skin during optical deep-tissue sectioning.Stem cells migrating along the hair shaft as well as in the wounded skin were also visualized (Fig. 21.5 (e)).
Nestin-expressing stem cells were found to have a different morphology than the main skin cell population in their physiological natural microenvironment.The typical size of stem cells was found to be about 7 µm, whereas the surrounding cells had a typical size of 15 µm [23,24].The visualized microenvironment consisted of the extracellular matrix (ECM) components elastin and collagen as well as a variety of nonlabeled cells.

Multiphoton surgery of stem cells
The same near-infrared femtosecond lasers used for multiphoton imaging can also be employed to manipulate cells/cellular organelles with higher precision (sub-100 nm) when applied at higher intensities [27,28].Rompolas et al. [29] combined two-photon imaging with laser-induced ablation to investigate stem cell behavior during physiological regeneration of hair follicles.Non-invasive high-resolution imaging allowed long-term observations of the nuclei of stem/progenitor cells in transgenic mice expressing histone H2B-GFP driven by the keratin 14 promoter and revealed oriented cell division and migration of stem cell progeny that accompany hair growth.Additionally, dermal papilla cells in Lef1RFP transgenic mice were targeted to eliminate the mesenchymal cells selectively in order to study hair regeneration.Mesenchyme was ablated with the same NIR femtosecond laser at 900 nm and observed in physiological conditions up to day 7 with multiphoton technique.Mesenchymal dermal papilla cells are required for stem cell activation and hair regeneration [29].GFP was excited with two-photon absorption at 940 nm, RFP at 1040 nm.

Imaging of GFP-labeled and unlabeled stem cells
MPT was used to compare GFP-labeled and unlabeled stem cell autofluorescence images in mouse whiskers.Autofluorescence multiphoton imaging revealed the same morphology of unlabeled hair follicle stem cells as GFP-labeled hair follicle stem cells [30].FLIM of stem cells can provide additional information on the differentiation process [31].

High-resolution non-invasive multi-photon tomographic cancer-cell imaging in living animals
MPT was used to visualize dynamics of cancer cells genetically-labeled with specific fluorescent proteins in intact mice as well as stromal cells by autofluorescence within deep heterogenic tumor tissue in live animals [32].Additionally, migrating rare metastatic cells were identified from nonmetastatic ones during consecutive longterm imaging [33].Furthermore, the combination of MPT with FLIM can gain insights into cancer cell metabolism due to autofluorescent molecules associated with cellular respiration such as NADH and flavin adenine dinucleotide [34,35].

Multi-photon imaging of tumor-targeting by Salmonella typhimurium A1-R
Our laboratory has developed a new substrain of engineered Salmonella typhimurium auxotrophs (S. typhimurium A1-R) to selectively eliminate cancer cells in viable as well as necrotic areas of tumor tissue but not in normal tissue [36].The use of GFP for imaging the bacteria allows real-time visualization of single bacteria in vivo which could lead to the selection of enhanced cancer-cell-targeting variants of S. typhimurium [37].We applied MPT to visualize S. typhimurium A1-R targeting Lewis lung cancer cells grown in the skin of nude mice.PMT1924 was used to detect signals from fluorescence and SHG channels.Filter sets LP409 and BP 395/14 were used for GFP and SHG, respectively.To separate Ds-RED fluorescence from GFP-and autofluorescence, filter sets BP593/40 and BP/510/42 were respectively used.
Collagen-extracellular matrix protein was imaged by SHG.Cancer cells expressing Ds-Red S. typhimurium A1-R expressing GFP, and stromal cells (autofluorescence) were simultaneously imaged by two-photon excitation (Fig. 21.6).Cancer cells were visualized in their natural microenvironment consisting of stromal cells and extracellular matrix collagen.Spatial distribution of single GFP-expressing bacteria and bacterial colonies inside the cancer cells and in the tumor tissue and tumor vasculature (inside and outside of capillaries) were detected several minutes after injection of bacteria in the tail vein (Fig. 21.6 (b)).Long-term MPT revealed that bacterially-infected cancer cells expanded and burst (Fig. 21.6 (c)).Vascular permeability of tumor-targeting bacteria could also be imaged to gain further mechanistic insights into tumor-targeting bacteria [37,38].
Previous studies have shown that fibrillary collagen is altered in various types of cancer and invading cells preferentially migrate along collagen fibers [39,40].Since MPT visualizes collagen in unperturbed physiological conditions, we applied highresolution SHG tomography in living mice to study extracellular matrix remodeling during tumorigenesis [41,42].The relationship between cancer cells and intratumor collagen, elastin fibrils was visualized in live mice with subcutaneously-grown Colo 26-GFP (GFP expressing colon cancer cells) colon cancer, breast tumor and Lewis lung carcinoma (Fig. 21.7).SHG of collagen fibrils and GFP of cancer cells were acquired simultaneously at an excitation wavelength of 790 nm.High-resolution multiphoton imaging revealed organized extracellular matrix collagen and elastin in tumor tissue of live mice.Well-aligned, parallel, closely packed collagen fibers were detected in Lewis lung carcinoma by SHG.Elastin fibers were seen as parallel structures to collagen structures which are detected by autofluorescence (Fig. 21.7 (a)).
Collagen detected in Colo 26-GFP colon cancer-bearing mice were organized in parallel structures with a wide interfiber spacing.Cancer cells aligned parallel to collagen fibrils.Multiphoton autofluorescence and SHG images of unlabeled breast cancer tissue revealed structures of collagen fibers as parallel, straight structures with interfiber spacing and with a different thickness.Breast cancer cell appeared well aligned parallel to collagen fibers.Breast cancer cells were visualized by autofluorescence and collagen by SHG.Our results suggest that collagen fibrils provide the scaffolding for cancer cells to anchor and acquire optimal shape in vivo.

Prospects and limitations of multiphoton tomography
Application of high-resolution MPT allowed the live imaging of stem cells up to depths of approximately 300 µm in nestin-GFP mice.However, localization of stem or cancer cells in deep organs requires future efforts to increase imaging depths.3D image recordings take a long time due to slow acquisition speed (typically 2-7 seconds per frame).Recording larger image areas is also time consuming.Moreover, animal motion induces artifacts in long-term imaging.Currently, biocompatible fluorescent probes and novel compact laser sources for the second optical window between 1000 nm and 1350 nm and even the third optical window from 1600 nm to 1870 nm are under development.This would allow much deeper tissue imaging than with current Ti:sapphire laser technology (600-1000 nm = first optical window) [44].Furthermore, there is a lack of information from nonfluorescent tissue components.Therefore a combination of multiphoton imaging tools with other optical label-free imaging technologies such as CARS imaging could provide complementary information on biochemical and

Fig. 21 . 3 :
Fig. 21.3: Non-invasive, subcellular imaging of drug response of dual-color mouse mammary cancer cells and GFP stromal cells in the live GFP nude mouse with and without doxorubicin.(a) Dual-color MMT cells were injected in the footpad of GFP transgenic nude mice, non-invasive image of untreated dual-color MMT cells in the footpad of a live GFP mouse.Spindle-shaped dual-color MMT cells are interdispersed among the GFP host cells.(b) Whole-body image of MMT dual-color cancer cells in a live GFP nude mouse 12 h after treatment with doxorubicin (10 mg/kg).The cancer cells lost their spindle shape, and the nuclei appear contracted [14].

Fig. 21 . 5 :
Fig. 21.5:(a) MPTflex with articulated flexible arm for non-invasive high-resolution imaging of living animals.(b) The tunable femtosecond laser (710-920 nm) is transmitted through an articulated mechano-optical arm with a compact scan head.The scan head contains galvoscanners and piezodriven optics for 3D imaging as wells as single-photon counting detectors for fluorescence and SHG imaging.Filters for fluorescence detection can be varied depending on fluorescent proteins (GFP or RFP).The magnetic interface between animal and scan head allows long-term imaging of live mice.(c) Intratissue hair follicle stem cells detected in live mice.Stem cells express nestin-GFP (green).Extra-cellular matrix (ECM) collagen was detected by SHG (false color coded in red).Typical stem cells in the hair follicles have an oval-shaped body with a typical size of about 7 µm and dendriticlike arms.Scale bar: 50 µm.(d) Long-term tracking of individual intratissue stem cells.Nestin-GFP stem cells moving into the field of view.(e) Nestin-GFP stem cells (bright green) in wounded skin.Autofluorescence of unlabeled cells from the skin tissue are also visible (images from[23,24]).

Fig. 21 . 6 :
Fig. 21.6:(a) In vivo multiphoton detection of genetically labelled Ds-Red-expressing cancer cells (red color) and autofluorescence of unlabeled stromal cells (green color) within the tumor tissue.In stromal-cells autofluorescence was mainly detected from organelles, e.g., mitochondria in the cytoplasm.Nuclei appear nonfluorescent.(b) Imaging of Ds-Red cancer cells (red), GFPexpressing capillaries (bright green), S. typhiurium A1-R-GFP (bright green), and autofluorescent stromal cells (yellow-green).Single bacteria invasion in the vessels of tumor tissue are seen (white arrows) minutes after bacteria injection in the tail vein of live tumor-bearing mice.(c) Bacteria killing of cancer cells monitored in the tumor of live mice.Cancer cells expressing Ds-Red were imaged by two-photon excitation and unlabeled extracellular protein collagen was imaged by SHG.Expanding and bursting cancer cells were detected after some hours of bacteria injection in the tail vein of live tumor-bearing mice (images from[38]).

Fig. 21 . 7 :
Fig. 21.7: High-resolution imaging of extracellular matrix collagen and elastin in tumor tissue of live mice.(a) Well-aligned, parallel, closely packed collagen fibers were detected in Lewis lung carcinoma by SHG (false color coded in red).Elastin fibers are seen as parallel structures to collagen structures which are detected by autofluorescence.(b) Collagen detected in Colo 26-GFP colon cancer-bearing mice.Cancer cells align parallel to collagen fibrils.Cancer cells express GFP and fluoresce green and collagen fibrils, visualized by SHG, are red (false color coded).The collagen fibrils are organized parallel structures with a wide interfiber spacing.SHG of collagen and GFP were excited simultaneously at an excitation wavelength of 790 nm.(c) Multiphoton autofluorescence and SHG images of unlabeled breast cancer tissue.Breast cancer cells were visualized by autofluorescence and collagen by SHG.Breast cancer cells appeared well-aligned parallel to collagen fibers (images from[41,42]).