Optical cell sorting using a Gaussian beam

3.3.1 Optical cell sorting through axial guiding to laminin coated coverslips

I have shown that on exposing CHO cells to a diverging optical field obtained through use of a low numerical aperture objective, cells with ingested microspheres have enhanced axial guiding (Figs. 3.12-13). Therefore, I sought to capitalize upon this feature to develop a novel optical cell sorting methodology for the separation of cells from a mixed cell population. To do this, I implemented the sorting of the cells with microspheres on a self-built inverted optical trapping setup, using analyte volumes of 100 µl as elucidated in section 3.3.1.2 to follow.

3.3.1.1 Experimental setup

A home built optical guiding apparatus was employed, where a diffracting Gaussian beam was directed upwards through a 10X objective lens (NA 0.28) (Comar, USA) towards a mixed population of CHO cells (with and without internalized microspheres) suspended between a hydrophobic glass bottom petri dish and a laminin-coated top coverslip. Only cells that had internalized microspheres were propelled axially to the top of the sample chamber and attached to the laminin-coated glass coverslips. Figure 3.14 below illustrates the home-built optical guiding setup used in this optical cell sorting experiment.

Figure 3.14: Cell sorting system built using a 1064 nm Nd:YAG laser, the beam (diameter = 1.5 mm) was emitted and the average power attenuated using a neutral density filter-wheel (FW), before expanding the beam via a two lens magnifying telescope system L1 and L2 (f = 50 and f = 200 mm) to a 6 mm diameter. Then it is reflected using members of a periscope mirrors M1 and M2 via a dichroic mirror (DM) onto the back aperture (diameter = 8mm) of low NA 0.28, 10X microscope objective (MO). The XYZ stage is the sample stage which was illuminated using Köhler illumination consisting of a light emitting diode (LED), three lenses (f = 20 mm, f = 20 and 65 mm respectively) and two diaphragm apertures. A tube lens (TL) (f = 100 mm) was positioned beneath the sample stage and focused the collected bundle of rays onto the sensor of a CCD camera through which the image forming data is transferred onto a data capturing computer.

3.3.1.2 Selective separation of CHO cells with internalized microspheres

A mixed population of CHO cells with a range of numbers of partially internalized 3 µm microspheres was trypsinised from a T25 culture flask and suspended in complete growth medium. These were strained through a 40 µm pore size filter (Millipore, UK) to obtain a mono-dispersed cell sample. The cell concentration used for the sorting experiment was calculated to be 6.2 X 105 cells/ml. A 100 µl of this cell sample per sorting experiment was used. Sample chambers used in these experiments consisted of 30 mm diameter type- zero glass bottom petri dishes (bottom compartment) (World Precision Instruments, Stevenage, K) and 22 mm diameter round glass type-one coverslips (top part) (BDH, Poole UK). To prevent cell adhesion to the 23 mm glass surface of the dishes, the dishes were coated with sigmacote (Sigma-Aldrich), a reagent that reacts with surface silanol groups on glass to produce a neutral, hydrophobic microscopic thin film. The coating procedure involved adding 1 ml of neat sigmacote per dish and incubating them at room temperature in a class II level Bio-hood (bio-hood) for 12 hrs, before aspirating and air drying the within the bio-hood. In contrast, the top coverslips were coated with 2 µg/cm2 laminin solution made up in sterile tissue culture grade water (Sigma-Aldrich) to promote cell adhesion upon close physical contact (25). To coat, the coverslips were incubated in the laminin solution overnight in a 37oC incubator and thereafter allowed to air dry in the bio-hood before use in experiments.

After sample preparation the sample chamber was placed on the sample stage of an inverted microscope optical setup. The sample was exposed to the beam and an average of 50 cells with ingested spheres per experiment were optically sorted and separated from the rest of the sample (figure 3.15).

Figure 3.15: Illustrates a 1064 nm laser Gaussian guiding beam less tightly focused through a low NA objective to propel cells with intracellular dielectric tags onto the top surface on the sample chamber. This image explicitly displays cells with 3 µ m spheres being optically transported onto the laminin coated glass slide whilst neat CHO cells remain on the bottom of the sample assembly without any optical treatment.

Optical guiding was achieved due to an increased axial scattering force exerted onto cells with internalized microspheres compared to those with no ingested spheres. The sorting occurred at a maximum rate 11 cells/min. Figure 3.16 shows the actual laboratory results of this experiment.

Figure 3.16: Sample chamber containing 100 µl of a mixed population (with and without ingested microspheres) of CHO cells was suspended between a hydrophobic glass bottom petri dish and laminin- coated top coverslip, while a diffracting Gaussian beam (red dotted circle) emerging from a 10X objective with NA 0.28 was illuminated toward the laminin-coated coverslip (video available). (A) and (B) CHO cells begin to migrate toward the centre of the beam. (C) CHO cells with ingested microspheres start to be axially propelled onto the top laminin-coated glass coverslip. (D) CHO cells with ingested spheres adhere to the top glass coverslip, and subsequently, can be cultured separately (reprint by permission from IEEE

Journal of Selected Topics in Quantum Electronics (24)).

The separated cells that adhered to the laminin-coated coverslip were then further cultured by placing the coverslips into 500 µl of filter-sterilized conditioning medium (collected from routine sub-culturing of CHO cells) in 30 mm diameter plastic petri dishes at 37oC with 5% CO2 , 85% humidity, and the medium changed every 48 h. After

four days of culturing, the optically sorted cells were trypsinised; their viability tested using the trypan blue exclusion dye method, and counted. A total cell count number of 1.2 X 103 cells/ml was obtained with 100 % viability. In figure 3.17, a brightfield image

Figure 3.17 Brightfield image of a sample chamber containing re-cultured CHO cells with ingested microspheres (dark spots) viewed with a 20X objective lens of NA 0.54 (reprint by permission from IEEE

Journal of Selected Topics in Quantum Electronics (24)).