When Measurements Must Not Miss a Moment
Fast dynamic processes often happen on time scales much shorter than the total duration of an experiment. A cell passing through a laser focus, a molecule changing its state, or a correlation signal evolving over time may only produce relevant photon events for a brief moment. In such cases, the measurement system must continuously capture consecutive data blocks without introducing blind gaps between them.
These data blocks can represent much more than repeated intensity values. Depending on the application, they may contain fluorescence lifetime histograms, time traces, correlation curves, or other time-resolved observables. PicoQuant time taggers support such continuous, gapless measurements through complementary hardware and software-based approaches.
Application Example: Fluorescence Lifetime Flow Cytometry
A good way to illustrate the need for continuous measurements is fluorescence lifetime flow cytometry. In this technique, cells or particles pass through an observation volume while their fluorescence decay is recorded. As illustrated, each object remains in the detection region only for a short time, producing a transient burst of photon events.
To analyze such signals, the photon stream can be divided into consecutive acquisition windows. Each window contains the photons detected during a defined time interval and can be used to build a fluorescence decay histogram for lifetime analysis. By arranging these histograms as a continuous sequence, the passage of individual cells or particles can be followed without losing information between measurement windows.
This application also shows why the implementation matters. At high photon count rates, software-based histogramming can become demanding because every detected event has to be transferred, buffered, and processed by the computer. Dedicated hardware histogramming provides a more direct route: the time tagger itself accumulates the histograms, allowing consecutive lifetime data blocks to be generated with very high throughput. This approach was developed in the context of fluorescence lifetime flow cytometry work with the group of Melissa Skala at the Morgridge Insitute for Research.
Continuous Hardware Histogramming
For applications that require rapid sequences of fluorescence decay histograms, continuous hardware histogramming provides an efficient solution. Instead of transferring every detected photon event to the computer for software-based binning, HydraHarp 500 accumulates histograms directly in the instrument. The resulting histogram blocks can then be read out consecutively and processed as a continuous measurement sequence.
In Conti Mode, the measurement is started once and the hardware automatically generates consecutive histogram blocks. Each block represents one defined acquisition window and contains the accumulated decay histograms for the enabled channels, together with timing information such as block number, start time, and duration. The software simply retrieves each completed block for display, evaluation, or storage while the next blocks are already being generated.
Conti Mode Workflow
- Configure the HydraHarp 500 and input channels.
- Set histogram parameters and acquisition time per block.
- Start one continuous measurement.
- Retrieve consecutive histogram blocks from the device.
- Display, evaluate, or store the histogram sequence.
Because the histogramming step is performed in hardware, the computer does not need to process the full raw time-tag stream before data evaluation. This makes Conti Mode particularly suitable for histogram-based applications at high count rates, such as fluorescence lifetime flow cytometry.
A C example demonstrating Conti Mode via the PicoQuant core DLL is available on GitHub.
Beyond Histograms: Gapless Software Sequences
Continuous measurements are not always limited to fluorescence decay histograms. In many experiments, other observables need to be followed over time, such as intensity traces, or correlation measurements. This is where snAPI provides a flexible software-based approach.
Using Sequence Mode, a measurement can be divided into consecutive segments instead of returning only one result for the complete acquisition. The sequence can be defined using either a time-based or count-based criterion, corresponding to the Timer and Counts options in Sequence Mode. Each segment is then evaluated as part of a continuous measurement sequence.
The key advantage is that this concept is not restricted to histograms. Sequence Mode can be combined with different snAPI measurement classes and manipulators, allowing users to record time traces, evaluate correlation measurements, such as FCS or g² data, or apply additional processing steps while preserving the sequence structure. This flexibility is illustrated in the examples below.
Choosing the Right Measurement Approach
Both approaches enable gapless measurement sequences, but they serve different priorities.
Conti Mode is ideal when the goal is a fast sequence of fluorescence decay histograms at the highest count rates. Here, histogramming is performed directly in the HydraHarp 500 hardware.
Sequence Mode via snAPI is the more flexible option when the analysis is not restricted to histogramming, but involves, for example, time traces, g², FCS, and workflows using manipulators.
Together, they allow users to choose between maximum histogram throughput in hardware and flexible time-resolved analysis in software.
Want to implement continuous measurement workflows in your own experiments? Explore how HydraHarp 500 and snAPI support gapless time-resolved acquisition and analysis.
