We developed and validated a new instrument, called Measuring Concept progressions in Acid-Base chemistry (MCAB) and used it to better understand the progression of undergraduate students' understandings about acid-base chemistry. Items were developed based on an existing learning progression for acid-base chemistry. We used the Rasch rating scale model for validation and to gain understanding of how students at different ability levels conceptualize the topic. We then used latent class analysis (LCA) to place students into concept classes. We found that three concept classes were sufficient to describe students' various response patterns and that these concept classes described a progression of understanding related to acid-base chemistry. All items fit well with the Rasch model. The final 33-item instrument measured students along a continuous scale with a reliability of 0.74 and categorized students with a precision of 0.89. All students possessed nonscientific understandings of pH and the pH scale. However, even the most novice students displayed basic scientific understandings about how acid-base indicators work and the Arrhenius model of acids and bases. We discuss the utility of Rasch and LCA procedures for analysis of learning progression-based assessments and suggest a simple algorithm that can be used to help chemistry instructors diagnose students' misconceptions.

Two-photon active green fluorescent protein -type chromophores were successfully synthesized following investigations directed toward a modified version of zFP538 chromophore, a structural analogue to the GFP-chromophore. A generalized approach for the chromophore synthesis via a well-studied cycloaddition reaction combining an iminoglycine methyl ester and a substituted benzaldehyde was developed allowing for flexibility in the incorporation of functional groups such as donorÀacceptor substituents and for additional groups to provide extended conjugation. Steady-state spectroscopy, fluorescence quantum yields, and time-resolved fluorescence lifetimes for synthesized chromophores were extensively investigated for the functionalized chromophores. Time-resolved fluorescence lifetimes were found to be biexponential generally with subpicosecond and picosecond components. The individual effects of substitution position of functional groups and relative bulk size were evaluated and found to be rather significant in changing the fluorescencedecay characteristics in the case of positioning, but ambiguous with respect to relative bulk. The GFP-type chromophores were found to possess modest to low two-photon absorption cross sections with the dimethylamino-substituted analogue possessing the largest value at nearly 40 GM. These molecules show promise as biological markers for application in the study of conformation changes and aggregation of amyloid peptides, known to play an important role in many neurodegenerative diseases.

The conformational changes associated with the aggregation of proteins are critical to the understanding of fundamental molecular events involved in early processes of neurodegenerative diseases. A detailed investigation of these processes requires the development of new approaches that allow for sensitive measurements of protein interactions. In this paper, we applied two-photon spectroscopy coupled with time-resolved fluorescence measurements to analyze amyloid peptide interactions through aggregation-dependent concentration effects. Labeled amyloid-beta peptide (TAMRA-Abeta1-42) was used in our investigation, and measurements of two-photon-excited fluorescence of the free and covalently conjugated peptide structure were carried out. The peptide secondary structure was correlated with a short fluorescence lifetime component, and this was associated with intramolecular interactions. Comparison of the fractional occupancy of the fluorescence lifetime measured at different excitation modes demonstrates the high sensitivity of the two-photon method in comparison to one-photon excitation (OPE). These results give strong justification for the development of fluorescence-lifetime-based multiphoton imaging and assays.

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the important calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.

The development of new sensitive methods for the detailed collection of conformational and morphological information about amyloids is crucial for the elucidation of critical questions regarding aggregation processes in neurodegenerative diseases. The combined approach of two-photon and time-resolved fluorescence spectroscopy described in this report interrogates the early conformational dynamics seen in soluble oligomers of amyloid-β(1-42). Concentration-dependent aggregation studies using two-photon absorption show enhanced sensitivity toward conformational changes taking place in the secondary structure of the amyloid peptide as aggregation proceeds. Fluorescence lifetimes and changes in anisotropy values indicate Förster-type energy transfer occurring as a function of aggregation state. The sensitivity of our two-photon methodology is compared to that of circular dichroism (CD) spectroscopy, and the results indicate that the two-photon absorption cross-section method exhibits superior sensitivity. A theoretical model is developed that, together with electronic structure calculations, explains the change in cross section as a function of aggregation in terms of interacting transition dipoles for aggregates showing stacked or parallel structures. This suggests that the two-photon method provides a sensitive alternative to CD spectroscopy while avoiding many of the inherent challenges particular to CD data collection. The implication of this finding is significant, as it indicates that a two-photon-based technique used in conjunction with time-resolved fluorescence might be able to reveal answers to conformational questions about amyloid-β(1-42) that are presently inaccessible with other techniques.