3i Slidebook

The newly redesigned SlideBook™ application comes complete with all of the necessary tools for imaging application ranging from simple 2-D and time-lapse imaging to multi-channel 3-D, multi-point, and 4-D imaging.

SlideBook™ 4.0 features the Data Slide architecture, which allows the user to store related images together and simplifies both data analysis and archive retrieval. The data structure in SlideBook also facilitates the accurate recording of image characteristics by automatically storing all of the information from image capture along with the individual images

In addition, SlideBook™ features a complete suite of analytical tools specifically designed for 2D, time-lapse, 3-D and 4-D microscopy data including elapsed time, velocity, area, volume, surface area, length, length along major axis, mean intensity, minimum and maximum intensity, integrated intensity, variance and standard deviation.

SlideBook™’s unique architecture also facilitates the processing of 2-D, 3-D and time-lapse data by providing No Neighbors Deconvolution, common filters and an open convolution kernel as part of the base application.

Macintosh OS X and Microsoft Windows XP Compliant

In addition to providing the tools for acquiring and analyzing 2-D and time-lapse data, SlideBook™ comes standard with the ability to control hardware associated with digital imaging. 

Images and Views

Images are observed in SlideBook™ by Views. Views can be used to show images in a variety of ways. Each view can be displayed in four modes: RGB, monochrome, inverse monochrome, and pseudocolor.

Views can be individually renormalized to display the exact range of data desired for each channel. The channel or channels displayed by an individual view are selectable. Although renormalization and channel selection can drastically change the look of a view, it has no effect on quantitative data stored by the image.

A default view of an image can be saved using the thumbnail feature. Any view can be exported as a TIFF file for final publication. The base SlideBook™ package contains a Main View. Main views support viewing along any of the major axes (X, Y, Z or T). Like all views, data displayed in a Main View can be renormalized and viewed in different modes. The Main View also supports 3D data selection as well as image zooming, distance measurements and angle measurements.

Slide Metaphor and Data Layout

SlideBook™ is designed around a slide data model. This means that all images collected from a single physical sample are stored in a single slide file.

An image in SlideBook™ is a single photographic data collection sequence. Images can be collected using SlideBook™'s image capture module or imported from other sources such as TIFF files or LSM data files. An image not only stores the quantitative pictures from a data collection run, but also records the entire optical pathway (i.e. objective lens, filters, light source, magnification changer, etc.) in addition to the capture time, date, and any operator comments. Images can contain 2D, 3D or time lapse data. They can be single channel or multi-channel.

A channel is data collected using the same configuration of filters. Channels can be collected using either the fluorescence (i.e. FITC, CY3, GFP etc.) or transmitted (i.e. bright field, dark field, DIC, or phase contrast) light paths. An image can contain as many channels as desired. Each data element of a channel is stored in 16 bits to accommodate the full data collected by the detector. Channels can be mathematically compared and manipulated for sophicated techniques such as ratio imaging and FRET.

Images in SlideBook™ are protected from manipulations that compromise the integrity of the data. All image operations are designed to improve the quantitative relationship of the data to the actual sample. These operations include simple corrections such as Flat Field and Dark Field as well as more sophisticated manipulations such as Deconvolution. Image data can be exported either as 3D TIFF files or as raw data.

2-D and 3-D Analysis

The quantitative data stored by images can be selectively accessed using masks. A mask is a 2D or 3D selection of locations within the image. Multiple masks can be created per image. Masks are stored with the image and can be created by segmentation (selecting a range of intensities or edge conditions), by manual delineation, or by interaction with an existing mask (AND, OR, NOT, XOR operations).

Statistics can be generated on the data delineated by masks. These statistics include integrated intensity by channel, average intensity by channel, volume, surface area, major axis, maximum intensity by channel, minimum intensity by channel, etc.

SlideBook™ can also break masks into submasks. Submasks are voxels defined within a mask that are contiguous in 3D. Submasks can be gated by size to select for certain objects or to eliminate noise. Submasks can be counted and individually quantified. This allows for operations such as cell counting and population variance description.

3-D Deconvolution

The 3D Deconvolution module adds a 3D deblurring and an image restoration algorithm to SlideBook. The nearest neighbor and constrained iterative deconvolution algorithms have been optimized for speed and performance. The module includes an interactive guide for measuring point spread functions (PSF) and a PSF database. The database can store multiple PSFs for each configuration, allowing easy selection of the appropriate PSF.

Nearest Neighbors DeconvolutionOur Nearest Neighbors deconvolution is a rapid way to “deblur” fluorescence data. The algorithm uses the plane above and below the plane of interest to compute and subtract the fraction of the data that is out-of-focus information. Constrained Iterative DeconvolutionOur Constrained Iterative (CI) deconvolution is a true image restoration tool. Based on the algorithm developed by David Agard at UCSF, our CI deconvolution can quantitatively reassign out-of-focus information in 3-D data while improving both axial and lateral data resolution. The module can use either measured point-spread functions (PSFs) or computed PSFs when measured PSFs are unavailable. The module includes a PSF collection guide and a PSF database. Given SlideBook’s knowledge of image collection optics, the correct PSF can automatically be applied to CI deconvolution without user assistance. With SlideBook 4.0, CI deconvolution includes extensive speed improvements taking full advantage of advances in computational algorithms, multiple processors and hardware acceleration for both Macintosh and Windows platforms. CI deconvolution has been improved so that it can handle deconvolution of very large data sets (up to 350 MB per wavelength for a system with 2 GB RAM). In addition, CI deconvolution now includes a number of advanced user options to compensate for a variety of imaging conditions, from single cells to tissue sections, so that an optimal deconvolution can be achieved for a particular experiment.

Particle Tracking Module

The evolution of digital microscopy has placed increasing emphasis on the quantitative information contained within collected images. The Statistics Engine for SlideBook™ has been completely reworked for version 4.0 to provide researchers with a complete suite of analytical tools specifically designed for quantifying data from microscopes.

The new Statistics Engine includes:

• Plug-in architecture for end-user writable features
• Automatic methods for generating and storing statistics to aid in gathering large amounts of data
• Advanced clustering techniques to help define populations of objects and images
• Many new statistics, including cross-channel colocalization and skeleton-based features

The all new 4D analysis and particle tracking module for SlideBook 4.0 contains powerful tools tracking individual 3D objects in 4-D datasets. The particle tracking engine is based on individual object paths. Individual objects will have a unique path through time and can therefore be followed.The particle tracking module can also correlate objects with respect to one another and generate statistics about each object at each time-point.

Object paths can be traced either automatically using computational algorithms or through manual delineation. The particle tracking module also offers complete flexibility for combining both automated and manual tools to optimize path detection for any given sample.

Once object paths have been traced, there are a variety of visualization tools to help explore the objects. In the OpenGL-based 3DpathView, for example, an individual path can be selected and individually rendered over time. This rendering can be rotated freely and played as a movie (in either direction) or in single frame increments. As users explore the data, they can manipulate the path, either to prune a split/merge as objects move, or get statistics for a given object.

The statistics generated by the particle tracker include everything from velocity and acceleration of objects to a description of randomness of an object's motion. These statistics can be automatically generated for all objects/paths or statistics can be generated on a path-by-path basis. In addition, all delineations, processes and analyses in the particle tracker are automatable. Each step in the tracking scheme can be stored and retrieved from a methods database, so that a series of commands can be easily written and retrieved for use in future experiments

FRET/Ratio/Densitometry (FRD) module

This module is specifically designed to handle data that is only meaningful in the context of other data. Examples of such data are FRET where the data are represented as a percentage of a donor molecule or FURA-2 where the data is meaningful as the ratio of emissions of two separate excitations.

The FRD module for SlideBook™ 4.0 is the first commercially available software specifically designed for FRET analysis. This revolutionary software was developed in collaboration with a number of leading biologists whose laboratories focus on FRET. The FRET module contains analysis tools for both sensitized emission FRET and acceptor photobleaching FRET. These tools include:

• Channel Bleedthrough Calculation
• Background Subtraction
• Corrected FRET Image Generation (Herman Equation)
• Single Pixel to Whole Object Energy Transfer Measurement
• Support for 2-D, 3-D and 4-D Data

The module can be combined with image capture and other on-line modules for real-time FRET analysis. In addition, SlideBook™ supports dual camera capture and the Multi-Spec Microimager device from Optical Insights for true real-time FRET image acquisition.   The measurement of free ion concentrations in living cells provides insight into cellular processes such as activation and compartmentalization. Ratiometric ion indicators change their excitation or emission profiles when bound to specific ions. These indicators allow the real-time measurement of ion concentrations in living cells. :This module supports a wide array of ion indicators including FURA-2, Indo-1 and BCECF. The module includes a variety of specific features including:

• Real-time ratio computation
• Real-time graphing of ratios and/or raw channels
• Real-time tracking of up to 20 ROI
• Simultaneous imaging of two ratio indicators
• Calibration guide for computing nM calcium concentration by pixel
• Analytical Graphing tools

TTL Synchronization

The rapid collection of 4-D data requires the precise timing and synchronization of all hardware involved in image capture. The TTL module for SlideBook™ 4.0 is a combination of hardware and software specifically designed to optimize the recording of 4-D data and synchronizing external devices such as perfusion systems and electrophysiology equipment.

Normally, hardware device control relies on serial connections between the computer and the specific piece of hardware. While serial control can accurately control single devices, it is difficult to simultaneously coordinate multiple devices through this interface. The Rapid 4D Synchronization module utilizes analog pulses of square waves (TTL pulses) to simultaneously trigger multiple devices for operation. The module comes complete with all of the necessary hardware for controlling devices through TTL pulses including multiple analog in and out connectors, PCI board and cables.