Multiphoton excitation spectra in biological samples

Multiphoton microscopy is becoming a popular mode of live and fixed cell imaging. This mode of imaging offers several advantages due to the fact that fluorochrome excitation is a nonlinear event resulting in excitation only at the plane of focus. Multiphoton excitation is enhanced by the use of ultrafast lasers emitting in the near IR, offering better depth penetration coupled with efficient excitation. Because these lasers, such as titanium:sapphire lasers, offer tunable output it is possible to use them to collect multiphoton excitation spectra. We use the software-tunable Coherent Chameleon laser coupled to the Zeiss LSM 510 META NLO to acquire x−y images of biological samples at multiple excitation wavelengths, creating excitation lambda stacks. The mean intensity of pixels within the image plotted versus excitation wavelength reveals the excitation spectra. Excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in much the same way that emission fingerprinting can be used to generate crosstalk free channels from emission lambda stacks using the META detector. We show how this technique can be used to eliminate autofluorescence and to produce crosstalk-free images of dyes with very close overlap in their emission spectra that cannot be separated using emission fingerprinting. Moreover, excitation finger- printing can be performed using nondescanned detectors (NDDs), offering more flexibility for eliminating autofluorescence or crosstalk between fluorochromes when imaging deep within the sample. Thus, excitation fingerprinting complements and extends the functions offered by the META detector and emission fingerprinting. We correct biases in the laser and microscope transmission to acquire realistic multiphoton excitation spectra for fluorochromes within cells using the microscope, which enables the optimization of the excitation wavelength for single and multilabel experiments and provides a means for studying the influence of the biological environment on nonlinear excitation

Introducing the Complete Case Study into an Australian Undergraduate Sociology Major

In recent years, higher education institutions have paid greater attention to establishing learning thresholds, benchmarks and outcomes for the teaching of sociology. This paper explores how course redesign using case studies can align with the recently established Australian Threshold Learning Outcomes (TLOs) for sociology, and assesses its advantages and challenges for students and lecturers. Case studies are common components of undergraduate courses in the social sciences; either as illustrative examples provided by teachers or as discrete participatory exercises for students. However, little has been written on integrating case studies into single-semester courses, or how teachers can navigate institutional and disciplinary requirements when delivering such courses. In this paper, we describe the development and delivery of a later-year undergraduate course in sociology that used a student-led ‘complete case study’ project to enhance students’ research skills and/or prepare them for higher-level research projects. We begin by explaining how our methods were inspired by the work of Pierre Bourdieu and the practice of ‘relational pedagogy’. We then discuss how these aims aligned with the emergent Australian TLOs with the participating university’s learning objectives of student engagement and questioning. We also reflect on the constraints and challenges presented by delivering case studies to undergraduate student