Examine datasets during and after acquisition with SlideBook’s 3D Capture Status and Multipoint Viewer. 3D Capture Status supports volumetric projection (via max-intensity projections) to observe dynamics during an experiment by examining more than one plane at a time. The Multipoint viewer allows for examination of multiple XYZT positions during acquisition, and post-acquisition the user may review all the XYZT positions in a capture.

3D Capture Status

Multipoint Viewer

The .sldy file format is an open-source-friendly file format designed for very large data sets (from 100GB to >1TB). The .sldy file is a distributed file format that points to a directory where each captured 2D or 3D image is stored in an independent file (by channel and timepoint) alongside metadata files describing the capture and microscope parameters. The distributed nature makes image manipulation operations – such as removing a channel or a selection of time points – nearly instantaneous. Images, masks, and histograms are stored as Python-compatible NumPy arrays, and the metadata is stored in the open-source YAML format, enabling users with coding experience to process images directly in Python. The default .sld file format is still available and appropriate for many users.

.sld is a single file that contains all images and metadata

.sldy is an open-source-friendly collection of individual image and metadata files in a directory. The .sldy file format allows for much faster execution of certain file operations, such as deleting a single image or timepoint.

The .sldy file format is also natively compatible with Python, allowing direct access to image data without the need for a special reader.

SlideBook supports compression of the .sldy file format (called .sldyz) with the Zstandard compression algorithm.  Zstandard is a lossless algorithm that compresses very large files with a balance of speed and compression ratios that outperforms standard lossless algorithms. Each NumPy image file is compressed individually, so the structure remains the same, but the overall size of the directory is smaller. Compressed files can be opened natively in SlideBook with no need to decompress them.

Conditional and Hierarchical Capture allows a MATLAB script to control experimental workflows. For example, a user can automate the selection and imaging of certain cells at higher magnification after a low-mag pass, or to stop a timelapse image after a particular event has been observed. By coupling with 6D Multicapture, users can customize highly specific and complex automated experiments.

SlideBook features a montaging tool for stitching 2D, 3D, and 4D data. This allows for better handling of large datasets in a constrained memory environment.  New features include:

• Test alignments on a subsection of the overall image
• Limit the alignment to a smaller Z-range in 3D and 4D datasets
• Subsample before stitching
• Output the montaged data to a new file
• Limit the number of CPU threads and run the job in the background to free resources for other operations

The multiwell interface is incorporated into the focus window to allow for easier plate alignment and selection of wells for imaging. The selected wells are seamlessly integrated into the normal SlideBook workflow, thus reducing the amount of time, mouse-clicks, and learning curve to perform multiwell imaging. There are several pre-set well plate layouts, and users can also define custom well plates of varying sizes up to 384 wells.

SlideBook’s interface for multiphoton systems assembles all frequently-used controls and status displays into a single window. The console features an advanced, intuitive tool for adjusting laser power delivery for different depths incorporating dynamic signal feedback.

For cleared tissue light sheet imaging, a console for AxL Cleared Tissue LightSheet is intuitive and feature rich, with features such as 2D and 3D prescan, ROI selection, automated lightsheet pattern generation, and axial chromatic aberration correction.

Bounding box in the 3D Volume View allows for cut-away viewing of projected volumes in all three dimensions and selection of subsets of the data for other operations. Users are also able to change the transparency and opacity of portions outside the bounding box. The bounding box functionality can be extended for use in making movies, calculating point spread functions and montaging.

StoryBoard is an all-new way to make movies from 3D and 4D datasets utilizing keyframes and custom framerates. Movie templates can be saved and re-used for multiple datasets. Incorporating spline interpolation, StoryBoard quickly renders beautiful and smoothly animated movies from large datasets.

SlideBook can link to ImageJ lookup tables (LUTs) and display 3D volumes with different colors depending on depth. The same lookup tables can be applied to other captures using the Pseudocolor Mode on a per-filter basis.

User-selectable color schemes with dozens of different options including light, dark and black modes.

Focus Surface allows users to correct for non-planar deformities by selecting multiple points of focus and interpolating a surface across them to automatically adjust focus as the system moves between points, wells, or tiles in a montage. This is helpful in situations where tissue may be warped or undulating from fixing and dehydrating, or where a multiwell plate is deformed due to slight imperfections in the plastic.

NVIDIA CUDA leverages parallel processing on the GPU to greatly speed up operations by executing many commands at the same time. Operations that have been CUDA-optimized in SlideBook 2022 include:

• Deconvolution (all modalities: No Neighbors, Nearest Neighbors, Constrained Iterative, Big Data, MLS Decon)
• MLS Localized Cross-correlation
• MLS POI Registration
• Adaptive Optics
• Software-based autofocus
• 3D montage
• Image deskew and rotations
• Max-intensity projections

3D Deconvolution

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 3D 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, the AutoQuant 3D Blind Deconvolution module may be used.

AutoQuant 3D Blind Deconvolution

This module allows the AutoQuant adaptive blind deconvolution algorithm to be run seamlessly within 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.

Stereology

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.

FRET

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.

Ratio Imaging

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.

FLIM

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.

TTL Synchronization

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.

Rapid 4D Streaming

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.

Scanning

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.

LightSheet

The LightSheet module facilitates control of Lattice LightSheet, Marianas LightSheet, and Cleared Tissue LightSheet alongside light sheet-specific analysis modules such as Localized Cross Correlation Reconstruction, deskewing, channel registration, and deconvolution.

Photomanipulation

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.

Computer Generated Holography

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, 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.