Welcome to LUMETRIC

LUMETRIC is a software tool for fluorescence microscopy that works together with Micro‑Manager. It lets you automatically capture images, select regions of interest (ROIs), and track fluorescence intensity or intensity ratios (e.g. in FRET experiments) in real time—all in one interface without switching between programs.

LUMETRIC extends fluorescence microscopy workflows by providing integrated, real-time feedback during image acquisition. It enables live visualization of ROI-based intensity traces and ratio signals directly during experiments, allowing users to monitor signal dynamics as data are being recorded.

In addition, LUMETRIC supports flexible acquisition protocols such as alternating excitation and photoswitching, enabling experimental designs that are widely used in practice. These capabilities are accessible within a unified graphical interface that combines acquisition control, real-time analysis, and live plotting.

By consolidating these functionalities into a single, open and transparent framework, LUMETRIC facilitates direct, reproducible monitoring of fluorescence signals and reduces reliance on fragmented workflows or external post-processing steps. This integration further enables users to perform advanced experiments independently of commercial, proprietary software environments.

LUMETRIC offers two ways to run image acquisition, depending on the required timing precision and hardware setup:

1. Software-controlled mode, which relies on standard Micro‑Manager control and is suitable for simple single wavelenght experiments without additional hardware
2. Hardware-triggered mode, which uses an external timing device to precisely synchronize camera exposure and illumination, enabling more complex and high-speed experiments

Despite these differences, both modes share the same interface, analysis tools, and data output, allowing users to switch between them without changing their workflow.

Intended use and scope

LUMETRIC is designed for fluorescence microscopy experiments where real-time feedback and reproducible data analysis are essential. It is particularly suited for workflows that require:

• Live monitoring of fluorescence intensity and intensity ratios (e.g. FRET signals)
• Multi-channel imaging and sequential acquisition steps
• Alternating excitation or photoswitching protocols

What LUMETRIC provides (independent of acquisition mode)

The following features are available in all versions of LUMETRIC, regardless of the selected acquisition mode (including LUMETRIC lite):

Image acquisition structure

• Set exposure time, frame interval, and number of frames
• Support for single-channel and split-image (e.g. opto-split) setups
• Automatic saving of images in TIFF format
• Automatic saving of acquired raw data

Real‑time analysis and visualization

• Live measurement of fluorescence intensity in user-defined ROIs
• Background correction using dedicated background ROIs
• Temporal and spatial ratio calculations (including FRET analyses)
• Simultaneous display of multiple live plots
• Ability to add event markers during acquisition

Data management and reproducibility

• Automatic creation of experiment‑specific output folders
• Export of raw and corrected ROI data as CSV
• Export of acquisition settings in human‑readable and hardware‑resolved formats
• Saving of ROIs in ImageJ‑compatible format
• Optional post‑processing of completed experiments without re‑acquisition

These features define the core LUMETRIC data and analysis pipeline and do not depend on the chosen acquisition backend.

Acquisition modes

LUMETRIC provides two acquisition modes, which differ in how precisely image timing and device control are handled. This allows users to choose between a simple setup without additional hardware and a more advanced setup for high-speed or complex experiments. Both modes share the same interface, live analysis tools, and data output, ensuring a consistent workflow independent of the chosen configuration.

Mode comparison

LUMETRIC lite (software‑controlled)

LUMETRIC lite uses Micro‑Manager’s software‑triggered acquisition mechanisms and does not require additional hardware.

Typical use cases:

• Single‑wavelength and low‑speed (>150 ms) experiments
• Live visualization of intensity or ratio signals
• Single-channel or FRET-Experiments

LUMETRIC Imaging System (hardware‑triggered)

The full LUMETRIC Imaging System extends Micro‑Manager with a Arduino‑based timing unit. Before acquisition, the complete experiment sequence is uploaded to the controller, which then executes the protocol autonomously.

Typical use cases:

• High‑speed acquisitions
• complex acquisition protocols with alternating excitation wavelength
• Photoswitching, photoactivation, and multicolor protocols
• Experiments requiring deterministic timing across thousands of frames