Simultaneous imaging of GFP, CFP and collagen in tumors in vivousing multiphoton microscopy
- Erik Sahai†1, 2,
- Jeffrey Wyckoff†2, 4Email author,
- Ulrike Philippar3,
- Jeffrey E Segall2,
- Frank Gertler3 and
- John Condeelis2, 4
© Sahai et al; licensee BioMed Central Ltd. 2005
Received: 18 February 2005
Accepted: 23 May 2005
Published: 23 May 2005
The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.
Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo.
Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.
The ability to image cell movements and dynamic changes in sub-cellular structures in living mammals will significantly enhance our understanding of biology. Many applications have been developed to investigate how tumor cells act in vivo [1–3]. In recent years multi-photon laser scanning microscopy has demonstrated that it has both the resolution and tissue penetration to significantly improve the analysis of tumor cell behavior in vivo [4–6]. A key requirement for multiphoton microscopy is the need to fluorescently label cells, sub-cellular compartments or proteins of interest. Green Fluorescent Protein (GFP) has been widely used to label cells; however the use of a single fluorophore, though successful, has been limiting. Tumor development and cell migration from intravasation to metastatic growth has been studied in vivo in orthotopic models and transgenic mice  using GFP, but limited by the fact that only one cell type can be examined without introducing a dye from an external source. GFP and RFP have been used to study separate cell populations in tumors using conventional imaging methods [8–10] and multiple flours have been used in cells in vitro . However, this combination of fluorophores are not compatible with multiphoton intravital imaging, since the high intensity pulsed infrared lasers commonly used for multiphoton microscopy produce light in the 720–980 nm range and are unable to excite RFP efficiently in deep tissue. While we and other groups have been able to use multiphoton laser scanning microscopy to image two or more chromophores in vivo, generally non genetically coded fluors such as Texas Red-labeled dextran or Hoechst are used [6, 12–15] in conjunction with GFP or other fluorescent proteins which prevents the application of multiphoton microscopy from being applied to the study of multiple cell populations in vivo. Therefore, here we describe methods to image the genetically encoded fluors GFP and Cyan Fluorescent Protein (CFP) simultaneously in a living tissue. Previous work by our group has correlated patterns in gene expression in cells with differing metastatic potential with differences in cell motility and polarization in vivo [6, 16]. Here we describe a method to compare the behavior of cancer cells in which expression of genes identified in these studies has been altered with the behavior of control cells in the same tumor micro-environment. We also describe a method for imaging two genetically encoded fluorophores in the same cell, thereby allowing imaging of sub-cellular compartments or proteins and the whole cell simultaneously.
Results and Discussion
To study two cell types in the same organ in vivo with differing fluorescent proteins, fluorescent pairs need to be chosen that can be excited equally at a common wavelength. We chose to simultaneously image GFP and CFP because their expression is well tolerated by most cell types and they are easily excited by standard Ti-sapphire lasers. Initial attempts to image RFP were not successful; both tetrameric and dimeric variants of dsRed formed aggregates which had deleterious effects on cell viability. The use of monomeric mRFP overcame these problems, however the power output of most Ti-Sapphire lasers drops off significantly at the wavelengths of light required to excite mRFP resulting in ineffective excitation of mRFP. While YFP has been used as part of a FRET pair in vivo , GFP was chosen over YFP because YFP is not effectively excited by wavelengths below 900 nm , YFP and GFP excitation closely mirror each other and are difficult to separate, and a large number of already available cell lines and transgenic animals use GFP.
To test if this method can be used to monitor protein localization, we transiently transfected cells expressing CFP with a GFP-ZYXIN expression construct. Figure 2 (right hand panels) shows one such transfected cell amongst other non-transfected cells, subtraction of the 450/80 signal from the 515/30 signal clearly reveals the focal adhesion localization of GFP-ZYXIN in the transfected cell while the untransfected cells are not seen. These results demonstrate that GFP and CFP fluorescence can be successfully separated even when simultaneously excited using an 880 nm laser beam. Furthermore, specific sub-cellular compartments or proteins can be visualized in cells simultaneously expressing GFP and CFP.
Additional File 1: Movie for figure 4A (avi. Format): Time-lapsed movie of high metastatic CFP labeled cells (white) and low metastatic GFP labeled cells (green) crawling on collagen fibers (purple) in vivo. Objective used 20×. (AVI 4 MB)
Our multi-fluor technique can also be used to explore metastatic ability. While fluorescently labeled cell have been used to explore metastatic and extravasive ability of differing cell types [9, 23, 24], our technique allows for multiphoton imaging at single cell resolution within the primary tumor of two cell populations and determination of differences in metastatic potential within the same animal. By examining of the lungs of animals with tumors formed by the injection of a mixture of CFP and GFP cells, extravasated cells can be counted to determine differences in numbers of cells metastasizing and compare this score to cell behavior differences in the primary tumor. Figure 4B shows how both cell types can be visualized in the lungs.
To visualize dynamic structures involved in cell motility we used a 60× LUMPlan/IR 0.9NA dipping objective. The morphology of single cells labeled with either CFP or GFP was easily visible on a background of the opposing color. Cell structures that were not well defined in prior in vivo experiments using conventional microscopy  can now be easily discerned. Cells are seen with filopodia (Figure 4C, left) and lamellipodia (Figure 4C, right) which when visualized as time-lapse movies (Additional file 2) can be seen protruding and retracting. Morphological changes of moving cells can also be resolved. Analysis of these time-lapse movies revealed that motile cells moved with amoeboid characteristics with the edge moving at up to 8 μm/min and making rapid changes in direction (Figure 3E and Additional file 3). The speed of the highly metastatic cells were twice what was previously reported for tumor cells with average metastatic potential in vivo . Furthermore, motile cells often followed the same path; such behavior has been inferred from the presence of chains of invading cells in fixed breast cancer samples  but this is the first direct observation of such behavior. These cells may follow the same path because it is physically favorable (i.e. lack of ECM blocking cell movement) or because of a chemotactic relay between the cells.
Recent advances in multiphoton imaging technologies have provided researchers with the tools to study cell-cell interactions and the microenvironment of living tissues [4, 5, 26, 27]. Our multi-fluor method described here will enable the direct comparison of the behavior of control and experimental manipulated cells in the same biological environment using multiphoton microscopy. This will be invaluable for determining the effects of genetic manipulations on cell behavior in vivo. In addition, the simultaneous imaging of GFP and CFP within the same cell will greatly aid the study of sub-cellular compartments and protein localization in living tissues.
Plasmids and Cell lines
ECFP-N1 (Clontech), GFP-CAAX (AP4-CAAX described ), GFP-zyxin . Standard retroviral and transfection methods were used to generate the following derivatives of the MTLn3 rat mammary carcinoma cell line: low metastatic MTLn3-pLXSN GFP, high metastatic MTLn3-ECFP N1,0 MTLn3-GFPCAAX + ECFP N1, MTLn3-GFPZYXIN + ECFP N1. Cells were maintained in αMEM + 5% Fetal Calf Serum. Pools of GFP and CFP expressing cells were isolated by FACS methods. For imaging, cells were grown in MatTek dishes and fixed with 4% formaldehyde in PBS.
To determine metastatic differences between the MTLn3 clones, a total of 106 cells were injected into the mammary fat-pad of groups of eight 5–6 week old female Balb/C SCID mice. After 4 1/2 weeks animals were euthanized and lung mets counted in H and E stained sections. To determine motility differences, cells were compared by using a Boyden Chamber chemotaxis assay. Both these experiments were performed as reported previously [6, 22].
106 MTLn3 cells were injected into the mammary fat-pad of either female 5–6 week old female Balb/C SCID mice for the comparison of cell motility and metastatic potential or 5–6 week old Fischer 344 rats to image the cell membranes. After 3–4 1/2 weeks animals were anaesthetized with isoflurane and small incision was made in the skin to expose the tumor [1, 6, 26]. The anaesthetic was maintained while the tumor was imaged using a Biorad Radiance 2000 multiphoton microscope with an inverted Olympus IX70, within a heated chamber maintained at 30°c, connected to a Spectra Physics Tsunami Ti-Sapphire laser. All images were collected using non de-scanned detectors. The objectives used were as follows: 20× Plan Apo 0.7NA (air), 40× LUMPlan/IR 0.8NA (water) and 60× LUMPlan/IR 0.9NA (water). The filters used were as follows: 450/40, 450/80, 480/30, 515/30 (all from Chroma). Time lapsed images were taken over the course of 30 mins and analyzed using ImageJ. Frequency and speed of motility were calculated as described previously .
We are grateful the members of the Analytical Imaging Facility for support and advice. This work was supported by NIH grant CA100324 (J.S. Condeelis) and NIH grant GM58801 (F. Gertler). E. Sahai was funded by a Unio Internationale Contra Cancrum Aventis Translational Cancer Research Fellowship. U. Philippar received a Ph.D. scholarship by the Boehringer Ingelheim Funds.
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