Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

The overall goal of this procedure is to obtain quantitative images of treated prostate tumor cells to measure the degree and extent of autophagy at different time points. This is accomplished by first treating cells with arginine, dase, or other experimental reagents to induce autophagy and or cell death. The second step is to decide whether to image live cells or fixed cells and to prepare samples accordingly.

Next, obtain 3D fluorescence images of either live or fixed cells using widefield, deconvolution or structured illumination fluorescence microscopy. The final step is to collect statistical data on autophagosome size distribution and colocalization with other intracellular structures. Using digital 3D image analysis software, this approach can obtain results that show not only the auto phage induction by arginine dase can cause prostate tumor cell death, but also that these cells exhibit structural changes that are morphologically distinct from changes associated with apoptosis and necrosis.

The main advantage of this technique over other currently existing methods is that quantitative 3D imaging can show when and where a specific molecular event occurring inside a living cell and then present it in a three dimensional format To begin grow human prostate cancer cells expressing green fluorescent protein, coupled light chain three on 35 millimeter poly de lycine coated glass bottom culture dishes as described in the text protocol cells should be plated at sufficient density to facilitate rapid proliferation, but not so much that cells are overgrown and clumped by the time of imaging. Treat selected cell samples with arginine dase or a DI in PBS to deplete cells of free arginine and induce metabolic stress in the cancer cells approximately one hour prior to imaging dilute 1.5 microliters of Lyo tracker red with 20 milliliters of RPMI containing 10%FPS and 1%antibiotics. Prepare solutions with a DI for the selected samples that were treated after warming all media to 37 degrees Celsius.

Add the appropriate media to each culture dish incubate cells with RPMI containing LYO Tracker red for 15 to 45 minutes at 37 degrees Celsius, approximately 30 minutes prior to imaging. Turn on the weather station environmental enclosure and allow equilibration to 37 degrees Celsius and 5%carbon dioxide wash cells with PBS and replace media with standard RPMI containing only 10%FBS and 1%antibiotics. Add a DI to samples as indicated.

Mount 35 millimeter cover glass bottom culture dishes in a customized adapter. Use immersion oil on the six DX magnification, 1.42 numerical aperture objective lens and position the mounted culture dish on the microscope stage. In this video, 3D microscopy is demonstrated using the Delta vision personal IDV applied position, deconvolution microscope and Associated Soft Works application suite.

To begin turn on the microscope system, including acquisition workstation and instrument controller. Open the resolve 3D computer control application to initialize the microscope stage and turn on the xenon light source. Allow 10 minutes for the light source to warm up and reach stable conditions.

Add a drop of immersion oil on the six DX magnification, 1.42 numerical aperture objective lens and center the slide specimen with cover slip face down over the objective lens using either brightfield or external illumination as well as the coarse focus. Adjust knob slowly raise the objective lens until the beat of immersion oil makes contact with the inverted cover glass. From this point, the cell layer should come into focus within a few turns of the fine focus adjust knob.

When working with fixed samples on the slides, it is recommended to avoid cells within or near any areas with bubbles. When viewing with live samples on the glass bottom dish, avoid moving the objective lens outside of the boundary of the glass cover slip. Select the desired field of view.

Ideally, cells should exhibit good fluorescent signal in all the appropriate channels and be sufficiently attached and spread out on the cover slip surface so that the intracellular contents are easy to visualize. Small adjustments in lateral X, Y, and Z focus can be controlled by the computer using the acquire 3D interface. Set benning to one by one or two by two depending on the desired field of view for a given image through the midsection of a cell, set the exposure parameters such that the maximum pixel intensity does not exceed 3000 counts.

Generally percent transmission should be set as low as possible without raising the corresponding exposure. Time over one second. Repeat this procedure for every fluorescence color to be imaged and for each separate field of view.

To obtain the highest quality images, set the upper and lower limits of the Zack by adjusting the focus just slightly over the top and bottom of the cell sample respectively. The total number of images in a given Z stack will thus be determined by these limits and by the spacing between layers. To minimize motion artifacts during image acquisition, the image acquisition mode can be set to wavelength.

Then ZS stack for fixed samples and ZS stack then wavelength for live samples. After all parameters are set, the fluorescence images can be acquired. Fluorescence images are then devolved using soft works and later analyzed using velocity.

The image sequence shown here shows the physical changes that occur in cwr 22 cells during the first 80 minutes of autophagy induction. In these images, the white dotted line represents the outline of the cell and the light shaded region represents the position of the nucleus. Over the course of 80 minutes, the images reveal displacement of the nucleus away from the cell center, reduction of focal adhesion points and general translocation of autosomes and lysosomes towards the center of the cell, displayed here in green and red respectively at later time points.

A small increase in colocalization was also observed between autosomes and lysosomes as indicated by the yellow color. The velocity Digital imaging application suite was then used to identify, count and collect statistical data on labeled autosomes and lysosomes in the images of the CWR 22 cells. Shown here the number and size of the autosomes after their initial formation at appearance do vary with time.

After 80 minutes of autophagy induction, there was a gradual button net decrease in the number of autosomes with a corresponding increase in the average size of the autosomes. In addition, there was a measurable increase in the colocalization of the autosomes and lysosomes based on Pearson's correlation coefficient analysis. Together, these findings suggested that upon stimulation of autophagy, numerous small autosomes fsed to form larger autosomes over time.

Shown here is a side-by-side comparison of images acquired and reconstructed in DV mode, simulated widefield deconvolution versus structured illumination mode. Using the OMX microscope lateral resolution was improved to up to 120 nanometers twice the resolution of conventional diffraction limited microscopy. Small scale colocalization of autosomes and lysosomes were clearly apparent with super resolution microscopy, whereas they were hardly evident with conventional fluorescence imaging.

One of the advantages of using deconvolution microscopy is to reconstruct all images into a 3D model that will reveal more accurate spatial information between different molecules. Shown here is an overall picture of a Cwr 22 RV one cell undergoing autophagy at a later time point where a significant amount of colocalization between autosomes and lysosomes begins to occur. The e coherent staining as indicated in white color, reveals the outline of the target cell in this movie.

The 3D reconstructed model provides more spatial details of the fusion between autosomes and lysosomes. As indicated in yellow color, the interaction between green light chain three signals and red lysosome signals is evident in the presence of the merged signals to produce yellow. So this approach will address two major challenges for researchers with studies autophagy.

First of all, we want to maintain a stress-free environment by using micro fluid, the profusion chamber to maintain its original physiological condition on the microscope stages. The second challenge is to collect statistically relevant quantitative image data and for the fixed cells we usually take a lot of images from different cells and compile them into useful information. However, with life cell, we just follow one cell over time to acquire equivalent information.

So we believe this technique will enable other researcher to study the function and the role of autophagy in different organ and different diseases.