Ablation of glass was accomplished with a home-built femtosecond Ti:Sapphire regenerative amplifier. The amplifier produced pulses with a typical duration and pulse energy of 40 fs and 30 μJ at 800 nm with a repetition rate of 1 kHz. The laser pulses were weakly focused (f/# = 9.2) to create an optimal zone of material removal for the desired channel dimensions. The lateral diameter of the ablated region per shot was 15 μm. Channels were created in a Pyrex window (ESCO Optics) by scanning the sample at a rate of 1 mm/s. The fluid channel was 3 cm long, 70 μm wide and ~55 μm deep. Fiber grooves were 2 cm long, 90 μm wide and ~70 μm deep. Under these conditions the total machine time for each channel or fiber groove was ~45 min. Placement of a fiber is shown in . The fibers laid several microns below the surface of the glass in order to ensure unhindered, direct contact between the silicon and the glass. Since the channels were machined from the top down, the ablated material was not trapped in the channel. Residual glass particles were rinsed away with water. We note that the channel depth measurements may include error arising from tip-substrate interactions when measuring channels of high aspect ratio with a standard profilometer. As such, the reported depths are lower limits.
Fiber placement in a femtosecond laser ablated groove.
The fluid channel is physically separated from the fiber grooves by a 50 μm thick glass barrier so as not to perturb the fluid flow. The fiber grooves terminate in short channels parallel to the fluid channel to provide a uniform surface at the optical interface. The interfaces scatter some of the light resulting in a constant background signal that is subsequently removed through electronic filtering. shows a white light image of the device. Multimode fibers (Polymicro Technologies, 0.22 numerical aperture (NA), 50 μm core, 55 μm cladding and 66 μm buffer) were placed by hand in the fiber grooves with the aid of a microscope. The device was then sealed by anodic bonding [24
] to a 270 μm thick silicon wafer (University Wafer). Anodic bonding seals the fluid channel and holds the optical fibers in the grooves without glue.
Fig. 2 (a) White light image of the fluid channel (horizontal) with four fiber grooves: one above at 14 degrees from normal and three below at −45, 0, and 45 degrees from normal. (b) Nanoport connectors were attached to either end of the fluid channel. (more ...)
The experimental setup is illustrated in . The incident laser light (2 mW at 532 nm) was coupled into the fiber with a 20×, 0.4 NA objective. (Power measurements before and after a loose fiber showed 60% coupling efficiency.) Scattered light was collected at 14 degrees as defined by the fiber groove direction and then collimated by an OFR LMU-15×-NUV objective and focused onto a photodiode (DET110, Thorlabs) by a 10 cm singlet. In subsequent implementations, this arrangement can be simplified by employing a fiber coupled diode laser on the illumination side and by directly aligning the collection fiber onto the face of the photodiode on the collection side, completely eliminating the need for external lenses and mounts.
An illustration of the experimental setup.
shows the overlap of the sensitive regions for the illumination fiber with the detection fiber. To characterize the overlap, the fluid channel was filled with a fluorescent dye. Light was alternately coupled into the illumination fiber and the detection fiber, and the fluorescence was imaged using a CCD camera. A lineout of the fluorescence intensity at the center of the fluid channel shows the overlap of the two regions.
Fig. 4 Overlap of the illumination region with the anticipated detection region was determined by filling the fluid channel with a fluorescent dye and then imaging the fluorescence. Lineouts along the center of the fluid channel (parallel to the x axis) show (more ...)
The analog signal was AC filtered to remove the large DC background and then amplified prior to being digitized (PCI-6251, National Instruments) at a rate of 200 kHz. The counter triggered at a set threshold value whenever passing cells deflected light into the collection fiber generating a positive voltage signal.
HeLa cells adapted for growth in suspension were used at a concentration of 2.5×104 cells/mL in Hank's Balanced Salts buffered to pH 7.4 with 20 mM HEPES (HHBSS) (Sigma Aldrich) with 1% Bovine Serum Albumin (BSA) to reduce sticking and 16% OptiPrep density gradient medium to prevent cell settling. The HeLa cell solution was placed in a pressurized reservoir and delivered to the microfluidic through tubing coupled to the device by Nanoport connectors (Upchurch Scientific). Applied pressures of 1.3 psi and 2.8 psi provided flow rates of 7.3 μL/min and 14.3 μL/min, respectively. Different aliquots of HeLa cells were run through the microfluidic device and counted independently by a commercial fluorescence-activated cell sorter (MoFlo FACS system, DakoCytomation) to test the accuracy of our microfluidic cell counter. FACS detection parameters were preset for our HeLa cells by calibration with a test sample of HeLa cells. The FACS measurement recorded the forward scatter and side scatter intensities for each cell and a total cell count.