The Deconvolution module adds both a nearest neighbor and a constrained iterative deconvolution algorithm to SlideBook. Included is an interactive guide for measuring point spread functions and a database for storing multiple PSFs. Either measured or computed PSFs can be used, with the correct PSF automatically applied to CI deconvolution without user assistance.
Nearest Neighbors deconvolution is a rapid way to deblur fluorescence data using images from the plane above and below the plane of interest to correct for out-of-focus information.
Constrained Iterative deconvolution is a quantitative 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.
For imaging with very low signal to noise or where measuring a PSF is not practical, please see our Autoquant 3D Blind Deconvolution.
This module allows the AutoQuant adaptive blind deconvolution algorithm to be run directly from SlideBook, with seamless integration with the SlideBook user interface. Blind deconvolution is extremely useful in situations where an objective’s point spread function cannot be accurately measured or practically captured. The PSF is iteratively reconstructed and applied to iterative reconstruction of the image data. At the expense of time, this method allows for statistically accurate data even at low signal to noise ratios.
CellNet allows SlideBook to be taught specific analysis by example and then apply the analysis to large datasets for a significant increase in analytical throughput. CellNet gives SlideBook the ability to recognize cells and subcellular features based on morphology, making it possible to locate objects that are hard to describe using only intensity information and tracing the extent of individual objects for subsequent statistical analysis. Employing proprietary neural network technology, CellNet builds a typical representation that identifies persistent features from a set of images and finds objects that possess similar features in new images. CellNet works on 3D data and can also provide the basis of segmentation for time-lapse and 4D particle tracking.
This module controls a phase-only spatial light modulator to produce holographic laser illumination patterns. When used in conjunction with Phasor™ or Phasor 2-Photon digital holography units this module produces simultaneous 3D multipoint and arbitrary 2D pattern photostimulation and photoactivation. Sequences of patterns can be defined and synchronized by time point, real time, or an external digital trigger.
The Fluorescence Lifetime Imaging module enables measurement of fluorescence lifetimes via frequency modulation of illumination and detection signals. FLIM is a powerful tool for molecular research in living cells and can be used to measure protein proximity (FRET), polymerization, relative concentration of different molecules, separation of different labels with spectral overlap, ion concentration, and remove autofluorescence. Lifetimes are measured at the sub-nanosecond level by using unique electronic timing circuitry for synchronized phase-shifted illumination. Near single molecule detection is possible. The 3i FLIM system can be used in combination with other multidimensional imaging techniques and images orders of magnitude faster than time domain systems.
The FRET module contains analysis tools for sensitized emission FRET and acceptor photobleaching FRET. Real-time FRET analysis is possible with simultaneous dual channel capture via image splitting devices or dual camera setups.
Graphs are displayed and updated during capture. Features include channel bleed through calculation, background subtraction, corrected FRET image generation (Herman equation), single pixel to whole object energy transfer measurement, and support for 2D, 3D and 4D data. Fluorescence anisotropy measurement is also possible and fluorescence lifetime FRET is available via the FLIM Module.
The Photomanipulation module allows control of both laser and widefield light sources used for applications such as FRAP, photoablation and photoactivation. Users can quickly switch from widefield or confocal imaging to photomanipulation to create a dynamic photon event in a region then return to imaging within milliseconds. The Photomanipulation module allows the user to define a region or regions of interest as a single diffraction limited spot or as user defined shapes. This module is typically paired with Vector for diffraction limited scanning or with Phasor for simultaneous stimulation of multiple points via digital holography.
Rapid 4D allows extremely high speed 3D image capture over time by combining free-run camera streaming with a piezo stage cycling continuously through a z focus range. The output of each camera image is recorded simultaneously with the Z location of the piezo stage. The Rapid 4D module enables both devices to run simultaneously at maximum speed by synchronizing their movements in hardware instead of relying on slower software synchronization. The result is full 3-dimensional z-stacks of data captured at 30, 60 or even 100 stacks per second continuously. This is an order of magnitude or two higher than typical z-stack capture speeds over time. The only limitation to Rapid 4D is camera readout speed and piezo Z performance.
SlideBook’s Ratio Imaging module allows calibration, acquisition, graphing, and analysis of data from ratiometric indicators such as Fura-2, BCECF, and genetically expressed Cameleons. Ratio data can be uncalibrated, calibrated using buffer solutions, or calibrated intracellularly. Ratio display and statistics are performed live using floating point arithmetic on the raw image data to minimize roundoff error and utilize memory efficiently. Background region can be adjusted after acquisition if spurious signal enters the originally selected background region.
SlideBook allows ratio channels to be collected alongside other fluorescence imaging channels allowing elaborate 3D and 4D experiments incorporating ratio information. It is possible to collect 4D data in GFP and select one plane of each stack to collect Fura-2 data. Ratio imaging can also be part of elaborate experiments which combine ratio acquisition with non-ratiometric labels, 3D and 4D imaging and synchronization with electrophysiology and automated perfusion.
The Scanning Module allows SlideBook to control dual galvanometer scanning systems using National Instruments data acquisition hardware. Scanners for such applications as photobleaching, point scanning confocal and multiphoton imaging can be controlled to scan points, lines, and user defined regions of interest via resonant scanning or standard x,y scanning.
The Stereology module employs unbiased stereological techniques for accurate estimation of total number, volume, surface area, and length of objects in a biological structure. Stereology systematically samples 3D volumes from a series of tissue sections in a random fashion. The module integrates support for a number of stereological tools within the standard image capture framework. Montaging can be used to outline the boundaries of a structure in a tissue section and then counts performed at high magnification using standard capture tools. Support for both transmitted light and fluorescence microscopy is included, and an offline mode the permits acquisition and counting to be performed separately.
Identify and track single molecules, reporting object statistics and path statistics.
Designed specifically for single molecule identification and tracking, Super-Resolution Particle Tracking employs a more advanced approach that allows for gaps in trajectories accounting for such phenomena as “blinking” Quantum Dots. Within this module, objects are automatically identified, their positions are tracked through time, and both individual object statistics and path statistics are reported. Trajectories are displayed in SlideBook and can also be exported to MATLAB for further analysis.
The Synchronization (TTL) module provides sophisticated electronic control of carefully timed devices via an electronic controller unit. The TTL controller can both create and receive triggering signals and orchestrate their interaction through an experiment. Timing that is not achievable through standard data connections to a PC is made possible by custom circuitry operating in cooperation with the computer and operating system.
The Synchronization module provides software control of analog/digital I/O boards, utilizing them to direct a variety of signals via TTL and other methods. Key to this functionality is pulse generation and recording. In addition to control of lasers, detectors, shutters and fine motion devices, the module can be used to synchronize external stimulation and data collection instruments such as electrodes, patch clamp recording devices and perfusion systems.
SlideBook is able to drive hundreds of devices including microscopes, stages, lasers, wheels, piezos, scanners, shutters and many more via serial commands and TTL pulses. SlideBook is capable of robust data acquisition in up to 7 dimensions, including z stacks in a native 3D format, time lapse, multiple color channels, multiple stage locations and fluorescence lifetimes. Configuration settings of acquisition channels allow capture of data easily and logically without requiring setting of each device for each experiment.
See and inspect specimen data through any numbers of portals, from single images to z-stacks, time lapse, color channels and 4D views. Correct raw data with intelligent processing via flatfield correction and filtering. Deconvolve 3D data via any of four algorithms to best match experimental conditions.
Analyze images and extract statistical data via a wide variety of algorithms while maintaining original data integrity. Segment image data via intensity, gradient and watershed algorithms to non-destructively cull out areas of interest for statistical analysis. Track objects in 2D and 3D via automatic or manual delineation for analysis based on time or pathway.
SlideBook includes a macro scripting system for analysis to enhance the flexibility and power for advanced users. Many processing commands have a macro command counterpart to automate sequences of operations and ensure that all images in each slide are processed identically. The scripting language is a simple sequence of text commands that let SlideBook know what operation to do, on which image or images to do it, and what the parameters are.
Present and export data easily as 16-bit TIFFs, 3D movies, graphs or spreadsheets. Data is directly portable to MATLAB® and Excel and adheres to Open Microscopy Environment standards.
The scripting function in SlideBook can directly control and port data to/from MATLAB. A SlideBook script can export channel and mask data directly to MATLAB without the user needing to access the program. The script can set variables and run a program in MATLAB to analyze the SlideBook data in a MATLAB matrix. Once analyzed the matrix data can be ported back to SlideBook as channels and masks for viewing and supplemental analysis.
SlideBook Reader is a free download that allows viewing of .sld files in the familiar user interface of SlideBook. In addition to viewing data in XY, XZ and YZ projections, Reader includes a handful of useful tools:
• Full renormalization and lookup table options
• Export 16-bit tiff images
• View and graph regions of interest
• Export graphs and graph data
• Perform length and angle measurements
• Display and edit annotations
SlideBook Reader is compatible with Windows 7 and Windows XP. Mac OSX is not available at this time.