2023.2.27 KENT BRASSEALE_THESIS.pdf (1.34 MB)
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posted on 2023-02-27, 15:04 authored by Kent Alan Brasseale IIIKent Alan Brasseale III


Fluorescent labeling and imaging techniques have become a standard for high-content screening (HCS), wherein autonomous spectroscopic imaging leads to detailed information related to multiple biological parameters.  However, the chemical labels these fluorescent methods rely on introduce significant challenges to analyzing live cells, such as perturbation to metabolic pathways, poor chemical selectivity, and limited detection channels. Nonlinear spectroscopic methods have historically been incapable of demonstrating competency in high-content screening due to some of the unique qualifications required for sample excitation and signal collection. Nonlinear optical sources of excitation, notably coherent anti-Stokes Raman scattering (CARS), is a rapidly developing field with a label-free nature capable of successfully addressing the shortcomings of fluorescence spectroscopy in many experimental environments, while also presenting unique impediments of its own. Among these are the short working distance incompatible with microplates and the requirement of transmission signal detection to achieve high sensitivity, restricting the application of CARS for HCS. Early attempts to construct the desired HCS pipeline using visible stimulated Raman scattering (SRS) were diverted due to an inability to balance detrimental photodamage with the need for increased energy input to maintain satisfactory signal generation. We developed a label-free CARS HCS platform using a pulse-picking technology and supporting imaging software to image live cells in the epi-direction with high resolution. This configuration allows the use of microplates and a surrounding incubation chamber for precision sample control and the simultaneous label-free chemical analysis of up to 64 conditions. This novel CARS-HCS platform allows automatic quantification and time-lapse monitoring of numerous conditions within a precision-controlled microenvironment.


Degree Type

Master of Science



Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Chi Zhang

Additional Committee Member 2

Garth Simpson

Additional Committee Member 3

Mike Reppert