VistaVision
VistaVision is a complete software package for instrument control, data acquisition and data processing. VistaVision enables control of the automated devices on all Alba instruments including shutters, filterwheels, XY stages, and light detectors. A convenient signal monitor displays the signal intensity from each channel in real time, and it is utilized during instrument alignment.
The software has been developed in modular components that can be installed independently when constructing a custom-built instrument that uses ISS modular components.
The VistaVision Instrument Control module includes the routines for instrument control (instrument alignment, shutter control, selection of the light detectors); control of the Imaging Devices (galvo-mirrors, piezo-controlled stages; stepper-motor controlled stages); laser launcher (laser intensity, laser modulation); and control of microscope automation features.
The VistaVision Imaging module for image acquisition, image processing and image display allows for the user to acquire single-point data (intensity, linetics, polarization, lifetime); line data; and images. The user interface includes the acquisition parameters (pixel dwell time, image size, and the image resolution) and the selection of image type (polarization, FLIM, N&B, RICS). Images stacks are acquired in different direction (XYZ, XZY). An array of time series is available (t, Xt, XYt, XZt) for both steady-state images and FLIM.
FLIM images are acquired using either the frequency-domain (DFD) technique or time-domain (TCSPC); the user selects the modality although the instrument can be fitted with both acquisition techniques. FLIM data are processed using the minimization technique (Marquardt-Levenberg algorithm) and the phaser plots. Data are exposed as lifetime images, images of pre-exponential factors, images of fractional contributions.
The software includes operations between images, smoothing, filtering, rotation, zooming, scaling and automatic threshold setting for image contrast enhancement. Images can be exported to ImageJ and MetaMorph; plots are exported to popular formats (png, jpeg, gif, tiff, bitmap, metafile). Movies are produced in avi format.
The VistaVision FFS module includes routines for 4-channel data acquisition and data processing of up to 3 components. Data are acquired in time mode or photon mode. VistaVision features a real-time display of the auto correlation function, G(τ) - apart from a nominal delay (less than one second) required for the computation of the function. A sequence of multiple data acquisition files can be acquired (for instance, when using a microwell plate on a computer-controlled XY stage) and displayed and stored automatically.
Data files are stored in several formats:
- VistaVision acquires and stores raw data files, which can be opened at a later time and re-correlated; sections of each raw data file can be purged from the presence of impurities (for instance, when an impurity traverses the observation volume) and re-analyzed.
- The software also stores the autocorrelation function, the cross-correlation function and the photon-counting histogram data files.
- The Time Tagged Time Resolved (TTTR) format is available when data are acquired along with lifetime data (FLIM).
Analysis is performed using autocorrelation function (FCS), cross-correlation function (FCCS) or photon counting histogram (PCH, proprietary technology of ISS) with different laser-beam point spread functions (one-photon and two-photon) . Custom model functions can be input by the user and fitted by the software; the custom functions are retained in a library for future use. Multiple files can be analyzed using global analysis with either autocorrelation functions or photon counting histograms, for up to 384 files simultaneously. Plots are exported to popular formats (png, jpeg, gif, tiff, bitmap, metafile).
The FFS module, in conjunction with the Imaging module allows for: the acquisition of the following measurements:
- FFS measurement at target XYZ locations in an image
- FLCS, fluorescence lifetime correlation spectroscopy
- Scanning FCS; the simultaneous acquisition of FFS data
- Number and Brightness (N&B)
Instrument Control Module
The VistaVision configuration file allows the user to select the proper driver for a variety of components.
| Data Acquisition Cards |
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| Scanning Mirrors Modules |
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| Piezo-controlled Stages |
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| Automated Microscopes |
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| Microscopes Stages |
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| Laser Launchers |
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| Two-detector unit |
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Imaging Module
Data Acquisition
| Single point measurements |
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| Line Measurements |
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| Steady-state images (single plan and z-stack) |
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| FLIM images (digital frequency-domain) (single plane and z-stack) |
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| FLIM images time-domain (single plane and z-stack) |
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| Raster Image Correlation Spectroscopy (RICS) | The laser beam acquires an image with a dwell time suitable for the molecular dynamics to be resolved. The technique for measuring molecular dynamics and concentrations from fluorescence confocal images |
| Scanning FCS (requires FFS module) | The laser beam rotates on a circle with diameter 100-200 nm (user determined) and FFS data are acquired at set angles. At each angle an FCS curve is reconstructed. |
| Number and Brightness (N&B) | The laser beam acquires an image with a dwell time suitable for the acquisition of local fluctuations. The measurement provides the presence of clusters and monomers/dimers. |
Image Acquisition (Raster Scan)
| Min Dwell Time | 4µs |
| Pixel Number | For steady-state images: User selectable from 2 to 4096
For FLIM images: User selectable from 2 to 2048 |
| Max Line Frequency | 12 KHz (on 20 points) |
| Min Line Frequency | 0.01 Hz |
| Max Frame Rate | 512 x 512: 1 second |
| Beam Park | The beam can be parked at any position for the acquisition of FFS data |
| Scan Modes | For kinetics studies: t, Xt, XYt, XZt, XYZt, XZt
For optical sectioning: XZ, XYZ |
Image Processing and Analysis
| Operations on Images |
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| FLIM Processing |
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| FLIM Display |
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| FLIM images (digital frequency-domain) (single plane and z-stack) |
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| Image Export Format |
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| Plots Export Format |
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| Movies Export Format |
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Data File Formats
| extension | format | description/application |
|---|---|---|
| ifi | 32-bit floating | Confocal images (steady-state)
[File header includes: image size, pixel time, intensity, etc.] |
| ifli | 32-bit floating | DFD (FastFLIM)
AFD (analog FD) [Includes: DC, phasor, G, S. The file header includes the image size, pixel time, etc.. The file is already corrected with reference.] |
| fbd | raw data (FIFO data) | DFD (FastFLIM)
no correction applied to the data [This file is saved simultaneously to the .ifli-format file. No header, image size, pixel time are not stored] |
| fbs | Experiment Info | Header for the fbd-format
[file header includes: image size, dwell time, padding info] |
| spc | Raw Data (FIFO Data) | TTTR, time tagged time resolved
Used with TCSPC |
| set | Experiment Info | Header for the spc-format
[file header includes: image size, dwell time, padding info] |
| tif | TIFF tagged images file format | Confocal images (steady-state) |
| bin | Binary 16-bit | LFD image file or FCS in time-mode
Confocal images (steady-state) RICS |
| int | 32-bit floating | LFD image file |
| ref | LFD ref file | DFD (FastFLIM), 256x256 pixels
already corrected with reference file |
Data File Formats Supported
| extension | format | description/application |
|---|---|---|
| ref | LFD ref file | DFD (FastFLIM), 256x256 pixels
already corrected with reference file |
| spc | B&H format for raw data [FIFO Data] | TTTR, time tagged time resolved
Used with TCSPC |
| set | B&H format for Experiment Info | Header for the spc-format
[file header includes: image size, dwell time, padding info] |
FFS Module
Data Acquisition
| Autocorrelation (FCS) | The FCS function gives the temporal correlation of the fluctuations |
| Cross-correlation (FCCS) | The FCCS function provides the temporal correlation of the fluctuations related to events occurring simultaneously on two or three channels. |
| Photon Counting Histogram (PCH) | The PCH function plots the distribution of the amplitude of the fluctuations. |
| FFS measurement at target XYZ locations in an image | The user selects the XYZ locations moving the cursor or entering the values in the software. The laser beam moves sequentially to each location to acquire FFS data that are then analyzed. |
| FLCS, Fluorescence Lifetime Correlation Spectroscopy | Using lifetime data (either from TCSPC or frequency domain), the technique allows for the separation of different FCS contributions. |
| Scanning FCS (requires FFS module) | The laser beam rotates on a circle with diameter 100-200 nm (user determined) and FFS data are acquired at set angles. At each angle an FCS curve is reconstructed. |
Data Analysis
| Statistical function utilized for FFS data analysis | Single set and Global fitting models available in the FFS software | Parameters determined by the FCS software |
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| Autocorrelation (FCS) |
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| Cross-correlation (FCCS) | ||
| Photon Counting Histogram (PCH) |
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| User Defined Equation |
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Data File Formats
| extension | format | description/application |
|---|---|---|
| fcs | Binary Format | FFS raw data file |
| csv | ASCII format (csv) | FCS
(correlation and autocorrelation function) |
| hst | ASCII format (csv) | PCH
photon counting histogram |
| nts | Text File | Notes on FFS files |
Screenshots of VistaVision™
VistaVision includes two operation panels: Device Control Panel and Data Acquisition Panel.
Device Control Panel
- 4 channels
- Gain of light detectors
- Automatic pinhole and lens alignment (for each channel)
- CMOS camera view
- XYZ image scan (scanning mirror or piezo stage)
Data Acquisition Panel
- Measurement selection: FFS, Scanning FFS, Single Molecule FRET, Steady-state Anisotropy, Imaging, RICS and N&B, FastFLIM, TDFLIM
- Time series, z stacks
- External trigger to start acquisition
The bottom left is the “Time Gating Selection”, for selection of the donor and acceptor decay times ranges. On the right, the 2D plot displaying FRET Efficiency on the x axis and stoichiometry on the y axis. The stoichiometry is defined as FD/(FD+FA_p), where FD is the Donor emission photon count within the donor decay window, the FA_p is the acceptor emission photon counts within the acceptor decay window; the higher the stoichiometry the less the acceptor exists. The Stoichiometry analysis panel also shows lifetime vs FRET efficiency or other parameters such as Anisotropy.
