Detection of gene expression at different stages of development
The zebra fish is a useful model to study gene expression and steps in embryo development. The time point at which a gene is expressed by the organism can be observed by imaging the transparent zebra fish embryo with a 2-photon microscope. For such an experiment, different genes have to be tagged with different fluorescent labels.
One method for labelling different genes is the in situ hybridization technique. This technique uses a fluorescent-labeled complementary DNA or RNA strand to localize a DNA or RNA sequence in the sample. The complementary strand can bound to its DNA/RNA sequence that is thereby labelled with the fluorescent tag.
In another method, transgenic genes can be marked with a fluorescent tag such as a fluorescent protein. The tagged transgenic gene is then expressed by the organism. With both methods, specific genes are labelled with different fluorescent dyes.
For imaging, the zebra fish embryo has to be mounted under the microscope. The use of low-melting-point agarose has proven to be useful to mount the embryo in the measurement chamber. To image live embryos, it is also necessary to keep the temperature constant and at a level at which the embryo can still grow. To monitor the time points of gene expression as well as their organization, regulation, and function, the zebra fish is imaged during its growing process over periods from several hours up to several days. The TriM Scope II provides all necessary components for this experiment. Multiple laser lines (Ti:Sa, OPO, tunable wide band lasers) can be coupled into the microscope to excite the different dyes. To detect the different emitted wavelengths, the Spectral Detector can be used.
This allows the detection of up to six different tagged genes at once. This means it is possible to detect the specific time points of gene expression. The ability to see the right gene in the field of view can be increased by using LaVision BioTec’s Horizontal 2-photon microscope. This new 2-photon imaging configuration uses a horizontally mounted objective to image the sample from the side on top of a small tip which can be rotated by 360°. This rotation allows the operator to easily find the best angle of view for the region of interest. For imaging, a zebra fish embryo can be embedded in a cylinder of agarose that is fixed on top of the tip. The same procedure is applicable to adult zebra fish. However, large samples can also be held by using a tweezer tip.
The setup then enables to image the fish from opposite sides by rotating the tip by 180°. This dual hemisphere measurement increases the imaging depth by a factor of two. Another application for the Horizontal 2-Photon Microscope uses the brainbow labeling strategy to observe the development of neurons in, for example the nervous system of zebra fish. Using brainbow labeling, individual neurons can be distinguished. A genetic construct is used that has different fluorescent protein genes implemented. After Cre-Lox recombination, different ratios of the fluorescent proteins are randomly expressed in neurons meaning each is labeled with a different color. Using the TriM Scope II or the Horizontal 2-photon microscope, the fluorescent proteins can be excited simultaneously. By detecting and distinguishing the different colors with the Spectral Detector, the formation of the nervous system can be investigated in detail.
Transgene Zebra fish embryo expressing gsc:GFP, and injected with H2b-mCherry at one-cell stage. The expression of gsc starts at 4 hpf, and remains at the membrane of the cell. The embryo has been imaged in two steps, at the angle 0° and at the angle 180°, with an overlap of 40µm for each view to facilitate the reconstruction. The two acquisitions don't exceed 2m30s to not miss a mitosis.
Studying processes in the vertebrate morphogenesisnt stages of development
The zebra fish is a suitable model organism for studies of cell adhesion, migration, and polarization. It allows observing the molecular and cellular mechanisms during the embryo’s development.
For example, the process by which zebrafish organs are formed and which cell shape changes or cellular rearrangements are involved in the formation can be investigated. The embryo is mounted in a low concentrated agarose solution in which the fish can still grow and is sufficiently fixed to be imaged successfully. To distinguish the different interesting parts in the sample, e.g. cell membrane and cell nuclei, these have to be labeled with different fluorescent dyes.
The TriM Scope II delivers all necessary components to excite the different dyes and detect their fluorescence with the Spectral Detector. The performance of time-lapse experiments allows the tracking of cells and observation of their development over time. Besides the time-lapse measurement, 3D stacks of images can be recorded to observe the cell movement in the whole tissue. To obtain images with comparable intensities at different depths in the tissue, the TriM Scope II software enables the adapting of the excitation intensity profiles to the imaging depth. These can be adapted with different profiles (e.g. linear, quadratic, exponential) depending on the type of tissue.
 Compagnon, J., Barone, V., Rajshekar, S., Kottmeier, R., Pranjic-Ferscha, K., Behrndt, M., Heisenberg, C.P., The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ, Dev Cell. (2014), 31(6), 774-83