Wide Field-of-view Microscopes and Endoscopes for Time-lapse Imaging and High-throughput Screening

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.

Mass spectrometry coupled to imaging techniques: The better the view the greater the challenge

These are definitively exciting times for membrane lipid researchers. Once considered just as the cell membrane building blocks, the important role these lipids play is steadily being acknowledged. The improvement occurred in mass spectrometry techniques (MS) allows the establishment of the precise lipid composition of biological extracts. However, to fully understand the biological function of each individual lipid species, we need to know its spatial distribution and dynamics. In the past 10 years, the field has experienced a profound revolution thanks to the development of MS-based techniques allowing lipid imaging (MSI). Images reveal and verify what many lipid researchers had already shown by different means, but none as convincing as an image: each cell type presents a specific lipid composition, which is highly sensitive to its physiological and pathological state. While these techniques will help to place membrane lipids in the position they deserve, they also open the black box containing all the unknown regulatory mechanisms accounting for such tailored lipid composition. Thus, these results urges to different disciplines to redefine their paradigm of study by including the complexity revealed by the MSI techniques.

Geomicrobiology of meromictic, metal-mine pit lakes in the Iberian Pyrite Belt and biotechnological applications

220 p.

Abundance of harbor porpoise (Phocoena phocoena) in three Alaskan regions, corrected for observer errors due to perception bias and species misidentification, and corrected for animals submerged from view

Estimating the abundance of cetaceans from aerial survey data requires careful attention to survey design and analysis. Once an aerial observer perceives a marine mammal or group of marine mammals, he or she has only a few seconds to identify and enumerate the individuals sighted, as well as to determine the distance to the sighting and record this information. In line-transect survey analyses, it is assumed that the observer has correctly identified and enumerated the group or individual. We describe methods used to test this assumption and how survey data should be adjusted to account for observer errors. Harbor porpoises (Phocoena phocoena) were censused during aerial surveys in the summer of 1997 in Southeast Alaska (9844 km survey effort), in the summer of 1998 in the Gulf of Alaska (10,127 km), and in the summer of 1999 in the Bering Sea (7849 km). Sightings of harbor porpoise during a beluga whale (Phocoena phocoena) survey in 1998 (1355 km) provided data on harbor porpoise abundance in Cook Inlet for the Gulf of Alaska stock. Sightings by primary observers at side windows were compared to an independent observer at a belly window to estimate the probability of misidentification, underestimation of group size, and the probability that porpoise on the surface at the trackline were missed (perception bias, g(0)). There were 129, 96, and 201 sightings of harbor porpoises in the three stock areas, respectively. Both g(0) and effective strip width (the realized width of the survey track) depended on survey year, and g(0) also depended on the visibility reported by observers. Harbor porpoise abundance in 1997–99 was estimated at 11,146 animals for the Southeast Alaska stock, 31,046 animals for the Gulf of Alaska stock, and 48,515 animals for the Bering Sea stock.

On modeling and management of capture fisheries: one view

Sustainability of benefits from capture fisheries has been a concern of fisheries scientists for a long time. The development of fisheries management models reflects the historical debate (from maximum sustainable yield to maximum economic yield, and so on) of what benefits are valued and need to be sustained. Social and anthropological research needs an increased emphasis on bio-socioeconomic models to effectively determine directions for fisheries management.