Microscopy > Software > VistaVision

VistaVision

VistaVision (Windows XP) 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
  • ISS FCS card (for FFS data acquisition)
  • ISS 3xDAC card (for controlling imaging devices)
  • ISS FastFLIM card (for frequency-domain FLIM)
  • NI 6052E
  • B&H SPC-830, SPC-150 cards (for TCSPC FLIM)
Scanning Mirrors Modules
  • ISS scanning mirrors module
  • Cambridge Technology Models
Piezo-controlled Stages
  • ISS XYZ piezo-controlled stage
  • MadCity Models Nanoposition
  • PI Models PiezoNano
  • Nanomotion, Model SC-AT
Automated Microscopes
  • Nikon Models TE2000-E, Ti-E
Microscopes Stages
  • ASI Model 2000
  • Prior Scientific Model H117P2IX
Laser Launchers
  • ISS Laser Launchers Series
  • Intensity control unit for Ti:Sapphire laser
Two-detector unit
  • Detector unit including two shutters, two filter wheels and one dichroic wheel used for LSM upgrade packages (Olympus, Leica, Nikon, Zeiss)

Imaging Module

Data Acquisition

Single point measurements
  • Intensity
  • Polarization
  • Kinetics
  • Lifetime
Line Measurements
  • Line Acquisition
  • Profile Acquisition
Steady-state images (single plan and z-stack)
  • Polarization
  • Ratiometric
  • Kinetics
  • Time-lapse Recording
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
FLIM images time-domain (single plane and z-stack)
  • Acquired in time-correlated single photon counting (TCSPC)
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
  • Scaling
  • Arithmetics
  • Smoothing
  • Zooming
  • Rotation
FLIM Processing
  • Minimization Technique (Marquardt-Levenberg algorithm)
  • Phasor Plots
FLIM Display
  • Lifetime Image
  • Fractional Contributions Image
  • Pre-exponential Factors Image
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
Image Export Format
  • ImageJ
  • MetaMorph
Plots Export Format
  • gif
  • tiff
  • jpeg
  • png
  • bmp
  • metafile
Movies Export Format
  • avi

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
Autocorrelation (FCS)
  • One or two species using:
    • 2D- or 3D-Gaussian PSF
    • 3D-Gaussian-Lorentzian PSF
    • one-photon excitation
    • two-photon excitation
    • presence of flow
  • Global analysis fitting
  • One or two species using:
    • Diffusion coefficient
    • Concentration
    • Triplet state decay time constant
    • Triplet function
    • Flow rate
    • Size of excitation volume
Cross-correlation (FCCS)
Photon Counting Histogram (PCH)
  • One or two species using:
    • 2D- or 3D-Uniform
    • 2D- or 3D-Gaussian PSF
    • 3D-Gaussian-Lorentzian PSF
    • one-photon excitation
    • two-photon excitation
  • Global analysis fitting
  • Number of molecules
  • Molecular brightness
User Defined Equation
  • Up to 50 different user defined equations kept in the panel list for user to choose.
  • Equation could include Sin, Cos, Exponential, etc… Except the integral unclosed form equation.
  • Global analysis fitting
  • Up to 30 parameters allowed in the equation.

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