AlliGator: Open Source Fluorescence Lifetime Imaging Analysis in G

Abstract

Fluorescence Lifetime Imaging (FLI) is a technique recording the temporal decay of fluorescence emission at every pixel of an image. Analyzing the information embedded in FLI dataset requires either fitting the decay to a predefined model using nonlinear least-square fit or maximum likelihood estimation, or projecting the decay on an orthogonal basis of periodic functions as in phasor analysis. AlliGator is a Windows open source software (BSD license) implementing these approaches in a user-friendly graphical user interface (GUI) and offering numerous unique features such as Förster Resonant Energy Transfer (FRET) stoichiometry analysis, or the creation of maps of lifetime-derived quantities such as membrane potential. It leverages the unique ability of the LabVIEW graphical programming language (G) to design feature-rich GUI, and supports user-developed plugins written in python to extend its native capabilities.

Keywords: LabVIEW; NLSF; Python; fluorescence lifetime imaging; phasor analysis.

Figure B.5:

Overview of the relationship between VI libraries comprising AlliGator. The libraries are identified by numbers in the figure, as follows. AlliGator-specific libraries: Accumulated Dataset (12), Action Engine (2), Dataset Information Window (10), Debug (6), Decay Analysis (9), Decay Fit Parameter Map (11), Decay Statistics (18), Dual-Channel Datasets (15), Files (13), Files Tests (8), Fit Method Benchmark (49), Global Decay Fit (5), Globals, Variables & Constants (20), Graphs (23), GUI (22), HDF5 (16), Image Profile Window (7), Intensity Corrections (30), Internal Variables (27), Lifetime (31), Local Decay Window (4), Notebook (38), Phasor Harmonics (1), Phasor Calibration (28), Phasor Graph (26), Phasor Plot (32), Phasor Plot Color Map (33), Phasor Ratio (29), Python Plugins (21), ROIs (34), Scripts (24), Settings (25), Shot Noise Influence on Average Lifetime (17), Source Image (36), Test Suite (3). Non-AlliGator-specific libraries: Arrays (46), Becker & Hickl Files (48), Boolean (14), Buttons (72), Comparison (54), Error (71), Files (61), Fits (51), Formula (57), Graphs (56), GUI (66), Histograms (37), Histogram Window (53), Image (40), Math (47), Menu (64), Notebook (55), openg_array (67), openg_error (74), openg_file (62), openg_string (70), openg_variant (73), openg_variant_configuration (63), Palette (44), Phasor (45), Phasor Explorer (52), PicoQuant (39), Plot Editor (43), PTU Files (42), Rich Text Box (59), Sound (60), String (65), SwissSPAD (19), SwissSPAD Live (41), Time (68), Variant (5), Utilities (68), Variant to Data (58), XY Graph Add-Ons (35).

Figure 1:

AlliGator’s main window during the final stage of an example of FLI analysis described in section 3. The left side of the window shows a dataset after overlay of the color-coded amplitude-averaged lifetime. The right side of the window show the Phasor Plot panel in which the pixel-wise phasor plot is represented. Two references (green and red dots) used for linear decomposition of each phasor are also represented, together with the segment connecting them and the region within which linear decomposition analysis is confined.

Figure 2:

Simplified architecture of the AlliGator software. The Main AlliGator Window virtual instrument (VI), whose simplified code is shown to the left, contains a user-event loop which registers user interactions with various UI objects and passes corresponding action requests via a queue (green Q icon) to the Action Loop VI (shown on the right) running in parallel. Ancillary windows opened by the Main AlliGator Window (not shown) also send action requests to the Action Loop VI. The action loop handles scripts (arrays of action requests) in a first-in first-out order, and can insert new action requests within a script. Actions are processed by a hierarchy of sub-VIs organized into specialized libraries (e.g. file loading, phasor analysis, NLSF, etc.). Update requests to ancillary windows are handled via custom user-events. Note that in the actual code, the two pieces of code shown in the two boxes are disconnected (run in parallel), the references to the main window’s objects being passed dynamically at launch time to the Action Loop VI. A hierarchical diagram of all VI libraries comprising the software is shown in Fig. B.5.

Figure 3:

Example of AlliGator code: AlliGator Use Phasor Plot UC-Principal Axis Intersections as References.vi. This snippet computes the minor or major axis of the phasor plot and determines its intersection with the universal circle (UC), the locus of single-exponential decays in the phasor plot, and uses the two corresponding phasors as references for subsequent mixture analyses.

Figure 4:

Example of phasor analysis using AlliGator. After loading the dataset (a) and defining ROIs (b), the phasor of each ROI is calculated after calibration with the appropriate instrument response function (c). A function of AlliGator’s Phasor Graph allows computing single-exponential references as the intersection of the universal circle (blue circular arc) and the major axis of the phasor scatter plot (green and red dots connected by a dashed line. These references can then be used to decompose each phasor into a linear combination of both of them, from which the amplitude-averaged lifetime 〈τ〉 can be computed for each phasor. The histogram in (d) is the result of a two-step analysis: first, the creation of a 〈τ〉 (I) plot and next, the calculation of the histogram of 〈τ〉’s and its fitting with a Gaussian distribution. All images and graphs are direct export from AlliGator to the clipboard. The raw data for each plot can also be exported as an ASCII file for further analysis by third party software.