Browsing by Subject "microscopy"

Cyanobacteria are an important part of the phytoplankton community and aquatic ecosystems. Cyanobacteria can form large mass occurrences, i.e. blooms, which can be toxic or cause other harm. Research and monitoring of cyanobacteria has been based on microscopy analysis. However, molecular-based methods, such as 16S rRNA sequencing are replacing microscopy analyses in the near future. The Finnish Environment Institute has stated that molecular methods are part of environmental monitoring before 2030. In this Master’s thesis the aim was to determine whether conventional microscopy analyses and 16S rRNA sequencing differ when comparing nano- and micro-sized cyanobacteria. The material was collected from a laboratory experiment of the Finnish Environment Institute’s (SYKE) MiDAS project, which was conducted in the summer of 2020. The results of the microscopy and 16S rRNA analyses differed from each other. The relative abundances of the cyanobacteria genera differed between sample types. Microscopy analyses estimated that the alpha diversity was higher compared to the results of the sequencing analyses. The main reason for the difference between the types of analyses was due to the differences in cyanobacteria belonging to the order of Synechococcales. Some of the Synechococcales species were observed only by the sequencing analyses, e.g. Snowella and some of the Synechococcales species were only observed by the microscopy analyses, e.g. Romeria and Woronichinia. It was observed that both methods are prone to identification errors. The differences between the 16S rRNA sequencing and the microscopy analyses are vastly different. It may affect on the review of long-term data of the phytoplankton community. Therefore, it is important to examine the differences between the types of analyses. Studying the dissimilarities between the types of analyses should be focused on the research of the small cell-sized colonial cyanobacteria, i.e. the species of Chroococcales and Synechococcales.

The aim of this thesis was to investigate the potential of label-free CARS microscopy as a new method for chemically-specific imaging of live cells and particle-cell interactions in a drug delivery context. Cells used to mimic the intestinal epithelium, Caco-2 cells and HT-29 cells and nano-/ microcrystal particle interactions with macrophages were studied. More information about drug absorption from intestinal and particle cell interactions are needed, since many novel drugs lack properties needed for good bioavailability. It would be beneficial if these events could be visualized without labels. CARS microscopy was found to be well suited to imaging live Caco-2 and HT-29 cells that were grown on PTFE Transwell inserts. CARS microscopy revealed lipid droplets inside these cells. The size of lipid droplets increased in Caco-2 cells a lot during a three week period so that at the end a large part of the inner part of the cell was filled with lipid droplets. It was also observed that Caco-2 cells and HT-29 cells can grow on top of each other on Transwell inserts and not just as a monolayer. These two facts could cause variations in drug absorption studies based on Caco-2 cell monolayers. CARS microscopy was able to detect nanocrystals as small as about 500 nm with label-free, molecular-specific CARS microscope inside RAW 264.7 macrophages after incubation of 120 min. This observation was important, since nanocrystal drug formulations are gaining interest in the field of pharmacy. Nanocrystals can be used in parenteral drug formulations as well as in oral dosage forms. In suspensions, nanocrystals can be used to cause long lasting drug release. Nanocrystals can be also used to enhance poor bioavailability of drugs. Whether these nanocrystals are used in parenteral formulations or in oral drug formulations it is evident that imaging techniques are needed to image interactions between these nanocrystals and cells. CARS microscopy could be one of those techniques, since it is suitable for live cell imaging and it can be used to image nanocrystals that are not labeled. The results in this thesis suggest that CARS microscopy could be used as fast imaging technique for nanocrystal particle cell interactions. Overall, CARS microscopy is a relatively new imaging method that shows much promise as a label-free chemically specific imaging technique for imaging cells and cell-particle interactions in a drug delivery context. As the technique becomes more widely available and undergoes some technical developments, it will become much more widespread imaging method in the future.

Glomus intraradices and Bacillus amyloliquefaciens are two commercially used plant growth promoting micro-organisms. They associate with plant roots to facilitate host plants to absorb nutrients, induce resistance against pathogens and pests, and regulate growth through phytohormones. Growth conditions for plants on green roofs are often unfavorable. In order to test whether growth and development of green roof plants could be enhanced via improving the microbial interface, G. intraradices and B. amyloliquefaciens were inoculated on experimental plots on a green roof in the summer of 2012. The experimental plots were marked as R (inoculated with B. amyloliquefaciens from Rhizocell), M (inoculated with G. intraradices from MYC4000), and C (control). The green roof was made of sedum-herb-grass mats. The plants included e.g. stonecrops, bluegrasses, yellow rockets, white clover, mullein, pennycress, and moss. The survival and development of G. intraradices and B. amyloliquefaciens were studied respectively from Poa alpina roots and soils in the summers of 2012 and 2013. G. intraradices was not detected in P alpina roots according to root staining and microscopy. Probable reasons for the lacking of G. intraradices include high phosphorus content in the soils, high soil temperature, and low soil moisture. PCR and qPCR were used to detect Bacillus content in green roof soils. The abundance of B. amyloliquefaciens was related to soil water content and soil temperature. During the last two measurements in 2012, 4 weeks of high moisture content in the soil resulted in large increase of B. amyloliquefaciens content in both M and R groups, but then decreased substantially due to drought and heat in 2013. In 2013, Only R group increased from the third to the last measurement, indicating probable resistance of the B. amyloliquefaciens strain from Rhizocell additive. The synergistic effect of B. amyloliquefaciens and G. intraradices might be responsible for the thousand-fold increase of Bacillus content in M group in 2012.