Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

2-photon excitation and emission properties of GFP and CFP expressed in cells. A) Stable cell lines expressing either CFP or GFP were imaged with the indicated excitation wavelength (in all cases the laser power was 0.95–1.0W). CFP fluorescence was captured using a 480/30 filter and non-descanned detection, GFP fluorescence was captured using a 515/30 filter and non-descanned detection. The amount of fluorescence captured using different excitation wavelengths is shown (relative to the maximum fluorescence). B) Stable cell lines expressing either CFP or GFP were imaged using an 880 nm excitation beam (in all cases the laser power was 0.95–1.0W). Fluorescence was captured using the indicated filters and non-descanned detection. The amount of GFP or CFP fluorescence captured using different filters is shown (relative to the maximum fluorescence).

Figure 2

Simultaneous capture of GFP and CFP. Stable cell lines expressing either GFP or CFP were co-cultured (left panels) or a stable cell line expressing CFP and GFP-CAAX (membrane targeted – middle panels) or a cell line expressing CFP transiently transfected with GFP-ZYXIN (right panels) were analyzed. Top panels show the fluorescence captured using a 450/80 filter and non-descanned detection. Upper-mid panels show the fluorescence captured using a 515/30 filter and non-descanned detection. Lower-mid panels show the result of subtracting the 450/80 signal from the 515/30 signal. Bottom panels show a merge of the top panels (CFP signal in blue) and the lower-mid panels (GFP signal in green). GFP-CAAX, which generally targets to the membrane can be seen labeling of intracellular structures in the merge and GFP images (middle panel; arrow). All images were taken with a 40× objective and a final magnification of 500× for the right 2 columns and 1000× for the left. Scale bar = 25 μm for the right 2 columns and 10 μm for the left.

Figure 3

Simultaneous capture of GFP, CFP and collagen second harmonic fluorescence in a living tissue. The panels on the left-hand side show images of an experimentally generated mammary tumour creating by injecting a mixture of cells either expressing GFP or CFP. The panels on the right-hand side show images of an experimentally generated mammary tumour with cells expressing CFP to mark the entire cytoplasmic volume and GFP-CAAX (membrane targeted). 880 nm laser light was used to excite the all the samples and the fluorescence was captured with indicated filters using non-descanned detection. All images were taken with a 40× objective and a final magnification of 500×. Scale bar = 25 μm.

Figure 4

Differences in motility and invasion can be determined by imaging cells with different metastatic potential in the same tumor. A) The panels show a time series of images of an experimentally generated mammary tumour with cells either expressing GFP (low metastatic in green) or CFP (high metastatic in white) with collagen fibres in purple. Arrows point to moving CFP cells and arrowheads to moving GFP cells. Supplementary movie shows this image sequence including intermediate frames (Additional file 1). Images were taken with a 20× objective and a final magnification of 250×. B) CFP-labeled cells (white) and control GFP labelled cells (green) are seen in the lung after extravasation and metastatic growth. Images were taken with a 20× objective and a final magnification of 250×. C) Left hand panel CFP cell (white) has a filopod (arrow) protruding into a field of GFP control cells (green). Right hand panel CFP cell (white) has a lamellapod (arrow) seen in the middle of a field of GFP control cells (green). Supplementary movie shows a lamellapod retracting over time (Additional file 2). Images were taken with a 60× objective and a final magnification of 1000×. D) The panels show a time series of high magnification images of an experimentally generated mammary tumor with cells either expressing GFP (in green) or CFP (in white) with collagen fibers in purple. Moving high metastatic cells are outlined in white and an orange arrow shows the path taken by both cells. Supplementary movie shows this image sequence including intermediate frames (Additional file 3). Images were taken with a 40× objective and a final magnification of 500×. Scale bar for A-E = 25 um.