Control latency limits the response time of all USB-connected microscope accessories, including cameras, stage controllers, filter wheels, and Lumencor light engines. Latency originates from the fact that the supervising PC operating system (usually Microsoft Windows) must allocate time to many competing tasks. As shown in the adjacent data plots, the impact on the user is that the duration of light output on the specimen is longer, and sometimes much longer, than the exposure time set in the acquisition software.
Duration of light output produced by a CELESTA light engine®, and detected by an external analog photodiode, in response to various exposure times set for a Hamamatsu ORCAFlash 4.0 camera controlled by MicroManager (v1.4.23) image acquisition software. Note that the discrepancies between set exposure time and light output duration are not speciﬁc (except in minor details) to any particular image acquisition software or light engine or camera model. Panel A shows data for light engine control via LEGACY mode USB communication. Panel B shows the same nominal exposure sequence controlled via STANDARD mode USB communication.
Two scenarios are shown, one using LEGACY mode USB communication (as implemented on the SPECTRA X and SOLA SE light engines), and the other using STANDARD mode communication (as implemented on the AURA III, SPECTRA III and CELESTA light engines). Because the USB data transmission rate in STANDARD mode is faster than that of LEGACY mode (115,200 vs 9,600 baud), it provides a signiﬁcant reduction of latency for exposure times on the order of 100 ms (and above). However at short exposure times (5–10 ms), the impact of the faster communication speed in STANDARD mode diminishes, as the response is dominated by the software processing speed.
To obtain light output durations less than about 50 ms, timing must be derived from a hardware controller instead of the PC operating system. The hardware controller supplies TTL timing signals to the light engine via a breakout cable (Table 1). Examples of millisecond-duration light pulses generated in this way can be found in the Performance section of our website. At the present time, the capacity to acquire time-lapse sequences of short duration exposures is limited by the camera (modern sCMOS cameras typically have a maximum frame rate of around 100/second), rather than by the light source.
To support the long-term stability of the laser light sources in CELESTA, SPECTRA and ZIVA light engines it is recommended that they should be operated only in environments where the dew point is below 15ºC. For reference, at a typical room temperature of 24ºC, a dew point of 15ºC corresponds to 57% humidity. The current dew point inside the light engine, calculated from onboard temperature and humidity sensors, is displayed on the settings page of the onboard control GUI.
Standby Mode, previously described in the October 2019 issue of Light Reading, is another control system designed to support the long-term stability of the laser light sources. Consequently, users writing their own light engine control software are strongly advised to NOT programmatically disable standby mode.
If operational situations arise where it is necessary to avoid the onset of standby mode during a data acquisition process, please contact firstname.lastname@example.org. We will be happy to work with you and our software engineering team to devise appropriate solutions.
Dichroic Mirrors and Filters for SPECTRA, CELESTA, and ZIVA Light Engines®
In fluorescence microscopy, the filter set, consisting of excitation and emission bandpass filters and a dichroic beamsplitter, plays a critical role. It performs the essential functions of directing excitation light from the light source to the sample and then separating it on the basis of wavelength, from fluorescence emitted from the specimen. The filter set consists of excitation and emission bandpass filters and a dichroic beamsplitter. Optimized filter sets are critical because fluorescence emission from a microscopic specimen ismany orders of magnitude (>106) weaker than the excitation from the light source. Recognizing optimized filter set specifications are critical for obtaining images with high signal:background contrast. Lumencor has developed uniquely high performance, multiband dichroic beamsplitters and multiband and single band emission filters for use with our SPECTRA, CELESTA, and ZIVA light engines®. These light engines are setting the standard for high performance, high brightness, turn-key solutions in solid-state lighting for life and industrial sciences.
All emission filters and dichroics have standard dimensions and are supplied in an unmounted form. Installation in filter cubes, filter wheels, or other instrumentation-specific mountings is required before use.
The multiband emitters and dichroics are optimized for compatibility with the electronically selectable excitation outputs of SPECTRA, CELESTA, and ZIVA light engines® (Figure 1). This enables fast multicolor imaging without the need for filter wheels or other positioning devices to execute filter interchanges. Single bandpass filters are offered for use in situations where fluorescence crosstalk (e.g. detection of FITC fluorescence derived from violet (DAPI) excitation) confounds identification of fluorescently labeled components of the specimen.
Figure 1. Transmission spectra of CELESTA/ZIVA VCGRnIR pentaband dichroic and emitter superimposed on the violet, cyan, green, red, and near-infrared output lines of the CELESTA and ZIVA light engines®.
Lumencor’s Earth Day Light Microscopy Imaging Competition is in full swing, offering the opportunity to win up to $10,000 worth of state-of-the-art, solid-state lighting! Submit your images today and help us celebrate and promote a brighter, greener planet through the use of mercury-free illumination. Qualifying images must be acquired using Lumencor light engines.
Lumencor can support your laboratory as you return to work. The challenges are numerous for researchers and instrument manufacturers alike- maintain best lab practices, manage constrained budgets, keep staff safe and productive. Let Lumencor help with illumination tools designed for the best data quality and best lab safety!