Jacky Ko

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Bone Marrow Imaging Stage

In vivo optical microscopy of cells in bone marrow in mice skull required a more functional imaging stage. Angular head shapes of mice made it difficult for scientists to get a high quality image due to varying depth of field from a non-flat surface. Movement of the mice during imaging due to minimal head stabilization also led to breathing artifacts during optical sectioning across the z-axis. I designed an imaging bed equipped with a fine adjustment pivoting system along with the necessary requirements for in vivo microscopy in the lab including temperature control, stabilization, anesthesia delivery, and vacuum of excess anesthesia.

Design Goals

  • Create a bed that can adjust its angle to match the imaging area.
  • Provide fine adjustments in tilting mechanism.
  • Deliver anesthesia to the animal during imaging.
  • Keep animal at body temperature of 37°C to prevent hypothermic shock in imaging lab
  • Fit the holder under the limited space between the objective and the imaging table while keeping the image in focus.
  • Restrict head movement of the imaged animal.

Pivoting Mechanism

The front part of pivoting mechanism acts as a tuning system where the user can determine the amount of force necessary to pivot. This is done by tightening or loosening the nut on the nylon nut, which increases or decreases the compressive force on the holder. A brass rod was machined and held in place by a spacer, and a gear with a pin. The gear design was created in Rhino to match the commercial off the shelf (COTS) gear at a 10:1 gear ratio for precise angles. An additional knob was fabricated to turn the brass rod.

Close up of the pivoting mechanism consisting of a rod and a custom gear.

Heating Element

A temperature feedback control system was created using an Omega temperature controller, a K-type thermocouple, and a heating pad. The K-type thermocouple sensed the temperature of the heated surface, which would then be sent to the controller. The controller compared the measured temperature with the sent temperature in order to decide whether or not to heat of the pad. The temperature was kept at a constant 37±1.2°C. I tested the temperature controller after I soldered the wires, and troubleshooted a broken temperature pad. The heating pad was temporarily applied using electrical tape, clear epoxy was used for the final design.

Testing the feedback control of the heating system.

Anesthesia Delivery and Head Stabilization

Fluid transfer was done using Luer locks, an industry standard in small-scale fluidic systems, attached to hoses. The bottom Luer lock delivered the anesthesia to the mice while the top fitting vacuumed excess anesthesia. The Luer lock could not be built into the model due to strength constraints. In previous projects, the design had led to failure due to repeated twisting of connection, so I solved the issue by tapping the acrylic, and using a much stronger nylon fitting.

Stabilization of the head was done in two parts. The Luer lock on the bottom had a second function as a bite bar. The front teeth of the mice would "bite" into the bite bar, preventing x-y movement. The rubber ear bars on the sides clamped onto the head during imaging to prevent rotational movement. Both the bite bar, and the ear bars were adjustable for different mice variations by using set screws to hold the bars in place.

Front part of the holder with stabilization bars and anesthesia system.

Results

The black shading occurring every few seconds are breathing artifacts from the mouse before the holder was put in place. Most of the breathing artifacts are removed with the new imaging stage. There is also an increase of focus for the imaged section with the use of the holder, which is most noticeable when examining the clarity of the cavities. For the "Before Stage" image, the red parts are fluorescent markers used for the experiment.