User manual LEICA TILE SCAN BROCHURE

[. . . ] Multiple positions, for example multiple cells in the same Petri dish, can be stored in advance of performing the experiment. Fig. 1: Mark & Find interface: Perform multi positioning experiments and define location specific stack sizes. Leica_Mark_&_Find 23. 09. 2005 10:00 Uhr Seite 2 How it works With the Mark & Find tool the user can define the current position of the sample and recall it later on from a list together with other previously stored positions. This enables the user to quickly step through several positions to compare them or record multiple time series in a pseudoparallel manner. The user observes the live image and moves the stage to an interesting cell or location. [. . . ] The chances of observing a process of interest in one single experiment are much higher compared to the conventional situation where only one cell can be observed at a time. In effect, Mark & Find can help the user to save time or observe a larger number of cells. If the aim is to obtain quantitative parameters from the experiment (i. e. size, migration speed, particle tracking) a gain in throughput will effectively improve the statistical significance of the data. Mark & Find can be applied over several rounds of cell cycles, too. Additionally, multiple cells can be observed and their behavior can be compared. Applications in developmental biology Since Mark & Find can be combined with both t-series and z-scans, very complex recording scenarios become possible such as following dynamic processes in large specimen at multiple positions over time, each with an independent z-stack. Applications in developmental biology are perfectly suited to such tools, for example to follow the development of the nervous system and transport systems of animal models. Figure 3 shows a real-world example of a time-lapse recorded using Mark & Find. NIH 3T3 mouse fibroblast cells expressing H2A-YFP stably and were incubated with Aphidicolin over night. The shown time series was observed 4. 5 hours after removal of the blocking agent. In total 10 positions were selected using Mark & Find over time, and z-stacks of 7-8 µm with a z-step size of 0. 3 µm were recorded for each cell. Out of those ten cells, only one could be followed through mitosis (left column), whilst the others did not divide during the observation time (right column). This is a typical situation in live cell imaging when biological processes are triggered using specific drugs. Mark & Find increases the chances of observing such a process in a single experiment. DIC (differential interference contrast) and fluorescence images of the cells are shown over time from the top to the bottom. All DIC images are single in focus slices of their respective z-stacks, H2A-YFP images shown as maximum projections over the whole stack after deconvolution. Magnification 63x objective. mitosis observed DIC H2A-YFP no mitosis observed DIC H2A-YFP t1 t2 t3 t4 t5 t6 t7 Fig 3: NIH 3T3 mouse fibroblast cells expressing H2A-YFP. Courtesy of Constantin Kappel, DKFZ, Heidelberg, Germany Leica_Mark_&_Find 23. 09. 2005 10:00 Uhr Seite 4 Tile Scan The Tile Scan module allows the user to automatically assemble an image from multiple smaller images. Large specimens that are bigger than the current field of view can be viewed as a whole using the Tile Scan function. [. . . ] 5: Drosophila salivary glands at 20x magnification with bright field and fluorescence channels overlaid. The whole image was assembled from 5 x 3 single images at full resolution (1392 x1040 per image). Image shows nuclei (DAPI) in blue, Neurons/Axons (Alexa 488/Cy3) in green and neuronal nuclei (Alexa 594) in red. Note; some crosstalk between Cy3 and Alexa 594 is visible, as the image was captured with a broad band pass BGR cube. [. . . ]