Chemicals, unless otherwise noted, were purchased from Sigma Aldrich (St. Louis, MO) at reagent grade or higher. Citric acid sheath buffer (25 mM, pH 2.25) was made by dissolving 5.25 g of C6H8O7•H2O in 1 L of ultrapure deionized (DI) water (Elga Purelab Ultra, Siemens Water Technologies, Warrendale, PA). Electrophoresis buffers were made by diluting a stock solution of 50 mM borate buffer, pH 8.8, prepared by dissolving 9.2 g of Na2B4O7•10 H2O and 3.0 g of B(OH)3 in 1 L of ultrapure DI water. For surfactant-containing electrophoresis buffers, 0.54 g of sodium dodecyl sulfate (SDS) was added to 50 mL of diluted borate buffer, pH 8.8, sonicated for 2 min to dissolve, and filtered with a 0.22 μm syringe filter (Nalgene, Rochester, NY). Serotonin (5-HT) (Alfa Aesar, Ward Hill, MA) and tyrosine (Tyr) were dissolved in 2.5 mM citric acid, pH 2.5, and sonicated on ice for 30 min. Tryptophan (Trp), NAS, 5-hydroxyindole acetic acid (HIAA), melatonin (MT), 5-hydroxytryptophan (HTP), 5-methoxytrytophol (MTOL), 5-methoxytryptamine (MOT) (TCI America, Portland, OR), tryptophol (TOL) (Research Organics, Inc., Cleveland, OH), 5-methoxyindole acetic acid (MIAA) (Gold Biotechnology, St. Louis, MO), and 5-hydroxytryptophol (HTOL) (Gold Biotechnology) were dissolved in 2.5 mM citric acid, pH 2.5, + 10% v/v acetone and sonicated on ice for 30–60 min. Standard buffers were prepared by diluting the sheath buffer 1:10 with ultrapure DI water. Fluorescein was prepared in ultrapure DI water. Standard stock solutions were diluted in either 1 mM borate buffer, pH 8.8 (1:50 dilution of stock borate electrophoresis buffer), or in high Ca+2/high Mg+2 modified Gey’s balanced salt solution (high salt mGBSS), pH 7.2, consisting of 3.0 mM CaCl2 (0.44 g), 4.9 mM KCl (0.37 g), 0.2 mM KH2PO4 (0.03 g), 22 mM MgCl2 (4.47 g), 0.6 mM MgSO4 (0.07 g), 138 mM NaCl (8.06 g), 27.7 mM NaHCO3 (2.33 g), 0.8 mM Na2HPO4 (0.11 g), 25 mM HEPES (5.95 g), and 10 mM glucose (1.80 g) dissolved in 1 L of ultrapure DI water. Biological samples were stored in either high salt mGBSS, as described above, or mGBSS, pH 7.2, consisting of 1.5 mM CaCl2 (0.22 g), 4.9 mM KCl (0.37 g), 0.2 mM KH2PO4 (0.03 g), 11 mM MgCl2 (2.24 g), 0.3 mM MgSO4 (0.04 g), 138 mM NaCl (8.06 g), 27.7 mM NaHCO3 (2.33 g), 0.8 mM Na2HPO4 (0.11 g), 25 mM HEPES (5.95 g), and 10 mM glucose (1.80 g) dissolved in 1 L of ultrapure DI water. Buffers were filtered by a 0.45 μm bottle-top filter system (Nalgene, Rochester, NY) and degassed under vacuum with stirring for 30–60 min. NaOH was prepared by dissolving one pellet (~0.002 g) in 0.025 L of ultrapure DI water.
All experimental procedures were conducted according to protocols approved by the Institutional Animal Care and Use Committee, University of Illinois at Urbana-Champaign. Animal care and experiments were performed in full compliance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Long-Evans/BluGill rats (University of Illinois at Urbana-Champaign), which have been demonstrated to be genetically homogeneous by high density genome scan, were sacrificed by rapid decapitation and the pineal glands isolated from the central nervous system. Males, 6–8 weeks, were sacrificed during the day, between circadian time 3:00 – 5:00, and pineal dissection and preparation was completed within 30 min. Glands were manually triturated for pinealocyte isolation and stored in high salt mGBSS on ice until analysis, typically between 30–60 min.
Rat pinealocyte suspensions were split into two groups. The first was incubated at room temperature for 60 min in 200 μM HTP, dissolved in high salt mGBSS. The second control group had an equal volume of high salt mGBSS and was incubated at room temperature for 100 min before analysis.
Preparation of the CE column inlet
The CE columns, 85–120 cm long, were made from 50 μm inner diameter, 360 μm outer diameter fused silica capillaries (Polymicro Technologies, Phoenix, AZ). The capillary inlets and outlets were etched with hydrofluoric acid (HF) in order to reduce the outer diameters and create sharply tapered tips with a 40° angle (see43
and Supplementary Information
OT design and construction
Unless otherwise noted, all custom-built components were designed and fabricated in-house. The optical axis is parallel to the optical table at a height of 20 cm until the beam is directed 90° vertically by a dichroic mirror. All of the optics are infrared (IR)-coated unless otherwise specified. The near infrared (NIR) beam is viewed with IR viewing cards (VC-1550 and F-IRC-HP, Thorlabs, Newton, New Jersey) and an IR viewer (IRV1-1700, Newport Corp., Irving, CA).
The OT design () uses a 1064 nm diode-pumped solid state Nd:YAG laser (Compass 1064-2500MN, Coherent Inc., Santa Clara, CA) with a maximum output of 2.5 W. The laser operates in TEM00
mode and has a wavelength stability of < 1 cm−1
. The beam has a nominal diameter of 0.4 mm, a divergence of < 3.5 mrad, a pointing stability of < ± 5%, and an ellipticity of < 1.1. It is air-cooled and turn-key operated. The beam is expanded by a 20× high energy beam expander (HB-20X, Newport Corp.) and directed by a pair of gold-coated mirrors (PF10-03-M01, Thorlabs) into a set of two plano-convex lenses (SPX029, Newport Corp.) in a 1:1 telescope configuration, used to steer and parfocalize the beam. The beam expander is housed in a precision gimbal optic mount (605-4, Newport Corp.) and translated in the x-, y-, and z-directions by a translation stage (UMR12.40, Newport Corp.) and heavy duty optical lab jack (L490, Thorlabs). Plano-convex lens 1 is mounted in a 3-axis optical mount (LP-1A-XYZ, Newport Corp.) located 1000 mm from the back aperture of the objective and plano-convex lens 2 is mounted in a 2-axis mount (LP-1A-XY, Newport Corp.) located 500 mm from the back aperture of the objective. This setup can be generalized as the distance between the back aperture of the objective and the steering optic is equal to 4f
, where f
is the focal length of the lens.44,45
In this case, the focal lengths of both of the plano-convex lenses are 250 mm. The beam is then directed into the epi-fluorescence port of the microscope (AxioObserver A1, Carl Zeiss, Jena, Germany) and directed into the back aperture of the objective (Objective C-Apochromat 63x/1.2 W Corr, 441777-9970-000, Carl Zeiss, Jena, Germany) by a dichroic mirror centered at 1064 nm (950dcsp-laser, Chroma Technology, Rockingham, VT).
Figure 1 The OT and MC-CE-LINF instrument design (both being interfaced to the same sample stage on the microscope). (A) Schematic of the optical trap. Emission from the Nd:YAG laser is expanded by a 20× beam expander to fill the back aperture of the objective. (more ...)
Polystyrene bead stock solutions (1 μm (PS04N/5749) and 10 μm (PS06N/6955) in diameter, Bangs Laboratories, Fishers, IN), were diluted 10-fold to 100-fold in ultrapure DI water and then used to optimize the optical trap. The beads were contained on a coverslip (2735-246, Corning Inc.) by a vacuum grease ring (Silicon High Vacuum Grease, Dow Corning, Midland, MI). Laser power measurements were taken with a PM10 sensor and a LabMax-TOP meter (Coherent Inc.).
MC-CE-LINF instrument design and construction
The injection port for the instrument is housed on a non-conductive breadboard platform on a microscope that is contained in a clear Plexiglas box. The capillary inlet has PEEK fittings (Upchurch Scientific, Oak Harbor, WA) and a FEP sleeve (Upchurch Scientific), attached to allow for fast and easy switching between the syringes and capillary holder.
The capillary is held in place in the instrument by a custom-built acetal resin (Delrin, E. I. duPont de Nemours & Co., Wilmington, DE) sheath flow cell. It enters at the top of the cell and is held in place by liquid-tight fittings (Upchurch Scientific). The sheath buffer enters the cuvette from the right side with respect to the optical table and exits from the bottom of the sheath flow cell. The quartz cuvette (Starna Cells, Atascadero, CA) used for excitation and detection of eluents is open on both ends and is attached to the top and bottom pieces of the sheath flow cell with Tra-Cast 3103 epoxy (Henkel Corp., Billerica, MA).
The instrument optics () were based on our prior instrument design.46
Deep UV radiation (224.6 nm) from an HeAg hollow cathode ion laser (HeAg70, Photon Systems Inc., Covina, CA) is spectrally filtered using a four-bounce mirror configuration, attached to the front of the laser head. The beam is directed via two UV-coated mirrors (Thorlabs) into a laboratory-built lightproof, non-conductive box and breadboard, which houses the detection optics and protects against spurious arcing. The collimated beam is nominally focused using a plano-convex lens (OptoSigma, Santa Ana, CA) to a 50 μm spot directly below the outlet of the capillary, which has been HF-etched to a cone-shaped tip and is housed in a custom-built sheath flow cell, as described above. As analytes elute from the capillary they are excited by the focused beam and emit fluorescence, which is collected and collimated by a 15× all-reflective objective (13596, Newport Corp.). The fluorescence is directed toward three photomultiplier tube (PMT) detectors (H6780-06, Hamamatsu, Middlesex, NJ) by two dichroic mirrors (310dcxxr-haf #110258 and 400dcxru #111563, Chroma Technology), with transition points at 310 nm and 400 nm, respectively. The first detector (PMT “blue”) measures emission from 250–310 nm, the second detector (PMT “green”) measures emission from 310–400 nm, and the third detector (PMT “red”) measures emission from 400 nm and above. The laser and PMTs are synchronized and controlled by software written in LABView and provided by Photon Systems Inc. Posts, post holders, and other optical mounts were purchased from Newport Corp., Melles Griot (Albuquerque, NM), or custom-built. Optical mounts for the focusing and collection optics are coated in Vinyl Liquid Electric Tape (Star Brite, Ft. Lauderdale, FL) and Scotch Super 88 electrical tape (3M, St. Paul, MN) to reduce arcing from the capillary outlet and tubing to the mounts.
Negative voltage for electrophoresis is applied to the sheath flow waste by a stainless steel cylinder connected to a power supply (PS/MJ30N0400-11, Glassman High Voltage, High Bridge, NJ) and laboratory-built control box. A 10 kΩ resistor and a digital multimeter (Fluke 76, Fluke Corp., Everett, WA) are part of the circuit and are used to measure the current across the capillary.
Sheath buffer is gravity-driven and flow can be adjusted by a right angle switching valve (Upchurch Scientific). High purity Teflon PFA Plus tubing and appropriate fittings were purchased from Upchurch Scientific. All tubing is further encased within FEP-lined polyethylene tubing (McMaster-Carr, Elmhurst, IL) to reduce static attraction and arcing during electrophoresis. Tubing between the optics box and the sheath box is also surrounded by four 16 oz. polyethylene containers and Scotch Super 88 electrical tape.
Interfacing the OT with the MC-CE-LINF instrument
The optical trap and the MC-CE-LINF system are interfaced at the microscope stage (Figure 1S, Supplemental Information
). The trap is located at the focal point of the objective, approximately 0.28 mm from the objective surface, including the coverslip thickness (0.13–0.16 mm). The capillary inlet is controlled by a computer-controlled motorized micromanipulator (MP-285, Sutter Instrument Co., Novato, CA), which has 1″ of travel in all three axes, two step sizes—coarse (0.2 μm/step) and fine (0.04 μm/step)—and a maximum speed of 2.9 mm/s. It has a tabletop controller and a rotary optical encoder for manual control; programmable robotic control is also available. The micromanipulator is mounted on a non-conductive optical breadboard, which is stabilized by two 1/4″-28-tapped beams that attach to the microscope stand on either side of the stage. The capillary is held in the micromanipulator by an acetal resin cylinder, which is 6″ long and has a 1/16″ diameter hole drilled in the center. This holder reduces the chance of arcing to the micromanipulator motors.
The sample is held on a coverslip holder, machined out of polycarbonate, with a lip to rest the coverslip edges on, and a 30° angled oval hole for holding the electrophoresis buffer vial, which consists of an Eppendorf tube (Hamburg, Germany) that has its top quarter removed at an angle. A platinum grounding wire (California Fine Wire Co., Grover Beach, CA) is placed in contact with the electrophoresis buffer, completing the circuit.
Trapping, manipulation, and injection of cells and beads are recorded by a monochrome CMOS camera (NT59-365, EO-1312M, Edmund Optics, Barrington, NJ) that is attached to the microscope housing with a 1× C-mount (Carl Zeiss, Jena, Germany).
An ancillary capillary was provided to clear the inlet to the separation capillary (and the trap) as needed by flowing high salt mGBSS at ~1 μL/min. The capillary position was controlled via another micromanipulator (Narishige Scientific Instrument Lab, Tokyo, Japan) placed to the left of the microscope; an HF-etched capillary (50 μm inner diameter, 360 μm outer diameter, and ~50 cm in length) was held by an acetal resin cylinder, similar to the one described above. PEEK fittings and a FEP sleeve were used to connect the capillary outlet to a syringe filled with high salt mGBSS. The pressure on the syringe was controlled by a syringe pump (model 601553, KD Scientific, Holliston, MA), located on top of the injection box.
Coverslip coatings and additives
Glass coverslips were used for all experiments except where noted. Several coatings and additives were tested to reduce adhesion of cells to the coverslip surface (as detailed in the Supplemental Information
). Treated coverslips were stored at ambient temperature and humidity in Parafilm M (Pechiney Plastic Packaging Inc., Chicago, IL)-covered glass dishes, unless otherwise noted, until use.
Single pinealocytes were injected into the CE column for analysis. A 2.5 μL droplet of sample was pipetted onto the coverslip. The selected cell is held in the OT. The capillary inlet was directed into the cell’s proximity by the micromanipulator, which was programmed to stop near the trap location and further position refinement was performed manually using the rotary optical encoder. Once the capillary was in place, the cell was released from the trap and hydrodynamic injection of the cell was performed by lowering the sheath waste outlet. Once injection was complete, the micromanipulator was used to bring the capillary inlet to the buffer vial, and the voltage and detectors were turned on. Injections were recorded using the CMOS camera on the microscope.
The sheath flow buffer was 25 mM citric acid, pH 2.25, and the flow rate was 0.2 mm/s for all experiments. The electrophoresis buffers and sample buffers varied as stated in the text and figure captions and both the capillary zone electrophoresis (CZE) and micellular electrokinetic chromatography (MEKC) modes were used. The voltage for all experiments was −30 kV unless otherwise stated. The injection volume varied as stated, but for bulk injections the volume was 14.7 nL for a 30 s hydrodynamic injection, which was achieved by lowering the sheath flow waste outlet by 32.5 cm. The typical laser pulse energy was between 1.5 μJ/pulse and 2 μJ/pulse.
The capillary was conditioned at the beginning of the day with 0.1 M NaOH for 15–20 min, followed by water for 5 min, and then electrophoresis buffer for a minimum of 5 min.
Data analysis was performed in IgorPro 5.05A (WaveMetrics Inc., Lake Oswego, OR). An automated data analysis script was written that reduces the user input to a single command. Output consists of four tables of calculated values with four corresponding color-coded graphs displaying the raw data, 6-point boxcar averaged data, normalized (with respect to the laser pulse energy) data, and both normalized and boxcar averaged data.
Limits of detection
The baseline range (30 points, 10 s) with the lowest standard deviation was determined and used to calculate the limits of detection (LOD) for each PMT channel. Ratiometric analysis (calculating the intensity ratio between peak maxima in each of the PMT channels) was also automated to aid in analyte identification.
Single-cell analyte concentrations were calculated with an approach that removes background signals from the media and differences in separation conditions, as described here. The ratio of the injection lengths between a sample analyzed under cell lysing conditions and the same sample analyzed under non-cell lysing conditions was used to normalize the analyte concentrations under non-cell lysing conditions. These normalized values were used to calculate equivalent background concentrations, which were subtracted from the concentrations calculated under cell lysing conditions. The analyte amounts were then adjusted based on an assumed cell volume of 4 pL to represent the actual analyte concentration within the cell.
LODs and concentrations of analytes were determined by generating calibration curves for each analyte under the appropriate conditions. Investigated analyte concentrations ranged from the micromolar to the low nanomolar range typical for biological systems. The criterion for calculating the LODs was three times the standard deviation of the baseline.