Investigations of the three-dimensional nuclear architecture has become a critical area of research with the realization of that chromatin position in the nucleus plays an important role in gene regulatory behavior. A number of approaches have been developed to investigate the presence of gene-enhancer looping, chromosome territories and the importance of active vs inactive compartmentalization. In this thesis I describe Femto-Seq, a new method that combines the strengths of confocal imaging and two-photon excitation with next generation sequencing to probe the three-dimensional architecture of the nucleus in a novel way. The method combines a DNA-binding psoralen-biotin reagent with targeted two-photon excitation allowing for localization of DNA-psoralen crosslinking and the covalent attachment of biotin to nuclear volumes as small as ~0.5 femtoliters. Psoralen forms inter-strand crosslinks in DNA when exposed to UV light, and by replacing linear UV excitation with nonlinear two-photon excitation at 700 nm, the crosslinking can be highly localized. Validation of Femto-Seq required instrument automation and image processing approaches to reduce the time needed to collect chromatin from a sufficient number of cells. To validate the method, Femto-seq was applied to two cell targets, a transgenic YFP U2OS cell line and a labeled Muc1 locus in MCF10A and MCFCA1A cells. Pulldown and sequencing of target regions shows an enrichment of reads in the target area consistent with the irradiation volumes used, with up to 30-fold enrichment of the locus using the minimal excitation volume. Directions for continued improvement of the technology include library preparation and sample handling of inter-strand crosslinked DNA, where typical approaches are hindered by the presence of Psoralen crosslinks that need to be removed using harsh conditions, and a closer look at beam positioning accuracy, which can introduce off target photobiotinylation. Future applications of Femto-Seq in complex 3D culture models of cancer or in tissue sections will provide more clinically relevant genomic exploration. Femto-seq provides the ability to isolate DNA from user selected sub-nuclear regions of interest in user-selected cells, making it a novel approach for probing the three-dimensional architecture of the nucleus.