Piercing CC-124 Chlamydomonas reinhardtii Microalgae Cells with Gold Microneedle Arrays

Ellen Sadri

University of Rochester, Department of Chemical Engineering

SEM Practicum OPT407

Introduction
Samples and Preparation
Imaging and Results
-Chlamydomonas reinhardtii cells
-Gold Microneedle Array
-Pierced C. reinhardtii cells
Conclusions
Acknowledgments
References

Introduction:

Genetic transformation of plant cells, like algae, would allow us to alter the organism’s genome to create more efficient biofuels or components of pharmaceuticals. However, the transformation of plant cells is challenging due to their cell wall, which makes it hard to insert DNA without killing them. Dr. Muakibo’s lab is testing a new method of transforming microalgae cells with an array of microneedles loaded with genetic materials. The cells are impaled on the needles using centrifugal force. The key feature of this approach is that we can control the impact of the gene delivery by simply changing the needle dimensions and/or centrifuge settings.

In order to confirm that the cells have in fact been pierced onto the microneedles, scanning electron microscopy is utilized. The cells and needles are too small to be seen with the naked eye thus high magnification is required. The backscatter application in the SEM uses the different atomic numbers of the cells and the needles to create a stronger contrast between them. It displays a x-ray image of the needle tip inside the cell

Samples and Preparation:

Steps to Fabricate Gold Microneedle Array

Figure 1. Schematic of the steps taken to create the gold microneedles following a procedure created by Dr. Hitomi Mukaibo in the Department of Chemical Engineering.

The gold microneedles were fabricated by first (1) etching conical pores into a track ion-etched PET (polyethylene terephthalate) films that are about 15um thick. This is done with a two chamber etching cell where one chamber holds etching solution (KOH/MeOH), the other holds a neutralizing stop solution, and they are separated by the PET membrane.

(2) These PET templates are coated with gold using electroless plating. The sample is mounted onto a copper slide and (3) the PET template is dissolved away with HFIP (hexafluoroisopropanol). Now we are left with free standing gold conical microneedle array.

Au needle array

Figure 2. SEM Image of an Au Microneedle Array; This image is taken at a 45 degree angle to show the morphology of the needles

C. reinhardtii cells were grown in TAP media for three days under a fluorescent lamp. In order to pierce these cells onto the gold microneedles, a ProPlate^TM is used for our setup. The gold microneedles were clamped into the ProPlate^TMand sterilized. An aliquot containing 1E07 cells was removed from the culture. These cells were pipetted into the ProPlate^TM well over a layer of Percoll solution. The Percoll is used to control the speed and direction at which the cells are accelerated onto the needles. This prevents the cells from being splattered onto the gold substrate.

The ProPlate^TM with the microneedles and cells is centrifuged to induce the piercing of the cells onto the needles. Immediately after the cells were pierced, they were fixed with glutaraldehyde and dehydrated with ethanol. The sample was then critical point dried to replace any water in the cells with carbon dioxide gas. Finally, the sample was sputtered with gold. These steps are outlined in the schematic below.

Figure 3. A schematic of the steps taken to pierce CC-124 C. reinhardtii cells onto gold microneedles

CPD (critical point drying) is a method of drying biological samples in order to prevent them from being destroyed in the vacuum of the SEM chamber. It replaces any water in the sample with ethanol then with carbon dioxide liquid. The CPD device then raises the temperature and pressure of the chamber to vaporize the carbon dioxide liquid into gas without experiencing the tension induced by phase change.

Figure 4. Phase Diagram of Carbon Dioxide Displaying Super Critical Drying Conditions

Gold sputtering was done to make the sample conductive and create a ground. The ground is necessary to create a pathway for the electrons from the beam to escape form the sample. Without a ground, the electrons would accumulate on the sample surface and cause blurring of the image. The sputtering also increases the mechanical strength of the cells.

Figure 5. The sputter coater in the SEM/TEM Prep Lab at the University of Rochester

Imaging and Results:

Figure 6. The Zeiss Auriga Crossbeam SEM System at the University of Rochester used for imaging

Chlamydomonas reinhardtii Cells

C. reinhardtii is a strain of microalgae commonly used in laboratories for research purposes. Dr. Mukaibo's lab use the CC-124 and CC-125 strains. These are the male and female wild strains of C. reinhardtii. They both have tough cell walls and are mobile due to their flagellum.

The genetic modification of microalgae will assist in the development of alternative energy. However the cell wall of the microalgae prevents transfection using traditional methods. Thus the idea of piercing the cells with conical microneedles coated in DNA was pursued.

Figure 7. SEM Image of CC-124 C. reinhardtii Cell

Gold Microneedle Array

When exposed, the gold microneedles are seen to be 'mounted' on a gold background allowing them to stand perpendicular to the surface. These needles average to be 5 microns in height and 1 micron in base diameter. They are randomly located across the gold base surface but project perpendicularly from the base.

Au microneedle

Figure 8. Colorized SEM Image of an Au Microneedle

Pierced C. reinhardtii Cells

Pierced Cell Image

Figure 9. Colorized SEM Image of C. reinhardtii Cell Pierced on an Au Microneedle

The C. reinhardtii cells are pierced onto the gold microneedles using centrifugal force. When pierced, the cells can receive the DNA on the surface of the needles and become transfected. The SEM images allow us to confirm that the cells were in fact pierced. Below is a series of images taken of the same pierced cell using varied imaging settings.

SE2 of pierced cell

Figure 10. An SEM Image of a Pierced Cell Using SE2 Imaging

In Lens Pireced Cell

Figure 11. An SEM Image of a Pierced Cell using In Lens Imaging

BSE Image
Figure 12. An SEM Image of a Pierced Cell Using BSE Imaging

The BSE image displays the difference in atomic number between the cell and the gold microneedle. This contrast displays the hidden tip of the needle. Due to this we can assume that the cell is pierced by the needle and not just resting on top of it.

Composition

The composition varies between the gold needle substrate and the cells. This allows us to see if a cell contains a needle tip or not. The composition can be studied with two tools in the SEM. These are the EDAX system and the elemental mapping. These use x-ray electrons to determine the composition of the region being analyzed. The x-ray electrons interact with the electrons in the element producing a signal that varies for every element.

EDAX shows the level of each element in the region the beam in focused on. What it detects is determined by the strength of the electron beam (accelerating voltage).

EDAX Image

Figure 15. EDAX of (A) C. reinhardtii cell, (B) Au Microneedle, and (C) A cell pierced on a microneedle; Note: Au is seen int he EDAX of the independent cell due to the gold sputter coating

Elemental mapping displays the spatial location of each element throughout the sample. In the elemental map below of microalgae cells on a gold microneedle array we see that the cells have a high concentration of carbon and the other noted elements are only found in the array.

Elemental map

Figure 16a. Elemental Map of C. reinhardtii cells pierced onto Au microneedles

SEM Image for Map

Figure 16b. SEM Image representing region of the sample the Elemental Map is displaying

When pierced, the cells can receive the DNA on the surface of the needles and become transfected. The SEM images allow us to confirm that the cells were in fact pierced. Below is a series of images taken of the same pierced cell using varied imaging settings.

Conclusions:

Using the many SEM settings I could determine that in fact the C. reinhardtii cells were pierced onto the gold microneedle arrays. The BSE setting made it most apparent that the tip of the needle was inside the cell.

Due to this confirmation, the microneedle array can now be coated with foreign DNA to transfect the cells. The cells will be tranfected with a florescent gene that once grown will demonstrate that the gene has been incorporated into the cells' genome.

Acknowledgments:

I would like to thank Brian McIntyre of the Optics Department for his lectures and instruction on the use of the imaging equipment. Also thank you to Dr. Hitomi Mukaibo for my position and mentoring.

References:

Choi, Seong-O, Yeu-Chun Kim, Jeong Woo Lee, Jung-Hwan Park, Mark R. Prausnitz, and Mark G. Allen. "Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Array." Wiley InterScience. 8.7 (2012): 1081-1091. Print.

Harell, C. Chad, Zuzanna S. Siwy, and Charles R. Martin. "Conical Nanopore Membranes: Controlling the Nanopore Shape." Wiley InterScience. 2.2 (2006): 194-198. Print.

Mukaibo, Hitomi, Llyod P. Horne, Dooho Park, and Charles R. Martin. "Controlling the Length of Conical Pores Etched in Ion Tracked Poly(ethylene terephthalate) Membranes." Wiley InterScience. 5.21 (2009): 2474-2479. Print.