The overall goal of this imaging method is to study subcellular organelles in a vertebrate in vivo. This method can help to answer key questions in the field of neuronal cell biology, such as dynamics of mitochondria, or centrosomes in an in-vivo context. The main advantage of this technique is that the entire experiment can be performed in an intact vertebrate, without the need for any surgical procedure.

This method can provide insight not only to subcellular dynamics under physiological conditions, but it can also be applied to studies of thesis models, such as Alzheimer's disease. After generating transiently-expressing embryos by microinjection, or obtaining embryos from stable transgenic lines, according to the text protocol, at the end of day one, screen for dead or unfertilized eggs. Using a plastic transfer pipette, transfer the viable embryos to Danieau's solution containing 1x PTU to prevent pigment formation, and maintain the embryos in Danieau's solution containing 1x PTU until screening for transgene expression.

On the day of the imaging experiment, use a fluorescence dissecting microscope to screen anesthetized embryos for transgene expression. Then transfer the selected embryos to a separate petri dish containing Danieau's buffer with 1x PTU and 1x tricaine. Once the embryos have been anesthetized, gently pipette a few into the low-melting agarose containing 1x PTU and 1x tricaine, transferring as little liquid as possible to avoid diluting the agarose.

Transfer the embryos with a small amount of agarose to a glass-bottom petri dish. Then, working relatively fast, use forceps to position the embryos in the desired orientation depending on the structure to be imaged. The embryos have to be orientated properly for example, flat on the side for imaging the retina or a Rohon-Beard neuron.

A precise orientation will ensure the best imaging quality and optimize the acquisiton speed. After allowing the agarose to solidify for at least 15 minutes, add Danieau's buffer with 1x PTU and 1x tricaine to cover the embryos. To carry out wide-field microscopy, after equilibrating a dish of mounted fish in a heating chamber at 28.5 degrees Celsuis, on the stage of a wide-field microscope, and choosing an area of interest, open the microscope software.

Under Illumination, click on Configuration settings to define the correct filter sets for the wavelength of interest. Under Camera settings, enter the exposure time required to acquire a suitable image. Then, choose a long working distance water dipping cone objective, ranging from 40x to 100x magnification, that has the highest numerical aperture and is chromatically corrected.

Click on Live to choose the field of view. In the case of an R-B neuron, image mitochondrial transport at the stem axon, emanating from the cell body or in the peripheral arbor. Then, in the ImageJ menu, click on the rectangular selection button and define a region of interest, or ROI.

And click on ROI in the micromanager window. After selecting the ROI for imaging, click Stop and Save to record the neuronal morphology in the YFP channel. To image mitochondrial transport, click on Multi-D Acquisition.

Then, in the window called Multi-dimensional Acquisition, select the number of time points and the interval between time points. In the Multi-dimensional Acquisition window, click on Channels, and add and define the wavelength for imaging and the time of exposure. Click on Save images to automatically save the files in the specific folder outlined in the directory route.

Now, click on Acquire at the upper right corner to start time lapse imaging. For confocal microscopy, after placing the mounted embryos in a heating chamber at 28.5 degrees Celsius on the confocal stage, open the microscope software and click on Trans Lamp or Epi Lamp. Then, via the oculars of the microscope, use either transmitted or fluorescence light respectively to identify the region of interest.

In the Acquisition setting window, verify that the objective chosen for imaging matches the objective that appears in the dropdown menu of the available objectives. Then, in the Image Acquisition Control window, click on the dye list button and select the appropriate fluorophores. Alternatively, click on the Light Path and Dyes button to manually set the parameters.

In the Acquisition Setting window, adjust the zoom factor and size aspect ratio of the scanned image to obtain the pixel size necessary to best resolve the structures being imaged. Set the pixel size to be about half the theoretical resolution of the objective, thereby following Nyquist's sampling criteria. To determine the pixel size of an acquired image, in the Image Acquisition Control window, click on the I button.

Next, set the scan speed to the fastest possible. Then, in the Image Acquisition Control window, click on Kalman line averaging and set to a factor of two or three to reduce noise. To select a sequential scanning mode for imaging fluorophores with overlapping spectra, click on Sequential and Line.

Then, adjust the power output of the relevant laser lines. To continuously scan the selected region while adjusting the detector settings for each channel, click on XY Repeat or the Focus x2 or Focus x4. Then, adjust HV, Gain and Offset for each channel to acquire images that have the highest dynamic range of gray values.

Now, press Control plus H to visualize the acquired images via a lookup table that identifies undersaturated pixels in blue, and oversaturated pixels in red, both of which should be avoided. Reevaluate the power output of the relevant laser lines, and the detector settings. To define the upper and lower limits of the volume to be imaged, in Acquisition Setting click on the End Set button at the upper limit and then focus to the lower limit and press Start Set.

To check the Z resolution of the objective, in Image Acquisition Control, click on the I symbol. Then select a step size that is half of the Z resolution for the given objective. In the TimeScan sub-window, enter the frequency at which Z-stacks should be acquired, and the number of times the images should be acquired, to set up a time series appropriate for the dynamics of the subcellular structure to be imaged.

Click on the Depth and Time buttons to confirm that the Z-stack and time series will be acquired. Finally, click on the XY Zt button to begin acquiring time lapse images. After image acquisition, on the software interface, click on Series Done.

And save the images in OIB format to record the images and associated metadata. This figure shows organelles and R-B neurons located on the surface of the embryo using wide fields and confocal microscopy. Here, time lapse imaging on a wide field microscope was used to track the movement of an individual mitochondrium in the peripheral arbor of an R-B neuron.

Confocal imaging is significantly slower and can lead to an underestimation of the dynamics of specific subcellular events. Images of retinal cells acquired by wide-field microscopy suffer from poor contrast, as fluorescent signals from a large volume of tissue obscures detail in the focal plane. Here, the optical sectioning capability of confocal microscopy noticeably improves the contrast.

The insets show details of a region of the inner nuclear layer, with labeling of cellular membranes and mitochondria. Driven by a sensory neuron's specific Gau-4 driver, the mosaic expression from the injections of UAS-mito-CFP and UAS MAY-FP permit the tracking of individual cells over days, as seen in this R-B sensory neuron at two and three days post fertilization. In addition to mitochondria, other subcellular organelles such as centrosomes, can be fluorescently labeled.

In this example, two UAS cassettes on a single contiguous construct drove the expression of centrosome-targeted YFP, and membrane-targeted cerulean in retinal cells. The insets show a cell in M phase. This entire procedure requires several days from mating, egg collection, injections, and then finally imaging on three or four days post-fertilization.

The actual imaging experiment on the microscope can take several minutes to an entire day. Following this procedure, other organelles like microtubules or peroxisomes can be labeled and imaged in order to understand their in-vivo dynamic. Don't forget that PTU is carcinogenic and precautions such as wearing gloves should always be taken while performing this procedure.