Two-photon flow cytometer
The two-photon flow cytometry apparatus () used a Ti:Sapphire laser (Coherent Mira), which generates 50-fs pulses at 800 nm with a repetition rate of 76-MHz. The femtosecond NIR laser beam was focused with a long-working distance objective (Olympus 40x) into a blood vessel of a mouse ear. A dichroic mirror directed the laser into the microscope objective while transmitting the fluorescence collected through the same objective. A secondary dichroic mirror and bandpass filters were used to separate the fluorescence from different labeling dyes into two channels, which were detected with two distinct photomultiplier tubes (PMT Hamamatsu HC7421-40) and recorded with two multichannel photon counting scalers (MCS Stanford Research SR430) which are capable of time resolution as low as 41 microseconds. The signals (number of photons counted per each 1.3 ms bin of sampling interval) were sent to a computer for data analysis.
Two-channel, two-photon flow cytometer for in vivo measurements.
For in vivo measurements, a custom-made heated stage was used to restrain anesthetized mice. An 8-mm diameter hole was drilled at the center of the stage, on top of which a glass slide was placed. The vasculature and blood flow of the mouse ear were visualized on a CCD camera via transmitted light from a fiber optic illuminator (EW-41500-50, Cole-Parmer).
A Matlab program was used to extract fluorescent peaks above the background noise level from the multi-channel photon counting scaler (MCS) trace signals. The particle detection threshold in a channel was set above the maximum signal level from control traces. The program scanned the trace signals for fluorescent peaks above the threshold. Once a peak was located, the peak characteristics, such as height (maximum fluorescent signal within the peak), width (number of consecutive bins above the background threshold) and location (the index of the maximum bin) were stored. For two-channel measurements, the data from each channel were analyzed separately. A double-peak event was treated as a single event if the fluorescent signal between the two peaks did not fall below the background threshold value.
The in vivo flow cytometry procedure
Five to six weeks old, specific-pathogen-free female immunocompromised nude (NU/NUCD-1 or NU/J Foxn1nu) or immunocompetent (CD-1) mice were purchased from Charles River Laboratories (Portage, Michigan) and housed in a specific pathogen-free animal facility at the University of Michigan Medical Center in accordance with the regulations of the University’s Committee on the Use and Care of Animals as well as with federal guidelines, including the principles of Laboratory Animal Care.
Prior to in vivo flow cytometry experiments, mice were injected with 0.5 ml of sterile saline heated to 37°C to maintain hydration. Mice were anesthetized with inhalation of isoflurane (4%) and then maintained on 1–2% isoflurane throughout experiments. For optical detection, mice were placed on a heated stage with the left ear on the glass slide window. Drops of glycerol were applied to the mice eyes to prevent drying. The femtosecond NIR laser beam was focused into the mouse ear with the objective from below. Microscope immersion oil was placed between the mouse ear and the glass slide to maintain the position of the ear during the cytometry measurement. When the short pass filter in the fluorescence collection optical path was switched off, the back-scattered light from the femtosecond NIR laser beam could be visualized on the CCD camera to align the excitation beam with the blood vessel. An ≈ 50 µm diameter arteriole flow was selected for cytometry measurements. The location of this arteriole was marked to allow subsequent measurements to be obtained from the same blood vessel.
Before injection of the solution of fluorescent-dye-labeled particles or cells, the background signals in both short wavelength channel (S-channel) and long wavelength channels (L-channel) were recorded as a control. Solutions containing the fluorescence-labeled particles were injected through the tail vein. In vivo flow cytometry typically began within 5 minutes of injection. The laser power used was below 20 mW at the focus, and no photodamage to the ear of the mice was observed after the experiments. During analysis, the thresholds were set such that no peaks existed in the control traces.
In vivo flow cytometry of fluorescent microspheres and DeepRed-labeled splenocytes
Two micron, yellow-green fluorescent (Ex505/Em515) microspheres (Molecular Probes, Inc. Eugene, OR) were washed, resuspended in 200 µl PBS (total number of beads 2.3 × 109), and injected via tail vein to CD-1 mice. Spleens were collected from euthanized animals and were disrupted in PBS to obtain a single cell suspension, after which the cells were washed in PBS and the red blood cells were lysed using ammonium chloride. To stain the splenocytes, the cells were treated with Mitotracker™ DeepRed 633 (Molecular Probes, Inc. Eugene, OR), incubated for 1 hour at 37°C, trypsinized, rinsed and resuspended in PBS, pH 8. A total of 3.6 × 106 DeepRed-labeled splenocytes were injected into a female NU/NU CD-1 mouse (chosen to prevent immune system recognition of the injected splenocytes). The two-channel fluorescence signals were recorded immediately after each injection for ten or twenty consecutive minutes and repeated at the same vessel location roughly 2 hours and also 1 day after the initial injection.
In vivo flow cytometry of DiD labeled red blood cells
Blood (25–50 µL) was collected from mice by retro-orbital puncture, and 12 µL were centrifuged to remove serum and the buffy coat as described above. Cells were washed once with PBS and then stained with 10 µL 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyllindodicarbocyanine perchlorate (DiD, a lipophilic dye that labels membrane bilayers) (Vybrant DiD, Labeling Solution, Invitrogen) in 100 µL PBS for 30 minutes at 37°C. Cells were washed once in PBS to remove unincorporated DiD and then resuspended in 100 µL sterile 0.9% NaCl. Samples (containing approximately 1 × 107 stained RBCs) were injected intravenously into a female NU/J Foxnlnu mouse via a tail vein for in vivo flow cytometry experiments. The long channel fluorescence signal was recorded at roughly the same vessel location several times over the next several weeks (0 minutes, 20 minutes, 5 hours, 24 hours, 48 hours, 4 days, and 17 days after injection). The frequency was calculated as the number of peaks in the L-channel within 214 seconds.
For measurements beginning on day 4, the location in the vessel was optimized manually in real time to maximize the amplitude and frequency of the detected events. This was possible since the large number of events enabled a frequency count every few seconds between manual adjustments of the objective. It is noteworthy that this improvement is only possible when the number of detected events is at least several per second, and thus cannot be used in one-color analysis of metastatic cancer cells or other rare populations of cells in the circulation.
Conventional flow cytometry of red blood cells
Blood samples (50–100 µL) were collected from mice via retro-orbital puncture using heparinized capillary tubes. Blood was transferred to heparinized microfuge tubes and centrifuged for 1 min at 10,000 RPM. Serum and the buffy coat were removed, and the cell pellet was resuspended in 750 µL FACS buffer (PBS with 0.1% sodium azide and 1% heat-inactivated fetal calf serum). Flow cytometry was performed on a FACS Calibur System (BD Biosciences), and data were analyzed with CellQuest software.
In vivo flow cytometry of two cell lines labeled with two color quantum dots
To simultaneously monitor circulating MCF-7 and MDA-MB-435 in the same mouse in vivo, cells were labeled with 15 nM or 7.5 nM of Qtracker quantum dots (Invitrogen) that emit at 585 nm (Qdot585) or 655 nm (Qdot655), respectively. Quantum dots were mixed with an equal volume of transfection reagent provided with the Qtracker kit according to the manufacturer’s protocol. Breast cancer cells were seeded at 4 × 106 cells/60 mm dish (MDA-MB-435) or 3 × 106 cells per dish (MCF-7) 6 hours prior to labeling with quantum dots mixed with 200 µL of cell culture medium. Cells were rocked every 15 minutes for 1 hour to distribute the quantum dots, and cell culture medium then was added to a final volume of 4 ml to continue labeling overnight. Cells were treated with 2 mM EDTA to release them from the culture dish, washed with PBS, and resuspended in sterile 0.9% saline for injection. Because initial studies with these cells suggested that fewer MDA-MB-435 cells could be detected immediately after injection, we injected twice as many of these cells: 1 × 106 435 cells were coinjected with 0.5 × 106 MCF-7 cells in 125 µL PBS. The two-channel fluorescence signals were recorded immediately after each injection and repeated at the same vessel location roughly 2 hours and 1 day after the initial injection. Another set of mice were injected with tumor cells labeled with the reverse pattern of quantum dots (MCF-7 labeled with Qdot585 and MDA-MB-435 labeled with Qdot655) to confirm that the particular fluorophore assignment does not influence the detectability of these cell lines in vivo.