Quantitative mRNA detection with advanced nonlinear microscopy

Jing Liu, Purdue University


Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The thesis presents the fundamental study on the probes used in SHaSM, the concept and instrumental layout of the two technologies, and the detection as well as quantification of mRNA transcript in cells and tissues by super resolution microscopy and background-free detection microscopy. The first part of my dissertation focuses on the introduction of available mRNA detection methods and nonlinear imaging techniques. In chapter 2, I mainly characterize the SHG emission behavior of individual BTO nanocrystals via time-resolved single molecule spectroscopy, correlation spectroscopy, and confocal microscopy. High-intensity stable emission is collected from individual BTO nanocrystals with a high signal-to-noise ratio; the polar-dependent emission behavior of individual BTO NCs was also investigated theoretically and experimentally; and the dynamics of individual BTO in turbid medium is studied by an improved autocorrelation spectroscopy. The third chapter develops a novel second harmonic super-resolution microscopy (SHaSM), which is capable of detecting individual BTO nanocrystals with the lateral resolution as high as 30 nm. Motivated by the capability of SHaSM to visualize single BTO nanocrystals beyond the diffraction limit, we develop a "dimer" configuration of BTO nanocrystals for detecting single mRNA transcript beyond the diffraction limit. We validate our SHaSM to resolve single mRNA transcript first in vitro. Preformed BTO dimers are detected and differentiated by the SHaSM and by the SEM as the control. Expression level and localization patterns of Her2 mRNA transcript in single SKBR3, MCF7, and HeLa cell are investigated with the SHaSM. SHaSM can successfully differentiate the Her2 mRNA from the nonspecific BTO monomers, and identify more than one transcript in a diffraction-limited spot for SKBR3 cells. Quantification results agree well with the theoretical estimation and the RNA FISH results, and in addition it shows that the SHaSM has more accurate quantification when detecting over-expressed mRNA transcript. Furthermore we applied the SHG probes and SHaSM to study the heterogeneity of Her2 mRNA transcript in breast cancer tissues. High-specific binding of the SHG probes is observed and high penetration detection can be realized. In addition to the SHaSM, I also develop a background-free method to detect and quantify mRNA transcript. A femto-second transient absorption microscopy (TAM) is developed in the lab. It starts with the theoretical description of the TAM process, and then introduce the fundamental optical properties of the gold nanoparticles in TAM. By chemically treating the gold nanoparticles and conjugating with ODN probes, the gold nanoparticles hybridize to the mRNA molecules and are visualized in the TAM, together with label-free images of cells obtained in the SRS microscopy. mRNA is quantified with single copy sensitivity and is validated by the FISH approach. Super resolution microscopy of Her2 mRNA transcript in single cells will provide more accurate quantification in single cells; what's more, it can be potentially employed to investigate the dynamics of single mRNA transcript beyond the diffraction limit, which is extremely significant in basic biology. TAM microscopy promotes the detection of mRNA transcript at a high speed without fluorescence background, which can be further utilized to investigate the dynamics of RNA regulation. Both these two methods will promote our understandings of the expression level and localization patterns of mRNA transcript in single cells, provide a route to employ mRNA transcript as a marker or indicator for cancer diagnosis and therapy.




Irudayaraj, Purdue University.

Subject Area


Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server