This application note describes new functions implemented in Version 9.78 of bh SPCM software. Version 9.78 especially addresses the needs of bh’s Fast-Acquisition FLIM system. It combines FLIM data from up to four TCSPC FLIM modules into a single FLIM data set, displays them online as gated intensity images or lifetime images, and displays decay curves selected from ROIs or POIs within the images online in a separate window.
Keywords: TCSPC, FLIM, Data Acquisition, Fast FLIM
New SPCM Version 9.78 Comes
With New Software Functions
Abstract: This application note describes new functions implemented
in Version 9.78 of the bh SPCM software [1]. Version 9.78 especially addresses the needs of bhs
Fast-Acquisition FLIM system [3]. FLIM data from up to four TCSPC modules can be
combined into a single FLIM data set, and displayed as gated intensity images
or as lifetime images. Moreover, decay curves can be selected from ROIs or POIs
within the images, and displayed online in a separate decay curve window.
Combination
of Data from Several SPC Modules
Many bh TCSPC and FLIM systems use several parallel TCSPC modules to record data in several wavelength channels, different polarisation channels, or from different source positions at an object of large spatial extension [1]. Multi-module systems are also used to increase the throughput rate of a TCSPC system. In this case, a single optical signal is either spread on several detectors the signals of which are recorded in parallel TCSPC channels [1, 5], or the photon pulses of a single detector are distributed into four parallel TCSPC FLIM channels [3, 4, 5]. SPCM Version 9.78 has a function to combine the data of these channels and to display intensity or lifetime images of the combined data. To activate the channel-combination function select the FIFO Imaging Mode and open the 3D Trace Parameters. Under Module select M1-4. Leave the other definitions as they are. Data Type is PS, Display Mode is F(x,y), see Fig. 1.
Fig. 1: Combination of TCSPC channels. Left: Measurement control section of the SPCM System Parameters. Right: 3D Display Parameters
Selecting a channel combination does not discard the data of the individual channels - it acts only on the display of the data and on how SPCM interprets the data. A Save operation will save the original channel data, independently of what is defined in the 3D trace parameters.
Intensity
Images from Combined Channels
SPCM is able to display (time-gated) intensity images or colour-coded lifetime images [1]. The result of the settings shown above is a main panel as shown in Fig. 2, with an image showing a (time-gated) intensity image of the combined data of SPC modules 1 to 4. Several such images can be displayed for different time gates, please see [1], [5] or [6] for details. In addition to the combined data, it is possible to display also the data of the individual channels. To do so, activate the active buttons for the individual TCSPC modules, M1, M2, M3, M4, in the 3D Trace definitions. Please see Fig. 5and Fig. 6.
Fig. 2: Main panel, display of the combined data of four TCSPC FLIM Channels
Lifetime
Images from Combined Channels
The channel-combination function works also in combination with online lifetime-image display [1, 2]. The trace parameters are shown in Fig. 3. Data Type is LIFET, the data of TCSPC Modules M1-4 are combined into one image.
Fig. 3: 3D Trace Parameters for lifetime image display of combined channels
The corresponding main panel is shown in Fig. 4. The Display Parameter panel is open on the right. In its lower part it contains the definitions for the lifetime range, the reference lifetime, binning, and brightness and contrast settings, please see [1] for details.
Fig. 4: SPCM Main panel with lifetime display of combined channels
SPCM is able to display up to eight images of different data type and/or from different TCSPC modules simultaneously. With the trace parameters shown in Fig. 5, a lifetime image of four combined channels is displayed together with the lifetime images of the individual channels. An SPCM main panel for this configuration is shown in Fig. 6.
Fig. 5: 3D trace parameters for display of combined channels together with images of individual modules
Fig. 6: Main panel with combined images of Channel 1-4 and images of individual channels
Display
of Decay Data in Point or Region of Interest
A right-mouse click into one of the image windows of SPCM opens a small panel from where you can access the image cursors, the display parameters, trace parameters, window parameters, etc. A click into Show Decays opens a decay-curve window as shown in Fig. 7, right.
Fig. 7: A right mouse click into an image and a click on Show Decays opens the decay curve panel shown on the right
The SPCM main panel with the decay curve window is shown in Fig. 8. The curves can be displayed for all active image windows, and for individual regions or points of interest (ROIs or POIs). The ROIs are defined by the image cursors, the POI by the Data Point. A right mouse click into the decay window opens a panel with the Trace Parameters for the individual curves. The panel can be seen in Fig. 8, lower right. You can add or remove curves, activate or de-activate curves, define colours for the curves, define whether a curve is from an ROI or a POI, and from which image display window the curve comes. The coordinates of the POI and of the ROI cursors are displayed on the right. As an example, Fig. 8 displays a lifetime images of the combined channels of a four-channel-fast acquisition system (large image), the lifetime images of the individual channels (small images), and the decay data in an ROI of the combined channels and the individual channels.
Fig. 8: SPCM Main Panel with lifetime images and decay curve window
The decay-curve display works for all images that contain decay data in their pixels. These can be intensity images, time-gated intensity images, lifetime images, combined images from several TCSPC modules, or images from different routing channels of a TCSPC module.
The display of the decay curves itself is controlled by the Display Parameters the same way as for decay curves recorded in the Single, Oscilloscope, or FIFO mode. The display scale can be linear or logarithmic, an autoscale function is available, and the curves can be displayed as individual data points, lines, or data points connected by lines. Please see software description in the bh TCSPC Handbook [1] or in the handbooks of the DCS-120 FLIM system [5] or of the FLIM systems for the Zeiss LSM 710/780/880 family microscopes [6].
Other
New SPCM Features
There are
other new SPCM features which have already been added in previous software
versions. For example, SPCM is able to control Ti:Sa lasers and the bh AOM
module [1]. Ti:Sa and AOM control is used in the
DCS-120 MP multiphoton FLIM microscopes, please see [5]. The features can, however, also
be used for user-built two-photon microscopes and for other Ti:Sa applications.
Control of a motorised sample stage has been implemented in SPCM version 9.76.
In combination with the bh DCS-120 confocal and multiphoton scanning FLIM
systems, the stage is used to record mosaics of FLIM images and to control the
microscope remotely. Please see [1] and [5].
References
1. W. Becker, The bh TCSPC handbook. Becker & Hickl GmbH, 7th ed. (2017). Available on www.becker-hickl.com