The overall goal of this procedure is to obtain fluorescence movies of growing single yeast cells and extract reporter expression time series for each cell. This is accomplished by first culturing a monolayer of cells in a microfluidic chamber. The second step is to perform time-lapse fluorescence microscopy for one or more reporters.
Next, the movies are processed using the graft software package to track and measure cells through time. The final step is to analyze the cell measurements and lineage assignments and to export reporter expression time series for each whole cell. Ultimately, an instantaneous transcription rate inferred from expression time series is used to show changes in transcription both over the cell cycle and in response to changing transcription factor levels.
This method can help answer key questions in the gene regulation field, such as how quickly do different promoters respond to the sudden addition or some subtraction of an active transcription factor. How variable is this response across a population of cells and how do different cellular characteristics including morphology and cell cycle phase influence the response? Moreover, this method can probe the effects of multiple additions and subtractions of an active transcription factor on gene expression.
Prepare yeast cells growing at a nutrient-rich, steady state at a density suitable for loading into the microfluidic device as described in the text protocol. Then prepare a Y zero four C microfluidic culture plate from cell ASIC by rinsing the wells with sterile water following removal of the shipping solution using the onyx perfusion system flow water through wells one through six at six PSI for five minutes to flush the channels, then flow from the loading well eight at six PSI for 10 seconds. Remove water from all wells and replace with 250 microliters of SC media in inlet wells, one through six and 50 microliters of the prepared cell culture in cell loading.
Well eight seal the onyx manifold to the plate and begin priming the channels and culture chamber by flowing from Inlet Wells one through six at six PSI for two minutes. Follow this with flowing from only the inlet well holding the starting media for at least five minutes. At six PSI flow this continually until the microscope step tape the plate to the stage mount to prevent its shifting throughout the experiment in order to improve cell tracking during data analysis, following preparation of the microscope as described in the text protocol, placed a few drops of immersion fluid on the objective and mount the microfluidic device securely.
In the stage of the inverted microscope, focus on the leftmost third of the first culture chamber using one of the embedded position markers. Turn off flow from the media inlet well and flow from cell loading well eight at six PSI In five second bursts. Move the stage to look around the culture chamber for cells.
Increase loading flow time and pressure until desired cell density is achieved, but avoid overloading and clogging the leftmost barrier where fresh media enters the chamber. Begin flowing from the media inlet well containing the starting media at six PSI. In Metamorph or other microscopy automation and control software.
Set up a multidimensional acquisition to take multiple images at multiple stage positions over time. Set the number of time points and interval between them. Then select several stage positions with approximately 10 cells.Each.
Set up the acquisition so that at each stage position and at each time point, the program will focus on the transmitted light brightfield image using metamorphs built-in autofocus method, implementing the autofocus as a custom written journal at the start of each stage. Position provides greater control over focus accuracy. Also set the program to acquire the brightfield image at plus one micron to the focal plane and to acquire a brightfield out of focus image at minus four microns to the focal plane.
Use custom written journals to change the focus as required between images. Finally, set the program. Acquire one gray scale image for each fluorescent reporter at the focal plane.
Using settings optimized for rapid acquisition with minimal photo bleaching. Prepare the flow program for the experiment in the onyx control software. Then simultaneously begin the acquisition program in Metamorph and the flow program in the Onyx control software.
Run the format data M file in MATLAB to open the data input graphical user interface or GUI. At the top, specify or browse to the folder containing the image files to set the data directory and then use the gooey to specify the data types and image files recorded in the experiment. Proceed to select the segmentation parameters as described in the text protocol.
Choosing the proper parameters for segmentation of brightfield images can be difficult and has a significant effect on the resulting time series data quality. We focus on identifying all cell regions with preference given to over segmenting rather than under segmenting regions. During visual data inspection, over segmented regions can be merged together to form whole cell regions, but under segmented regions cannot be split Later.
Set the different segmentation parameters and test the segmentation quality by clicking test Successfully. Segmented regions will be green with magenta borders. Be sure to use the slider to check multiple time points in the movie when the segmentation is satisfactory.
Select done to return the segmentation parameters to the format data gui. After selecting the color parameters as described in the text, push the auto threshold button to automatically determine the threshold. Using tzu's method, the image will highlight the areas preserved above the threshold.
The threshold can be manually adjusted using the edit box. Use the scroll bar to confirm the threshold is appropriate for all time points. When satisfied, push done.
To return the threshold to the format data gui, press add and segment. To create the color mask, press format data to read the image files, process the data and create a dot mat file in the specified folder. This file will serve as the input to the process time series gui.
Run the process time series M file in MATLAB to open the tracking gui. After opening the gui first set the chamber height. This indicates the height of the chamber in which the cells are trapped.
It is used as a constraint in approximating cell volume as a 3D ellipsoid based on the major and minor axis of the cell region. Then set micrometers per pixel. This is the conversion factor used to calibrate pixel to micrometer distance in the images so that cross-sectional area and volume can be calibrated in square micrometers and cubic micrometers respectively.
Next, use the filters panel to set the minimum and maximum parameters to filter out junk regions in the mask. The area is the region area in pixels. ECC is the eccentricity factor, which is more circular as the value approaches zero.
And SF is the shape factor, which is more circular as the value approaches one click load images in the file input output panel and select the dot mat data file of interest output by the format data gui. The region mask will be applied to each color channel and a number of different measurements are made. The data file is then saved after selecting the track cells parameter as described in the text.
Click track cells to track regions from one frame to the next using the region OIDs to assign lineages for newly appearing buds and to save the data, change the parameters in the pop-up windows as necessary. Lineages are automatically assigned by minimizing distances between putative buds and potential mothers. In each frame.
Click the button above the selected cell information panel and curate poorly segmented regions and mistakes in ID or lineage assignments using the various gooey tools or shortcut keys which are described in the text protocol to properly curate the data. Merge any over segmented regions, delete any regions not capturing the whole cell and be sure mother bud relationships are properly assigned. Also, eliminate any green IDs by merging regions.
Fixing the id, assigning the region to a mother, assigning no mother or deleting the region. Since bud data will be added to that of the mother during the final, final analysis. Do not merge a bud region to its mother.
Visually inspect data for each frame up to the last time of interest. It is not required of the entire time series. If not using the later frames, be sure to save changes once all cell regions have been inspected over the time span of interest data for all fluorescence channels and how they change over time are ready to be output.
Push time series analysis to analyze the time series for whole cells over time. Calculate cell cycle information and take derivatives. A window will open allowing selection of the measurements of interest and input of analysis parameters Enter the last time point curated in the gooey.
The default smoothing and cell cycle assignment parameters work well for haplo and diploid strains imaged in five minute intervals, but these may need to be varied for best results. When all parameters are set, select analyze to export single cell time series data as described in the text protocol, A successfully performed and analyzed experiment will yield mostly continuous time series for single whole cells with realistically assigned but emergence and division times for a halo yeast strain expressing an integrated copy of Ian fluorescent protein or CFP driven by the constituent of a DH one promoter. Whole cell measurements over time were obtained from the time series analysis.
The brightfield images show the segmented mother cell in blue and its buds in green. With the contiguous whole cell outlined in red, a dependence on the cell cycle emerges for both volume and expression. Here, grated regions show when the cell is in SG two M and has a bud budding begins at the G one to S transition from white to gray.
Shading and cell division occurs at the M to G one transition from gray to white shading after budd formation. The total combined volume increases more quickly than before, but emergence while protein concentration or average intensity decreases slightly. The rise in combined integrated protein also accelerates after budding comparing to the mother region alone.
These results demonstrate the importance of properly incorporating bud contributions to the whole cell measurement and highlight the need for proper bud formation and division time assignments. The differentiated time series can be used to calculate an instantaneous relative mRNA level and transcription rate, which both also increase after budding the ability to generate stable time. Derivatives of measurement time series also benefits kinetic studies using the switchable expression system described in the text protocol.
A time series analysis shows when the transcription factor is localized to the nucleus and when the target gene starts and stops. Transcription shown here are micrographs from the four indicated acquisition channels for a cell with a switchable YFP fused PHO four Tet R trans activator. The trans activator localizes to the RFP marked nucleus in response to low phosphate and drives expression of a seven X Tet O promoter CFP reporter.
The inferred transcription rate changes with localization on average and at the single cell level. Following this procedure, other analyses of the single cell multidimensional time series data can be performed in order to answer additional questions, including how does expression vary across the population over time? Are the effects of extrinsic fluctuations inherited over multiple generations?
Do gene activation kinetics change upon reactivation? Does the cell cycle play a role and many other questions requiring gene expression growth and lineage trajectories of single cells.
