The three aquatic model organisms mentioned above, zebrafish, medaka, and Xenopus, are often used in molecular and developmental biology. An adult zebrafish is shown below.
In molecular and developmental biology, these aquatic vertebrate model organisms are widely applied to study molecular processes of development and as disease models. To study these molecular mechanisms, proteins of interest are fluorescently labeled and observed in the developing organism at the cellular or sub-cellular level over the course of hours or days .
All three model organisms described here can be easily bred and maintained in a laboratory, have short life cycles, and are amenable for genetic modifications. Examples of these modifications are the deletion of a gene (knock-out) or the introduction of a gene (knock-in). If an exogenous gene is introduced into the genome, the result is a so-called transgenic organism. Below, we will focus on this method .
In addition, zebrafish have a specific trait that also make them useful for developmental neuroscience: the larvae of zebrafish are semi-transparent so the activity of multiple neurons can be measured simultaneously during development .
There are three common steps when doing routine work with aquaticmodel organisms, such as zebrafish:
- fluorescent screening
- functional imaging.
A more detailed description of each work step is given in the section below. Efficient and reliable microscopy is needed for each of these. This sequence of steps will be referred to as "workflow" in this report.
Most countries have well defined regulations for animal safety when used for scientific experiments. Switzerland has such regulations as well . To adhere to these regulations, it is advantageous to have efficient and fast screening of transgenic embryos and rapidprocessing of the adult zebrafish which generated the embryos.
As individual adult zebrafish cannot be permanently labeled, at least not at the present time, males and females that are cross-bred, to assess their embryos while screening for transgenics, need to be kept in individual holding tanks until their embryos are well characterized. The faster the embryos’ traits can be determined:
- the sooner the adults can be put back into proper housing tanks
- the number of individual tanks in the facility, and the amount of work for the staff, can be minimized
- and only zebrafish with the desirable traits would be maintained, avoiding the need to keep unnecessarily high numbers of fish for experimental work.
Faster, accurate characterization of the zebrafish embryos leads to a more efficient, cost-effective way to maintain these model organisms.
There are two key factors for fast and accurate embryo characterization:
- efficient fluorescence detection of sometimes dimly glowing transgenes
- and an efficient, convenient way of imaging the embryos for screening.
In practical terms, fluorescence microscopes that detect weak florescence signals and make them visible to the eye in an uncomplicated way are the optimal tools to achieve this goal.
Genetic modifications in zebrafish, medaka, and xenopus are typically carried out by microinjection of DNA, RNA, or dyes (as for plasmids, mRNAs, morpholinos, siRNAs, etc.) . These manipulations are efficiently supported by the optical magnifications achievable with a stereo microscope, such as the Leica M50, Leica M60, or Leica M80 . If DNA is injected into a cell and incorporates into the genome (transgene), this results in a transgenic animal.
As the organism develops into the larval stage, successful integration (into the genome) and expression of the transgene is evaluated. A part of the transgene is usually a gene for a fluorescent protein, such as green, red, or yellow fluorescent protein . Therefore, screening of potentially transgenic larvae is commonly done with a fluorescence stereo microscope, such as the Leica MZ10 F , Leica M165 FC, or Leica M205 FA .
An example of functional imaging is electrophysiological investigation via Ca2+ signaling in various types of cells. Injection of an organism with synthetic Ca2+ indicators , frequently using a micromanipulator, enables studies of neuronal activity in neurons and glial cells. Calcium indicators can also be genetically expressed and imaged in intact or semi-intact organisms due to the semi-transparent nature of developing zebrafish larvae. These experiments are frequently done using multiphoton fluorescence microscopy .
During development, the organism is often imaged with a stereo microscope and, in some cases, manipulation and preparation for further experiments is performed with it as well. When only a 2D view is required, imaging is performed using a macroscope, such as the Leica Z6