OVERVIEW

Femtonics microscopes are controlled and work together with our measurement control and analysis software package called MES. It is designed with day-to-day lab experiences in the field of cellular and network imaging. Its main goals are the user-friendly, convenient, and fast control and automation of measurements, real-time data display and analysis. To fit different hardware and user needs, the software has a highly modular design, and it can be extended with many modules.

LABMASTER

MES is capable of performing all standard microscopy functions and it fully integrates the control of all hardware units in the microscope:
  • scanner, PMTs, light path actuators, electrical devices,
  • focusing, camera handling,
  • auxiliary digital and analog channels to interface stimulators,
  • behavior control devices,
  • sample stage or patch pipette devices.
MES stores all metadata of the aboves in a complex file containing multiple measurement units along with your comments.

FLEXIBLE ENVIRONMENT

The MES software is written in the MATLAB high level programming environment. This enables rapid code development and opens the data to the users to the deepest levels. Data can be interactively accessed at MATLAB command prompt or through self-developed script or GUI tools beyond the wide opportunities of the existing program package.

REAL-TIME DISPLAY AND ANALYSIS

MES offers a wide range of specialized tools. Integrated, quantitative intensity-based calculations allow following real-time fluorescence changes and simultaneous analysis (e.g. ∆F/F calculation). To extend the function, MES can adapt electrophysiological recordings to displaying Ca2+ and to electrical signals side-by-side. In addition MES supports several export and import functions with other programs.

BATCH PROCESSING

The analysis module of MES contains efficient tools to analyze entire multi-ROI measurement sets conveniently. Sophisticated curve analysis tool allows fast exploration of the measurement data. Having direct access to the data, any user created analysis methods can be developed.

2D ROI SELECTION

Numerous software tools support all kinds of 2D scanning pattern generation: points, straight or curved lines and areas with various shapes can be selected improving the efficiency of imaging.
  • Stimulus mapping and analysis allows easy placing of predefined shapes, such as spirals which is useful for photostimulation.
  • TravellingSalesman creates the shortest pathway between the scanning points increasing the velocity of the scanning.

3D ROI SELECTION

MES manages the control of piezo objective positioner and focus tunable liquid lens supporting 2D systems to become capable of 3D imaging.
MES works together with Femto3D-AO microscope too. New software tools with custom designed user interface support the most flexible 3D scanning pattern selection of multiple points, multiple areas or 3D trajectories as the basis of the advanced scanning modes. Various software tools help selection, such as 3D cell localization, automatic pattern generation and XYZ adjustment.

EXTENSION MODULES

MES interacts with numerous software during the processing and it can be extended with many software modules to support various measurement protocols. It can adapt electrophysiological recordings and integrate the control of numerous devices (pipette manipulators, Luigs&Neumann sample devices). It involves import and export functions with other programs such as MS Excel, Imaris, Amira, Huygens.

EXTERNAL STAGES/MANIPULATORS

PIPETTE MANIPULATION

Robotic positioning of patch pipettes in the sample
This module implements semi-automatic two-photon image guided tip positioning in the sample. It realizes the software control of the following micromanipulators:
  • Luigs & Neumann: SM-5 to SM-10
  • Sutter Instrument: MP-285

SAMPLE STAGE

Sample stage control
This module adds a GUI for controlling sample stages. It allows storing actual stage positions in each new measurement unit and and later recalling (up to four stage positions).
Currently supported hardware:
  • Luigs & Neumann
  • Sutter Instrument
  • Thorlabs APT
  • All Femtonics stages and movable microscope bodies

FIX WITH STAGE

Sample fixation using a bright spot and the sample stage
The module implements a sample position stabilization algorithm with using bright spot or cell that is repeatedly 2p scanned and re-centered in the viewport by commanding the sample stage.
  • Slow timescale XY stabilization
  • User selectable fixation point or region (e.g., a bright glial cell)
Prerequisite: Sample Stage module

SPECIAL MEASUREMENT CONTROL

STIMULUS MAPPING AND ANALYSIS

Specialized functions for photostimulation mapping
This module allows performing photostimulation mapping experiments at a range of locations and shapes. The locations can be picked manually one-by-one, along a line or in a raster, or automatically by the Cell3DFinder module. Various stimulation shapes (patterns) can be selected like point, spiral, x, zigzag, or 3D shapes for RC and AO scanning, and advanced point and pattern management functions are available. Analysis tool for stimulation mapping can create (multichannel) map images formed by the elicited responses after a mapping experiment. It forms datasets quickly by evaluating fluorescence changes (images) or electrophysiology (curves) at defined time intervals, or imports datasets from the clipboard, working together with the Line-scan and Curve analyzer tools.
Recommended for uncaging, special photostimulation needs, synaptic mapping experiments, and connectome mapping.

META-PROTOCOL

Microscope data capture automation
This module automates line-scan, frame-scan, and Z-stack measurements on the timescale of seconds by letting you run freely composed sequences of Image acquisition, XYZ-scan, Line-scan, AO line-scan, and Fix with stage measurements.
Recommended for periodic measurements, long physiological studies, time-lapse imaging (even at multiple locations), LTP and LTD protocols.

TRAVELLING SALESMAN

Fast 2D galvo point scanning mode
TravellingSalesman creates the shortest pathway visiting user defined points for galvo scanning by approximating the traveling salesman problem. The short roundtrip time results in a high measurement speed reaching 100 Hz for a number of points arbitrarily dispersed in the field of view.
Recommended for 2D optimized random access point scanning and high S/N network measurements.

DOCALLBACK

Running custom user scripts during measurements
It enables you to define custom MATLAB functions to be called before or after a measurement is performed. Useful for pre-measurement signaling or post-measurement modification of data, annotation, or analysis.
DoCallBack includes sample code for e.g. waiting for a trigger, parallel camera acquisition, and automatic TIFF export.
Recommended for behavioral studies.

CELL3DFINDER

3D localization of cells in Z-stacks
This module finds cell centers in 3D image stacks as a set of points. The generated point sets can be used for measurements or for the photo stimulation of cells in a random access fashion.
Recommended for the fast selection of a large number of ROIs for network imaging with the AO, RollerCoaster, and TravellingSalesman scanning modes.

ROLLERCOASTER

This module handles the 3D scanning functionalities relying on piezoelectric objective positioning.
Scanning modes:
  • 3D trajectory, stripe, tilted frame and volume scanning,
  • Control of 3D measurements,
  • Analysis of 3D measurements: 3D spatial normalization and projection.

DATA ANALYSIS

BATCH ANALYSIS

Batch line scan, area scan, and curve analysis functions
This analysis module contains efficient tools to analyze entire multi-ROI measurement sets conveniently. It implements the grouped analysis of imaging data at multiple regions and multiple measurement repetitions, and the handling of the resulting calcium transients, electrophysiology time series, and metadata.
  • Batch export to TIFF files
  • Line-scan analysis
  • Curve analysis
  • Property collection and statistics tools

3D SLICER

Z-stack slicing tool
This module implements XZ or YZ sectioning and projection of the Z-stacks and allows 2D visualization of 3D stacks projected to any of the three axes. The user can define depth for better apparent S/N. Maximum, mean, and minimum projections are available.

EXCEL INTERFACE

Embedding measurement data into Excel worksheets
This module allows moving data and images between Curve analysis and Annotation windows and Microsoft Excel worksheets in both directions. Producing measurement reports and performing data analysis becomes easy and easy to track, until nearly publication ready datasets are formed.

RATIO IMAGING

Implements two-channel ratio imaging analysis calculations
This module offers different types of dG/R and G/R calculations in the measurement viewer windows and batch analysis tools:
  • real-time ratio metric analysis of two channel measurements (dG/R),
  • possibility to calculate dG/R images of the raw line-scan data,
  • adds dG/R analysis tools for batch analysis panels,
  • selectable source channels,
  • automatic background calculation algorithms for both channels,
  • running averaging before or after division.

AUXILIARY BIOLOGY

VISUAL STIMULATION PLAYER

Integrated visual stimulus player
This module allows loading and sequentially playing videos or images for visual stimulus and other stimuli can be triggered such as tactile, whisker, auditory or odor stimuli. The stimuli can be triggered by software or TTL signals.
Recommended for behavioral studies and for intrinsic imaging.

INTRINSIC IMAGING

Intrinsic imaging feature
Intrinsic software module helps to follow the evoked activity-related intrinsic signals: evokes, measures, integrates, and displays the activated brain regions. The evoked stimulus can be:
  • visual (when used together with the Visual Stimulation Player module), and/or
  • tactile, whisker, auditory, odor, etc., triggered by TTL signals (user supported devices needed).
It uses the cameras supported by the microscope.
Recommended: behavioral studies, localizing of brain regions such as higher order visual areas, barrel cortex, sensomotory cortex or auditory cortex.

ELECTROPHYSIOLOGY SOFTWARE MODULE

Simple electrophysiology functions commanded from MES
This module acquires electrophysiology signals using the A/D converters of the microscope. The telegraphing function communicates the conversion parameters from the amplifier to the A/D converters controlled by MES. Convenient acquisition of electrophysiology signals time is aligned with the imaging data. The electrophysiology GUI includes a seal test window, too.
Supported amplifiers: MultiClamp700B

EPHYS IMPORTER

Importing electrophysiology data
With this module, user can imports externally recorded electrophysiology curves into MES measurement units. Imaging with MES works simultaneously with the external digitizer by triggering each sweep of electrophysiology recording. An easy-to-use tool helps matching sweeps in the electrophysiology files and MES measurement units and also imports electrophysiology curves to the optical data. Hundreds of curves can be loaded at the end of the measurement with a few clicks. The module allows importing electrophysiology data directly into the Curve analysis window of MES.
Supported file types:
  • .abf: Molecular Devices (Axon), Digidata 1440, pCLAMP10
  • .rhd: Intan Technologies
  • .cnt: Neuroscan

OVERVIEW

OVERVIEW

Femtonics microscopes are controlled and work together with our measurement control and analysis software package called MES. It is designed with day-to-day lab experiences in the field of cellular and network imaging. Its main goals are the user-friendly, convenient, and fast control and automation of measurements, real-time data display and analysis. To fit different hardware and user needs, the software has a highly modular design, and it can be extended with many modules.

LABMASTER

MES is capable of performing all standard microscopy functions and it fully integrates the control of all hardware units in the microscope:
  • scanner, PMTs, light path actuators, electrical devices,
  • focusing, camera handling,
  • auxiliary digital and analog channels to interface stimulators,
  • behavior control devices,
  • sample stage or patch pipette devices.
MES stores all metadata of the aboves in a complex file containing multiple measurement units along with your comments.

FLEXIBLE ENVIRONMENT

The MES software is written in the MATLAB high level programming environment. This enables rapid code development and opens the data to the users to the deepest levels. Data can be interactively accessed at MATLAB command prompt or through self-developed script or GUI tools beyond the wide opportunities of the existing program package.

REAL-TIME DISPLAY AND ANALYSIS

MES offers a wide range of specialized tools. Integrated, quantitative intensity-based calculations allow following real-time fluorescence changes and simultaneous analysis (e.g. ∆F/F calculation). To extend the function, MES can adapt electrophysiological recordings to displaying Ca2+ and to electrical signals side-by-side. In addition MES supports several export and import functions with other programs.

BATCH PROCESSING

The analysis module of MES contains efficient tools to analyze entire multi-ROI measurement sets conveniently. Sophisticated curve analysis tool allows fast exploration of the measurement data. Having direct access to the data, any user created analysis methods can be developed.

2D ROI SELECTION

Numerous software tools support all kinds of 2D scanning pattern generation: points, straight or curved lines and areas with various shapes can be selected improving the efficiency of imaging.
  • Stimulus mapping and analysis allows easy placing of predefined shapes, such as spirals which is useful for photostimulation.
  • TravellingSalesman creates the shortest pathway between the scanning points increasing the velocity of the scanning.

3D ROI SELECTION

MES manages the control of piezo objective positioner and focus tunable liquid lens supporting 2D systems to become capable of 3D imaging.
MES works together with Femto3D-AO microscope too. New software tools with custom designed user interface support the most flexible 3D scanning pattern selection of multiple points, multiple areas or 3D trajectories as the basis of the advanced scanning modes. Various software tools help selection, such as 3D cell localization, automatic pattern generation and XYZ adjustment.

EXTENSION MODULES

MES interacts with numerous software during the processing and it can be extended with many software modules to support various measurement protocols. It can adapt electrophysiological recordings and integrate the control of numerous devices (pipette manipulators, Luigs&Neumann sample devices). It involves import and export functions with other programs such as MS Excel, Imaris, Amira, Huygens.

MODULES

TRAVELLING SALESMAN

Approximates the traveling salesman problem to find an optimal visit order of the locations namely this module adds the ability to create the shortest pathway between points for galvo scanner. The short jumping time results higher measurement speed which can reach 100 Hz in a number of points locating dispersed in the field of view.

Recommended: 2D optimized random access point scanning, high S/N network measurements

CELL3DFINDER

This module finds cell centers in 3D image stacks and adds a new panel to the XYZ viewer window. The generated point sets can be used later to scan or photostimulate cells in a random access fashion.

Recommended: fast selection of a number for RoI scanning and network imaging

ROLLERCOASTER

This module handles the 3D scanning functionalities relying on piezoelectric objective positioning.
  • 3D trajectory, stripe and tilted frame scanning
  • control of 3D measurements
  • analysis of 3D measurements: 3D spatial normalization and projection

LINEMAGIC

This module adds further automation capabilities based on the free line-scan modes. Advanced scanning modes: spiral, zigzag, helix, etc.

Recommended: uncaging, special photostimulation needs, mapping experiments

STIMULUS AND MAPPING ANALYSIS

This software module can create (multichannel) map images or smoothed maps if the uncaging experiment series was done in a raster, or it can display intensities by circles drawn onto a background image.
Mapping software tool is able to form datasets quickly by evaluating fluorescence changes (images) or electrophysiology (curves) at defined time intervals, or it is able to import datasets from the clipboard, thus working together with the Line-scan and Curve analyzer tools facilitating manual control and in-depth data analysis.

Recommended: uncaging, photostimulation mapping experiments

EPHYS IMPORTER

Molecular Device’s patch amplifiers and the digitizer Digidata 1440 works side-by-side with MES to record electrophysiology signals parallel with the optical data collection. This module allows importing .abf files from pCLAMP10 electrophysiology digitizing control software into the Curve analysis window of MES and also has a tool to match by creation time sweeps in abf files and measurement units.

PIPETTE MANIPULATION

This module adds hardware support for software control of the Luigs&Neumann controlled manipulators. The module implements a semi automatic two-photon image guided tip positioning method.

RATIO IMAGING

This module allows dG/R, and G/R calculations in the measurement viewer windows and in the batch analysis tools.
  • real-time ratio metric analysis of two channel measurements (dG/R),
  • possibility to calculate dG/R images of the raw line-scan data,
  • batch analysis tools for bulk dG/R analysis on multiple line-scans,
  • automatic background calculation algorithms for both channels.

BATCH ANALYSIS

The analysis module of MES contains efficient tools to analyze entire multi-ROI measurement sets conveniently. Sophisticated curve analysis tool allows fast exploration of the measurement data. Having direct access to the data, any user created analysis methods can be developed.

3D SLICER

This module allows XZ or YZ sectioning and projection of the z-stacks. See the XYZ viewer window description for further details.

Recommended: project / visualize 3D stacks

METAPROTOCOL

This module creates the ability to automate measurements on the timescale of seconds. You can run Image acquisition, XYZ-scan, Line-scan, AO line-scan and Fix with stage sequences .A sequence of measurements (with different protocols) can be programmed.

Recommended: periodic events, long physiological studies, timelapse imaging even at multiple locations, LTP and LTD protocols

EXCEL INTERFACE

With this module you can save and load the data and pictures of Curve analysis windows or Annotation windows into/from Excel. This way you can easily produce measurement reports and analysis steps and results until publication ready datasets.

STIMVISUAL AND INTRINSIC IMAGING

This complex module allows setting and controlling visual and other stimuli in MES and follow the evoked activity-related intrinsic signals. StimVisual allows loading visual stimulus and other stimuli can be triggered such as tactile, whisker, auditory or odor stimuli. Intrinsic module helps to determine activated brain regions. StimVisual is available as a stand-alone module too. Recommended: behavioral studies, localize brain regions such as higher order visual areas, barrel cortex, sensomotory cortex or auditory cortex.