Poster 96 - Investigating the Impact of Laser Power on PC-12 Cell Thickness Using Robotic Laser Microscopy and Quantitative Phase Imaging

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sadhika Sadhikak.7777@gmail.com

High School Student
University of California, San Diego: Institute of Engineering in Medicine
San Diego, California, United States

Co-Author(s)

Zachary Z. Wang (he/him/his)

Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, United States
Grace He (she/her/hers)

Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

YY

Yatish Yanamala

High School Student
University of California, San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

Bryce Liu

Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, United States
CY

Calvin Yang

High School Student
University of California, San Diego: Institute of Engineering in Medicine, United States
KC

Kevin Cheng (he/him/his)

High School Student
University of California, San Diego: Institute of Engineering in Medicine
Irvine, California, United States
Jeffrey Takahashi

High School Student
University of California, San Diego: Institute of Engineering in Medicine
Torrance, California, United States
LD

Laksh Desai

High School Student
University of California, San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

JK

Jaren R. Kishimoto

High School Student
University of California, San Diego: Institute of Engineering in Medicine
SAN DIEGO, California, United States

Co-Author(s)

Sarah Kocian-Rudenberg

Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, California, United States
JZ

Junxiao Zhou

Postdoc
Electrical and Computer Engineering, United States
ML

Ming Lei

Ph.D. Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, United States

Primary Investigator(s)

Linda Shi, PhD (she/her/hers)

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Last Author(s)

ZL

Zhaowei Liu

Professor
University of California, San Diego: Department of Electrical & Computer Engineering, United States

Introduction: Laser-induced cellular manipulation is a critical technique in biomedical research, with applications ranging from targeted ablation to precise cellular surgeries. Understanding the effects of laser parameters, particularly laser power, on cellular structures is essential for optimizing these techniques to minimize unwanted damage and enhance therapeutic outcomes. The PC-12 cell line, derived from a pheochromocytoma of the rat adrenal medulla, is commonly used in neurobiology and cellular responses to external stimuli, making it an ideal model for investigating the effects of laser-induced damage.

This study investigated the impact of varying laser powers on the thickness of PC-12 cells to understand how different energy levels influence cellular integrity and structural alterations. Utilizing the advanced capabilities of the Robotic Laser Microscope System (RoboLase) and the innovative quantitative phase imaging (QPI) methodology, specifically FOSSMM (Fourier-Space Scanning Microscopy Method), the study provides a detailed analysis of how laser ablation affects cell thickness.

Materials and

Methods: Before plating the PC-12 cells, the cover glasses were pre-coated with 0.1% PDL. Then, the PC-12 cells were plated using DMEM high glucose,1% penicillin/streptomycin, 10% horse serum, and 5 % fetal bovine serum. The cells were stored at 37℃ a 5% CO2 incubator.

The quantitative phase imaging (QPI) methodology, FOSSMM, achieved 3D QPI to measure the PC 12 thickness. The FOSSMM comprises two lenses, one metasurface, and a polarized camera compatible with traditional bright-field microscopes. The utilization of the FOSSM has shown promising outcomes in investigating the cells' amplitude and phase information, as demonstrated in [1]. By incorporating sample scanning and reconstruction techniques, attaining 3D quantitative phase information becomes feasible.

We used the Robotic Laser Microscope System (RoboLase) to image the PC-12 cells in the X-Y plane and generate a precise laser ablation damage on the live cells. This system incorporated a Mai Tai femtosecond laser (set to the wavelength of 780 nm) guided into a Zeiss inverted microscope via a sequence of highly reflective coated mirrors and the 40x LUCPLFLN Bright field air objective. The ablation was applied precisely along a drawn line, and images were captured using a QuantEM camera. The ablation process followed a predetermined line, with images captured before and after cutting the PC-12 cells using a polarized camera.

Results, Conclusions, and Discussions: The experiment investigates the effect of varying laser power on the thickness of PC-12 cells. Three laser powers—244mW, 281mW, and 311mW—were tested at the focal point to determine the extent of cellular damage and corresponding changes in thickness.

The results indicate that cell thickness was minimally impacted at the lowest power, 244mW, with only a 0.50% change observed, shown in Figure 1A. This suggests that the laser energy was insufficient at this power level to induce significant structural alterations in the cells, likely because the energy absorbed was below the threshold needed to cause noticeable expansion through the ablation.

At a laser power of 281mW, a substantial increase in cell thickness was observed, with a 77.55% expansion occurring immediately after laser ablation, shown in Figure 1B. This change indicates that the laser power at this level was sufficient to induce damage, increasing cell volume, likely due to thermal effects or the generation of intracellular stress and swelling.

The most pronounced effect was observed at the highest power, 311mW, where the cell thickness increased by 230.53% following laser ablation, shown in Figure 1C. This increase suggests a high degree of cellular disruption, potentially due to the formation of cavitation bubbles or other damage mechanisms induced by the higher energy input. The results imply that as the laser power increases, the extent of cellular damage and subsequent expansion becomes more significant.

The study demonstrates a clear correlation between laser power and changes in PC-12 cell thickness, with higher laser powers leading to greater cellular expansion. The minimal change at 244mW suggests that this power is below the threshold for significant damage. In comparison, the marked increases at 281mW and 311mW indicate that these powers are sufficient to cause substantial structural changes in the cells.

These findings are crucial for understanding the effects of laser ablation on cellular structures. They inform future studies to optimize laser parameters for applications in cellular manipulation, laser surgery, and other biomedical fields. Further research could explore the underlying mechanisms driving these thickness changes and examine the long-term effects of laser exposure on cell viability and function.

Acknowledgements (Optional): This research was made possible through the generous support of the Beckman Laser Institute Inc. to LS and National Science Foundation - MRI project # 2320437 to Z.W. The high school students (*) participated in the UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional): [1] J. Zhou, F. Tian, J. Hu, Z. Shi, V. G. Godinez, D. P. Tsai, Z. Liu, Eagle-Eye Inspired Meta-Device for Phase Imaging. Adv. Mater. 2024, 36, 2402751. https://doi.org/10.1002/adma.202402751

[2] S. Barber, V. Gomez-Godinez, J. Young, A. Wei, S. Chen, A. Snissarenko, S. S. Chan, C. Wu, L. Shi, J. Biophotonics 2024, 17(3), e202300370. https://doi.org/10.1002/jbio.202300370