CellEraser™ calibration

Prior to operation, the device of the present invention is preferably calibrated. The calibration procedure is illustrated in Figure 3. The calibration procedure is performed to ensure that the millimeter wave radiation level is high enough to heat the cells in the region of exposure to the desired temperatures (for example 48-50°C). The calibration procedure starts by preparing a calibration plate 302 that is similar to the cultureware. The internal surface 304 of the calibration plate 302 is preferably coated with a thin film (about 10 µm thick) of long-chain alcohol (LCA) with a known melting point temperature (LCA itself does not absorb millimeter wave radiation so melting happens because of heating of water adjacent to the thin film of LCA and/or water trapped inside this porous film). For example, 1-Heptadecanol (1-HD) has a melting point (MP) temperature 51-55°C, 1-Hexadecanol (1-HXD) MP is 48-50°C, 1-Octadecanol (1-OD) melts at 58°C.

Next, a millimeter wave radiation at a frequency in the range of 75-110 GHz with the time of exposure of about 2-3 sec is applied to the coated calibration plate 302 via a waveguide 308 with an end face 310 positioned below the calibration plate 302. This leads to melting of an area 312 in the 1-OD microfilm 306 (see Movies). The melting area 312 may be visualized with phase contrast microscopy; the images B-C of Figure 3 show a melted area of approximately 0.3-0.4 mm.

By adjusting exposure parameters such as the radiation power and exposure time of the millimeter wave radiation, and positional relationship of the end face 310 of the waveguide 308 and of the calibration plate 302, a bigger or smaller melting area 312 may be attained. Images D-E of Figure 3 show a melted area 312 of approximately 0.7 mm in diameter (upper area on images D-C, the field of view was shifted up) obtained with an increased power of millimeter wave radiation. A 2X air objective lens with a large working distance and a small numerical aperture (not shown in the drawing) was used to observe the microfilm 306.

Lower millimeter wave radiation power results in a smaller diameter of the melting area 312. Thus, smaller melted microfilm area 312 may be obtained when the source of millimeter wave radiation is adjusted to a lower power via a variable attenuator (not shown) and/or when the end face 310 of the waveguide 308 is positioned farther from the external surface 314 of the calibration plate bottom 316. For precise single cell ablation the diameter of the region of exposure may be decreased to 0.25 mm or less. The maximal size of the melting area 312 (about 1 mm in diameter) is determined by the wavelength of millimeter wave radiation in the W-band. Thus, the equipment can be calibrated to ablate cells in areas with different diameters.

For best results, the calibration procedure is preferably performed under conditions very similar to the conditions at which the cells would be treated: the calibration plate 302 with the microfilm containing the same volume of the same culture medium as the cell cultureware with the cells placed therein. In the absence of the culture medium in the calibration plate 302, no melting of the LCA microfilm is observed (data not shown). This is consistent with the fact that, absorption of millimeter wave radiation by LCA, higher alkanes or oils is negligible. The LCA film 306 melts when the culture medium closely adjacent to the film 306 is heated via application of the millimeter wave radiation. The heated culture medium 318 moves upward creating a convectional flow 320; therefore the cells adjacent to the region of exposure are not heated (Figure 3).

Thus LCA/alcane microfilms may serve as local sensors of the temperature in a narrow layer (~ 10-15 µm thick) of cell culture medium at the glass/plastic bottom of the calibration plate 302. The LCA microfilm may also be formed directly in the cultureware having cells in a culture medium placed therein to allow performing the calibration procedure in the same plate, in close proximity to the cells.

Time-lapse movie of LCA microfilm melting  (diameter of melted area depends on power of millimeter wave radiation (2x magnification)

10x magnification

Movies

Electromagnetic Field was firstly applied to square E (melt a hole in LCA microfilm) and then to square F (melt second hole).

Electromagnetic filed power is  adjusted to minimal level so the diameter of heated area become less than 120 µm (grid is 500 µm).

New calibration procedure
The new calibration procedure is illustrated in Figure 4. The calibration procedure might be performed simultaneously with melting of LCA films or without them. The temperature near cultureware bottom is measured directly via thin flexible thermocouple microprobe with claimed tip size 0.009" (230 µm). Real tip size is less that 150 µm (see image with tip and 1 mm/0.1mm grid). The thermocouple controller shows the temperature in the center of hot spot when stage move to waveguide position and the millimeter wave radiation starts to heat water solution. in the region of exposure to the desired temperatures. LCA film (in this experiment LCA is 1-Pentadecanol with MP = 40°C) melts around probe tip (see Figure 4). Thus, the calibration procedure become very simple and robust.

Figure 4

Movie shows temperature changes within thermocouple probe tip (in the center of circle on monitor). Lower line on oscillograph monitor shows when MMW is turned on. Upper line detects voltage on thermocouple. The thermocouple controller shows the temperature dynamics.

Calibration for cell isolation 

When CellEraser™ is calibrated for 50°C you have a tool for cell isolation. The diameter of melted area is a size of this tool. Just choose desired spot in cell culture and start the program that will move automatic stage around this spot. The ring around this spot will be irradiated by electromagnetic field that heat cells to 50°C but cells inside of this ring will not be heated. As an example the ring is melted in LCA with melting temperature 50°C. The desired object is shown by arrow. The diameter of melting spot is 0.6 mm  (600 µm) - melted spot is shown on bottom image. Internal diameter of the ring is 0.5 mm (500 µm). The process of ring melting is not visible on inverted microscope but might be observed in real time on upright microscope (gold rectangle is waveguide end).

Calibration for 3D spheroids embedded in Matrigel 

Multicellular three-dimensional (3D) tumor spheroids are recognized as superior models for the preclinical evaluation of anti-cancer therapies due to their better and more advanced representation of tumors in vivo. The organization of cells within the 3D spheroidal structure means that they are in direct contact with each other and the secreted extracellular matrix (ECM), allowing them to utilize alternative cell growth and survival signaling mechanisms not readily observed in 2D monolayers. Similar to in vivo cancers, 3D spheroids exhibit differential rates of cell proliferation throughout the spheroid, and physiologically relevant gradients of oxygen, nutrients, and waste products are also observed.

If you are going to use CellEraser™ for 3D spheroids growing in Matrigel the calibration process should be done in similar cultureware where LCA film on the bottom is covered with Matrigel. LCA melting temperature should be same or close to the temperature that you want to heat your 3D spheroids. We have observed that the diameter of melted spot in LCA film in gel is at least 2 times bigger than in LCA melted in water-based solutions (see image below). However, when the thermocouple is inserted in the middle of the spot melted in gel it detects same temperature as it was measured in water-based solution. Moreover, we have detected that the diameter of spot melted in gel increases if time of radiation exposure is increased (this is never observed for LCA melted in water-based  solution) (see image below). 

We explain this phenomena by an absence of vertical convection in gel (because of too high viscosity of gel matrix). Instead of vertical convection the lateral one is happening in gel when liquid moves in narrow layer between gel and substrate. Since lateral convection in gel is not very effective the temperature in the middle of the spot will increase 2-3°C if exposure time is increased to 10-20s (instead of 5s). The temperature calibration will work for 3D spheroids that embedded in Matrigel close to a substrate (glass or plastic). The measurements with thermocouple showed that the temperature in the spot sharply declines in Z axis. It explained by a short penetration depth of millimeter waves in water and an absence of vertical convection in gel.