2.1.

Cell Cultures

Human embryonic kidney 293T cells (Open Biosystems) and MDA-MB-231 human breast cancer cells (ATCC) were cultured in DMEM (Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (Hyclone Thermo Fisher Scientific, Waltham, MA), 1% glutamine, and 0.1% penicillin and streptomycin.

2.2.

Fluorescent Proteins and Plasmids

We used plasmids for the following fluorescent proteins: mTagBFP, EBFP, ECFP, cerulean, mTurquoise (gift of Joachim Goedhart), EGFP, AcGFP, YFP, citrine; mOrange2, TagRFP-T, tdTomato, mCherry, mPlum (gifts of Roger Tsien); mKate2, katushka, FP650, and mNeptune (gifts of Dmitriy Chudakov).^16–27 All plasmids used a CMV promoter to drive expression of fluorescent proteins. Spectral properties of fluorescent proteins used in this study are shown in Table 1.

Table 1

Spectral properties of fluorescent proteins used in this study.

2.3.

DNA Transfection and Lentiviral Transduction

We transiently transfected 293T cells with plasmids for various fluorescent proteins using calcium phosphate precipitation as described previously.²⁸ All transfections were performed with the same amount of plasmid DNA (0.5 µg) per well in 12 well plates. We prepared lentiviral vectors for mTagBFP, mTurquoise, citrine, mOrange2, and mCherry by transient transfection of 293T cells.²⁹ Lentiviruses were used to stably transduce different populations of MDA-MB-231 cells with these fluorescent proteins.

2.4.

Cell-Based Imaging

293T cells expressing fluorescent proteins were transferred to 35 mm dishes one day following transfection and used for imaging experiments the next day. As a control for cellular autofluorescence, we also used mock-transfected cells that did not express a fluorescent protein. For imaging studies, cells were switched to modified Earle’s balanced salt solution (MEBSS)³⁰ and placed on a warming plate (Tokai Hits, MATS-U5050S) set to 37°C. We used a FV1000 MPE Twin system (Olympus) for microscopy. The system has a Mai-Tai DeepSee laser (Spectra-Physics) with tuning range from 690 to 1040 nm. All imaging was performed with a $25\times$, 1.05 NA, water immersion objective (Olympus). To generate excitation spectral profiles for various fluorescent proteins, we increased the wavelength of excitation light by 10 nm increments between 690 and 1040 nm. Over this range, power at the laser source decreased from 3.2 to 1 W. The percent of laser power delivered to the cell sample was held constant over the full range of excitation wavelengths for a given fluorescent protein with values typically at 4% to 6%. Emission light was separated by dichroic mirrors into three detection channels: 1. 420 to 460 nm; 2. 495 to 540 nm; and 3. 575 to 630 nm. Percent of laser power delivered to samples and detector settings were adjusted minimally to avoid saturated pixels and reduce background noise for each experiment, and these settings remained constant for a given fluorescent protein. We quantified data for fluorescence intensity in each detection channel at individual excitation wavelengths by region of interest analysis (Fluoview software, Olympus), and we normalized fluorescence intensity measurements to account for differences in laser power at each wavelength. Background fluorescence intensity was 100 to 120 units for all experiments. We graphed mean values for normalized fluorescence intensity for each fluorescent protein ($n\ge 10$ cells per fluorescent protein).

2.5.

Intravital Microscopy

The University of Michigan Committee for Care and Use of Animals approved all animal studies. $1\times {10}^6$ MDA-MB-231 breast cancer cells were implanted orthotopically into the 4th inguinal mammary fat pads of NOD/SCID mice (Taconic).³¹ We performed intravital microscopy when tumors reached approximately 4 to 5 mm diameter, which occurred two to three weeks after implantation for all studies. We anesthetized mice with 1% to 2% isoflurane and maintained mice on 0.5% to 1% isoflurane throughout the procedure. We surgically exposed the mammary fat pad tumor with minor modifications of a previously described protocol.⁸ Briefly, we incised the abdominal skin without disturbing the underlying peritoneal membrane or internal abdominal organs. We rotated a skin flap containing the intact mammary fat pad tumor xenograft away from the abdomen to minimize transmitted respiratory motion and pinned the skin flap to a 3 to 4 mm thick piece of polydimethylsiloxane (PDMS). We glued a 2 to 3 mm thick ring of PDMS around the exposed tumor to contain sterile phosphate buffered saline as an aqueous interface for the microscope objective. We covered the exposed skin surface and peritoneal membrane with sterile 0.9% saline. We placed mice directly on a 37°C warming plate for imaging procedures. At the end of imaging, we closed the surgical incision with sterile wound clips.

We acquired images near the center of each tumor in the $X$-$Y$ plane at $Z$-axis depths listed in figure legends. We imaged each tumor with a range of excitation profiles as described for cell-based imaging studies and quantified fluorescence intensity from individual MDA-MB-231 cells by region of interest analysis as described above. For analyses of fluorescence from mTurquoise and mOrange2 proteins in tumor xenografts, we corrected fluorescence intensity for differences in laser output at 760, 770, and 780 nm and then normalized values for each cell to the value obtained at 760 nm.

3.1.

Live Cell Imaging

To optimize selection of fluorescent proteins for multi-color two photon microscopy in living cells, we transiently transfected 293T cells with 18 different fluorescent proteins with emissions ranging from blue to far red. We selected 293T cells for these experiments because these cells transfect readily, allowing us to efficiently test a large series of fluorescent proteins. For blue and cyan fluorescent proteins, we analyzed mTagBFP, EBFP, ECFP, cerulean, and mTurquoise, respectively. For 420 to 460 nm emitted light, mTagBFP was excited effectively with a broad range of light ranging from 700 to 890 nm, peaking at 760 to 810 nm [Fig. 1(a)]. EBFP and mTurquoise also emitted readily detectable fluorescence in the 420 to 460 nm detection channel. Both proteins showed peak fluorescence with 800 to 810 nm excitation, and fluorescence from mTurquoise was produced by excitation wavelengths up to 920 nm. All proteins produced fluorescence in the 495 to 540 nm detection channel with a rank order of fluorescence intensity of $\text{mTagBFP}>\text{mTurquoise}>\mathrm{EBFP}=\mathrm{ECFP}>\text{cerulean}$ [Fig. 1(b)]. Peak fluorescence emission for all proteins occurred with 800 to 810 nm excitation, and mTagBFP, mTurquoise, and ECFP were excited by wavelengths up to 900 nm. There was substantially reduced light in the 575 to 630 nm range for all five proteins, although both mTagBFP and mTurquoise produced detectable light above background signal in this detection channel [Fig. 1(c)].

Fig. 1

Two photon excitation wavelengths and fluorescence emission intensities of blue and cyan fluorescent proteins expressed in 293T cells. Fluorescence intensities were measured by region of interest analysis of light detected at (a) 420 to 460 nm, (b) 495 to 540 nm, and (c) 575 to 630 nm. Panel d shows autofluorescence intensities in the same three detector channels from mock-transfected 293T cells not expressing a fluorescent protein.

We also measured autofluorescence from mock-transfected cells that did not express a fluorescent protein. Cellular autofluorescence peaked at 750 nm excitation with comparable emissions in 420 to 460 and 495 to 540 nm channels and relatively less fluorescence detected at 570 to 630 nm. This autofluorescence profile previously has been attributed to emission from pyridine nucleotides NAD(P)H.³² Fluorescence emission from mTagBFP at 420 to 460 nm was substantially higher than autofluorescence at 750 nm excitation. For other blue and cyan fluorescent proteins, fluorescence emission in the 420 to 460 nm channel was minimally above background autofluorescence intensity, but these proteins were clearly detectable above background in the 495 to 540 channel except for cerulean.

Using selected variants of green and yellow fluorescent proteins (EGFP, AcGFP, citrine, and YFP), only EGFP with 700 to 800 nm two photon excitation produced light detectable in the 420 to 460 nm channel [Fig. 2(a)]. The 420 to 460 nm light from EGFP was maximal at 750 nm excitation. As expected, fluorescence from these proteins was detected best at 495 to 540 nm with relative maximum fluorescence intensities of $\mathrm{EGFP}>\mathrm{AcGFP}>\text{citrine}=\mathrm{YFP}$ [Fig 2(b)]. Two photon excitation from 750 to 920 nm produced relatively uniform, high level fluorescence from EGFP, while AcGFP was excited by a narrower range of laser light between 800 and 920 nm. Citrine and YFP also were excited by a wide range of two photon laser light from 750 to 950 nm, albeit producing lower intensities of fluorescence than EGFP or AcGFP. We also detected fluorescence emission from EGFP and AcGFP at 575 to 630 nm using excitation wavelengths comparable to those measured for emission between 495 to 540 nm [Fig. 2(c)].

Fig. 2

Fluorescence intensity measurements for green and yellow fluorescent proteins imaged in intact 293T cells in response to a range of two photon excitation wavelengths. Intensity measurements were recorded in detector channels for light emission at (a) 420 to 460 nm, (b) 495 to 540 nm, and (c) 575 to 630 nm.

Recent studies have developed several fluorescent proteins with red and far red emissions to capitalize on enhanced transmission of these wavelengths of light through tissues.^25,27,33 We analyzed several of these new fluorescent proteins (mOrange2, tdTomato, TagRFP-T, mCherry, katushka, mKate2, mPlum, mNeptune, eqFP650) in living 293T cells. Only mOrange2 produced minimal fluorescence emissions at 420 to 460 nm and 495 to 540 nm, respectively, with peak fluorescence intensity produced by 800 nm laser excitation [Fig. 3(a) and 3(b)]. tdTomato and mKate2 also produced modest fluorescence emission in the 495 to 540 nm channel with two photon excitation at 800 nm (tdTomato) and 880 nm (tdTomato and mKate2). Under our experimental conditions, all of the tested red and far red fluorescent proteins produced 575 to 630 nm light following two photon excitation with 760 to 780 nm laser light [Fig. 3(c)]. At these excitation wavelengths, mKate2 produced notably higher fluorescence intensity at 575 to 630 nm than other fluorescent proteins. These data reproduce observations made with purified red fluorescent proteins showing efficient two photon excitation at relatively shorter wavelengths due to higher energy transitions.³⁴ mKate2 also showed a second peak of higher fluorescence emission when excited from 870 to 960 nm, and this protein was detected readily above background through 1040 nm. Similar to results in the 495 to 540 nm detection channel, tdTomato and mOrange2 also produced a second smaller peak of 575 to 630 nm fluorescence emission at 880 nm excitation.

Fig. 3

Two photon excitation and fluorescence emission profiles for red fluorescent proteins imaged in 293T cells. Fluorescence intensities were quantified for light (a) 420 to 460 nm, (b) 495 to 540 nm, and (c) 575 to 630 nm.

3.2.

Intravital Microscopy

Detection of fluorescent proteins by intravital microscopy is complicated by factors including autofluorescence, light scattering, and preferential transmission of longer wavelengths of light through tissue due to absorption by molecules including hemoglobin and lipids.³⁵ To investigate effects of living tissue on two photon emission profiles of selected fluorescent proteins, we used an orthotopic tumor xenograft model of human breast cancer. For these experiments, we used MDA-MB-231 breast cancer cells, which have the advantage of efficiently forming orthotopic tumor xenografts in immunocompromised mice. We stably transduced MDA-MB-231 breast cancer cells with mTagBFP, mTurquoise, citrine, mOrange2, or mCherry, respectively. These proteins were selected as representative blue-cyan, green-yellow, and red fluorescent proteins that could enable multi-spectral imaging with minimal cross-talk between emission channels based on live cell imaging.

We imaged orthotopic tumor xenografts containing MDA-MB-231-mTagBFP, MDA-MB-231-citrine, or MDA-MB-231-mCherry transduced cells at depths of 100, 150, and 200 µm below the tumor surface and measured fluorescence emissions in the same three detector channels used for cell-based imaging following two photon excitation from 690 to 1040 nm. For tumors with MDA-MB-231-mTagBFP cells, we detected fluorescence in all three emission channels when imaging 100 or 150 µm below the tumor surface [Fig. 4(a) and 4(b)]. Fluorescence was highest in the 420 to 460 nm detection channel, although substantial amounts of light also were detected in the longer wavelength channels. Similar to live cell imaging, two photon excitation at 800 nm produced the highest fluorescence emission at 420 to 460 nm. When imaging tumors at 200 µm depth, detected fluorescence was comparable in all three output channels. Since near infrared light used for excitation has greater tissue penetration than blue light, it is likely that the inability to detect mTagBFP at 200 µm depth is due to attenuation and scattering of emitted fluorescence rather than excitation light not reaching this protein [Fig. 4(c)].

Fig. 4

Two photon intravital microscopy of orthotopic tumor xenografts of MDA-MB-231 breast cancer cells stably transduced with mTagBFP. Two photon excitation and fluorescence intensity measurements at (a) 420 to 460 nm, (b) 495 to 540 nm, and (c) 575 to 630 nm were measured at $\approx$ (a) 100, (b) 150, and (c) 200 µm depths within a tumor.

At 100 µm, tumors with MDA-MB-231-citrine cells predominantly produced fluorescence in the expected detection channel of 495 to 540 nm light [Fig. 5(a)]. Peak fluorescence emission occurred with 810 nm two photon excitation. With greater imaging depths, there was no change in wavelength of two photon laser light that produced peak fluorescence emission from MDA-MB-231-citrine cells [Fig. 5(b) and 5(c)]. However, relative differences between fluorescence emitted in the 495 to 540 nm versus 575 to 630 nm channels decreased on images obtained at 150 and 200 µm.

Fig. 5

Two photon excitation and three detector channel fluorescence intensity measurements of citrine stably expressed in MDA-MB-231 tumor xenografts. Measurements were obtained $\approx$ (a) 100, (b) 150, and (c) 200 µm below the tumor surface.

Tumors with MDA-MB-231-mCherry cells showed a similar pattern to MDA-MB-231-citrine tumors on progressively deeper images. We detected peak fluorescence from mCherry in the 575 to 630 nm detection channel using 760 nm two photon laser excitation on images obtained 100, 150, and 200 µm below the tumor surface [Fig. 6(a) to 6(c)]. Fluorescence emission at 575 to 630 nm was the predominant signal detected from MDA-MB-231-mCherry cells on images at 100 and 150 µm. At 200 µm, mCherry still produced highest fluorescence in the 575 to 630 nm detection channel, although signal intensity was only modestly higher than that detected in the other two detection channels.

Fig. 6

Fluorescence intensity measurements of mCherry in MDA-MB-231 tumor xenografts in response to a range of two photon excitation wavelengths. Fluorescence measurements at (a) 420 to 460 nm, (b) 495 to 540 nm, and (c) 575 to 630 nm were determined $\approx$ (a) 100, (b) 150, and (c) 200 µm below the tumor surface.

To illustrate dual color imaging and the trade-offs in measured fluorescence intensity produced by shifting excitation wavelengths, we implanted MDA-MB-231 cells expressing either mTurquoise or mOrange2 into the same tumor xenograft. Based on data from cell culture experiments, we focused on excitation wavelengths of 760 to 780 nm. At an imaging depth of 50 μm, increasing excitation wavelength from 760 to 780 nm significantly increased fluorescence intensity from mTurquoise in the 495 to 540 detection channel and decreased measured fluorescence output at 575 to 630 nm from cells expressing mOrange2, respectively ($p<0.05$ and $p<0.01$ for 770 and 780 nm, respectively) [Fig. 7(a) and 7(b)]. Relative to fluorescence intensity measured at 760 nm excitation, fluorescence from mTurquoise increased by 55% and mOrange2 decreased by 20%, respectively. These results show that small increments in excitation wavelength produce notable changes in fluorescence emission from two different proteins used for intravital microscopy.

Fig. 7

Intravital microscopy of tumors comprised of MDA-MB-231 cells stably expressing mTurquoise or mOrange2. (a) Representative fluorescence images at 50 μm depth within a tumor showing fluorescence from mTurquoise (495 to 540 nm detection channel), mOrange2 (575 to 630 nm detection channel), and merged images at 760, 770, and 780 nm excitation. (b) Graph depicts $\text{mean values}+\mathrm{SEM}$ for relative fluorescence intensity from each protein normalized to laser power output and fluorescence intensity at 760 nm excitation.…*, $p<0.05$; **, $p<0.01$.