examples: add droplet fusion analysis

docs/examples/droplet_fusion/droplet_fusion.rst

@@ -0,0 +1,252 @@
+.. warning::
+    Disclaimer: this Notebook serves as an example; the steps will likely have to be adjusted to optimize them for your personal workflow.
+
+Droplet fusion
+==============
+
+.. only:: html
+
+    :nbexport:`Download this page as a Jupyter notebook <self>`
+
+.. _droplet_fusion:
+
+Analyzing a droplet fusion event
+--------------------------------
+
+The data in this notebook were acquired by moving two fluorescently labeled RNA droplets together at a constant speed.
+The droplets were held by optical tweezers and the right tweezers is moving while the left one is static.
+As the droplets come close together, they fuse to form one, larger droplet.
+The relaxation time of the fusion event, 𝜏, together with the radii of the droplets can reveal something about the material properties of the droplets;
+when plotting 𝜏 vs the average droplet radius for many droplets, the slope is given by 𝜂/𝛾, viscosity (Pa*s) /surface tension (N/m), assuming a Newtonian fluid [2]_. 
+The ratio 𝜂/𝛾 is also known as the inverse capillary velocity.
+
+Both the PSD signal (usually used to determine force exerted by the optical tweezers) and a scan were recorded during the experiment.
+The PSD signal has a much higher time resolution than the images, therefore it is best to use PSD signal to determine the relaxation time of the fusion event. 
+In this Notebook, we will first obtain the relaxation time from the PSD signal and then estimate the size of the droplets from the scan.
+
+Download the droplet fusion data
+--------------------------------
+
+The droplet fusion data are stored on zenodo.org.
+We can download the data directly from Zenodo using the function :func:`~lumicks.pylake.download_from_doi`.
+The data will be stored in the folder called `"test_data"`::
+
+    filenames = lk.download_from_doi("10.5281/zenodo.12772709", "test_data")
+
+Relaxation time of fusion event
+-------------------------------
+
+First, plot the PSD signal using Pylake. 
+Since the assumptions underlying force calibration are not met during the fusion event, the absolute value of the force is not reliable, and we label the y-axis as 'PSD signal'::
+
+    f = lk.File("test_data/Droplet_fusion_data.h5")
+    plt.figure()
+    f["Force HF"]["Force 2x"].plot()  
+    plt.ylabel("PSD signal (a.u.)")
+    plt.show()
+
+.. image:: force_signal.png
+
+The jump in the signal after 5 seconds shows the typical exponential relaxation for a droplet fusion event.
+
+Select data for fit
+^^^^^^^^^^^^^^^^^^^
+Below we are selecting the force and trap data at the fusion event. When fitting the fusion relaxation time, 
+it is important that the traps holding the droplets are either both static, or one of the traps is moving at a constant speed. 
+We plot the trap position over time to check which of these conditions is met::
+
+    start = "5.4s"
+    stop = "5.9s"
+
+    force_selection = f.force2x[start:stop]
+    trap_selection = f["Trap position"]["1X"][start:stop]
+
+    plt.figure()
+    plt.subplot(2, 1, 1)
+    force_selection.plot()
+    plt.ylabel("PSD signal (a.u.)")
+    plt.subplot(2, 1, 2)
+    trap_selection.plot()
+    plt.ylabel(r"x-coordinate ($\mu$m)")
+    plt.tight_layout()
+    plt.show()
+
+.. image:: selected_data.png
+
+Model for fusion
+^^^^^^^^^^^^^^^^
+
+The force data during the fusion event is fitted with the following equation: :math:`f(t) = ae^{-t/\tau}+bt+c`
+
+The term :math:`bt` accounts for the movement of the trap, assuming a constant trap speed [1]_. 
+(When both traps are static, the term :math:`bt` should be removed from the model.)
+The parameter of interest is :math:`𝜏`, the relaxation time scale of the fusion event::
+
+    from scipy.optimize import curve_fit
+
+    def relaxation_model(t, tau, a, b, c):
+        return a * np.exp(-t / tau) + b * t + c
+
+Fit the data and plot the result::
+
+    time = force_selection.seconds
+    force = force_selection.data
+
+    popt, pcov = curve_fit(relaxation_model, time, force, [0.1, force[0], 0, 0])
+    plt.figure()
+    plt.plot(time, force)
+    plt.plot(time, relaxation_model(time,*popt), label=fr"$\tau$ = {popt[0]:0.2f}s")
+    plt.ylabel(r"x-coordinate ($\mu$m)")
+    plt.xlabel("Time (s)")
+    plt.legend()
+    plt.show()
+
+.. image:: fit.png
+
+The array :math:`popt` contains all the fitted parameters::
+
+    >>>> print(popt)
+
+    [ 0.04754597  5.39347958 -4.5188036  -6.2716303 ]
+
+The first parameter in :math:`popt` is :math:`𝜏` and the other 3 parameters are :math:`a`, :math:`b` and :math:`c` respectively, as defined in the model above.
+The matrix :math:`pcov` is the covariance matrix and the standard deviation errors in the fitted parameters can be obtained as:: 
+
+    >>>> np.sqrt(np.diag(pcov))
+
+    [0.00021354, 0.01104559, 0.0218276 , 0.00779505]
+
+The relaxation time obtained from the fit is 0.0475 +- 0.0002 seconds. 
+
+Now, we will proceed to determine the size of the droplets before the fusion event.
+
+Droplet size
+------------
+
+First load the scan and print the relevant metadata::
+
+    >>> for name, scan in f.scans.items():
+    >>>        print(f"num frames: {scan.num_frames}")
+    >>>        frame_duration = (scan.frame_timestamp_ranges()[0][1]-scan.frame_timestamp_ranges()[0][0])/1e9
+    >>>        print(f"frame duration: {frame_duration} s")
+
+    num frames: 27
+    frame duration: 0.4977664 s
+
+Plot a frame before the fusion event::
+
+    framenr = 2
+    plt.figure()
+    scan.plot(channel="green", frame=framenr)
+    plt.show()
+
+.. image:: frame2.png
+
+Plot a frame after the fusion event::
+
+    framenr = 6
+    plt.figure()
+    scan.plot(channel="green", frame=framenr)
+    plt.show()
+
+.. image:: frame6.png
+
+If the droplets are in focus, the size of the droplet can be estimated from the 2D scan. 
+The estimate has limited precision because the sphere edges in the scanned images are not very sharp.
+For experimental data such as the one used in this notebook, we would expect an error on the order of ~10%.
+
+The first step, is to use image segmentation to identify the two droplets in the image. 
+The threshold may need to be optimized for your data::
+
+    from skimage.measure import label, regionprops
+
+    framenr = 2  # Choose a frame before the fusion event on which you want to identify and measure droplets
+
+    image = scan.get_image(channel='red')[framenr]
+    image = image / np.max(image)
+    threshold = 0.5
+    blobs = image > threshold
+    label_img = label(blobs)
+
+    plt.figure()
+    plt.subplot(2, 1, 1)
+    plt.title("original, normalized image")
+    plt.imshow(image)
+    plt.subplot(2, 1, 2)
+    plt.title(f"Identified objects")
+    plt.imshow(label_img)
+    plt.tight_layout()
+    plt.show()
+
+    if (blobs := len(np.unique(label_img))) != 3:
+        raise RuntimeError(f"Expected 2 blobs, found {blobs - 1} instead! Maybe adjust the threshold?")
+
+.. image:: image_segmentation.png
+
+For this scan, the fast axis is along the horizontal coordinate (you can check the direction of the fast axis by typing :attr:`scan.fast_axis <lumicks.pylake.scan.Scan.fast_axis>`). 
+Therefore, we estimate the size of the droplets by looking at the width of the identified objects:::
+
+    def get_center_and_width(scan, mask, axis):
+        """Grabs the center and width along the fast scanning axis"""
+        widths = np.sum(mask, axis=axis)
+        max_width = np.max(widths)
+
+        # Grab the position
+        coordinate_weighted_mask = np.indices(mask.shape)[axis] * mask
+        centers = np.sum(coordinate_weighted_mask, axis=axis) / np.clip(np.sum(mask, axis=axis), 1, np.inf)
+        
+        # Since some scanlines can have the same width, we'd want the vertical position to be the average of these
+        max_scanline = int(np.mean(np.nonzero(max_width == widths)[0]))
+
+        if axis:
+            center = (centers[max_scanline], max_scanline)
+        else:
+            center = (max_scanline, centers[max_scanline])
+        
+        return center, max_width
+
+
+    def plot_width(scan, center, width, axis):
+        plt.plot(center[0], center[1], "ko")
+        if axis:
+            plt.plot([center[0] - 0.5 * width, center[0] + 0.5 * width], [center[1], center[1]])
+        else:
+            plt.plot([center[0], center[0]], [center[1] - 0.5 * width, center[1] + 0.5 * width])
+
+    droplet_radii = np.array([]) 
+
+    fig, ax = plt.subplots()
+    ax.imshow(image, cmap=plt.cm.gray)
+    plt.xlabel("x (pixels)")
+    plt.ylabel("y (pixels)")
+    axis = 1 if scan.fast_axis == "X" else 0
+    center, width = get_center_and_width(scan, label_img == 1, axis)
+    droplet_radii = np.append(
+            droplet_radii, 0.5* width)
+    plot_width(scan, center, width, axis)
+    center, width = get_center_and_width(scan, label_img == 2, axis)
+    droplet_radii = np.append(
+            droplet_radii, 0.5* width)
+    plot_width(scan, center, width, axis)
+
+    plt.title("Width along the fast axis")
+    plt.show()
+
+.. image:: droplet_size_pixels.png
+
+The array `droplet_radii` contains the radii of both droplets in the image, in pixels. 
+Below we are multiplying this array by the pixel size in micron to obtain the radii in micron::
+
+    droplet_radii_um = droplet_radii * scan.pixelsize_um[0]
+
+The radii for the droplets in micrometers are::
+
+    >>> print(droplet_radii_um)
+
+    [1.15 1.1]
+
+We now determined the relaxation time as well as the droplet radii. The next step is to measure these two quantities for many different fusion events, plot 𝜏 vs average radius and determine the slope.
+
+
+.. [1]  Patel A. *et* al, A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation, Cell (2015).
+.. [2]  Brangwynne C.P. *et al*, Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation, Science (2009).

docs/examples/index.rst

@@ -21,4 +21,5 @@ For all of the examples, it is assumed that the following lines precede any othe
     reca_fitting/reca_fitting
     cas9_kymotracking/cas9_kymotracking
     hairpin_fitting/hairpin_unfolding
+    droplet_fusion/droplet_fusion
     bead_coupling/coupling