Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.

Localization of sources of human evoked responses

The evoked response, a signal present in the electro-encephalogram when specific sense modalities are stimulated with brief sensory inputs, has not yet revealed as much about brain function as it apparently promised when first recorded in the late 1940's. One of the problems has been to record the responses at a large number of points on the surface of the head; thus in order to achieve greater spatial resolution than previously attained, a 50-channel recording system was designed to monitor experiments with human visually evoked responses.

Conventional voltage versus time plots of the responses were found inadequate as a means of making qualitative studies of such a large data space. This problem was solved by creating a graphical display of the responses in the form of equipotential maps of the activity at successive instants during the complete response. In order to ascertain the necessary complexity of any models of the responses, factor analytic procedures were used to show that models characterized by only five or six independent parameters could adequately represent the variability in all recording channels.

One type of equivalent source for the responses which meets these specifications is the electrostatic dipole. Two different dipole models were studied: the dipole in a homogeneous sphere and the dipole in a sphere comprised of two spherical shells (of different conductivities) concentric with and enclosing a homogeneous sphere of a third conductivity. These models were used to determine nonlinear least squares fits of dipole parameters to a given potential distribution on the surface of a spherical approximation to the head. Numerous tests of the procedures were conducted with problems having known solutions. After these theoretical studies demonstrated the applicability of the technique, the models were used to determine inverse solutions for the evoked response potentials at various times throughout the responses. It was found that reliable estimates of the location and strength of cortical activity were obtained, and that the two models differed only slightly in their inverse solutions. These techniques enabled information flow in the brain, as indicated by locations and strengths of active sites, to be followed throughout the evoked response.