FLIM - the Lambert Solution
Lambert Instruments (“Lambert”), is a well-known manufacturer of high-speed, intensified and fluorescence imaging systems, with extensive experience in fluorescence lifetime imaging microscopy (FLIM). Their cutting edge FLIM systems and components are used in a broad range of research applications, including cell biology, cancer research and high throughput screening. The quality and performance of Lambert FLIM systems are trusted by researchers in the field worldwide.
Lambert FLIM systems are designed to accurately measure and analyse fluorescence lifetimes using frequency -domain techniques. Using high modulation frequencies allows for precise measurement of short fluorescence lifetimes, enabling researchers to study dynamic processes. The LIFA FLIM systems provide high lifetime resolution, single-photon sensitivity, precise timing measurements, a wide dynamic range, integration capabilities, and real-time imaging, all of which contribute to improved accuracy and versatility in fluorescence lifetime measurements.
What is FLIM?
FLIM is an acronym for Fluorescence Lifetime Imaging Microscopy. It is a technique used in fluorescence microscopy to measure the lifetimes of fluorophores, molecules that absorb light at a specific wavelength (excitation) and subsequently emit light at a longer wavelength (emission). FLIM provides information about the fluorescence lifetime of the fluorophores, the average time a fluorophore stays in an excited state before returning to the ground state and emitting light.
FLIM takes advantage of the fact that the lifetime of a fluorophore can be influenced by its microenvironment. For example, if a fluorophore is near another molecule, its lifetime may change due to energy transfer between them. FLIM measures these changes in lifetime to provide information about the local environment. By analysing the fluorescence lifetimes, FLIM can provide valuable information about various biological processes, such as protein-protein interactions, membrane dynamics, and cellular metabolism. Traditionally, FLIM has applications in fields like cell biology, neuroscience, and medical research, enabling scientists to study dynamic molecular events and gain insights into complex biological systems. However, FLIM’ s ability to rapidly provide researchers with lifetime images of their samples or study their dynamics, makes it a promising technology for areas outside the field of biology.
The Lambert Solution
Lambert’s LIFA is a camera based FLIM system. Combining excellent light sensitivity, easy image acquisition and data analysis with easy integrates into any fluorescence microscope, Lambert’s solution provides a plug-n-play experience that allows for flexibility to switch between setups quickly and easily, simplifying experiments for researchers and imaging centres. The systems are coupled with advanced tools and software for data acquisition, analysis, and visualisation of fluorescence lifetime data.
A standard Lambert LIFA system consists of a camera, light source, capture software and computer with USB connection. The camera, in combination with the light source, makes it possible to carry out frequency domain FLIM measurements. Acquisition and analysis of the FLIM data takes place through the capture software, which supports features as timelapse and multi-frequency recordings.
Utilising a fluorescence microscope, the sample of interest is illuminated with a modulated light source, such as a laser or LED. The emitted fluorescence from the sample is collected by a specialised camera. Lambert’s Capture software then takes the recorded modulated signal and calculates the phase and modulation lifetime per pixel. This data is used to generate two lifetime images, one for the phase lifetime and one for the modulation lifetime.
LIFA Cameras
The LIFA vTau camera features an image sensor with excellent temporal resolution, in the sub-nanosecond or even picosecond range. This high time resolution enables accurate measurement of short fluorescence lifetimes, which can be crucial for studying fast dynamic processes or distinguishing different fluorophores with similar emission wavelengths but different lifetimes.
Pixel resolution Readout Noise
512 x 512 px < 7.04e-6
Pixel size Framerate
16 µm 300 fps
Dark current
0.0154 /s
LIFA TRiCAM is a compact intensified camera designed for applications that require low-light imaging. TRiCAM is capable of ultra-short exposures through fast gating and frequency-domain imaging. With a gated TRiCAM camera, LIFA records a series of images and automatically shifts the timing between the light pulse and the camera exposure for time-domain FLIM on Widefield microscopes
Pixel resolution Readout Noise
1920 x 1200 px < 6.5e
Pixel size Framerate
7.6 µm >100 fps
Dark current
<7 e-/s
Light Source
The Multi-LED is a versatile pulsed excitation light source which contains up to 4 LEDs that provide non-phototoxic illumination levels, have a low cost and a long economic lifespan for FLIM imaging. Seamlessly integrated with the LIFA software, available wavelengths cover the range from 360 to 640 nm.
LIFA Software
Focusing on user-friendly solutions, Lambert’s FLIM systems are designed with intuitive interfaces and easy-to-use software, making them accessible to researchers with varying levels of expertise. They are compatible with various microscope platforms and easily integrated into existing setups.
The LIFA software guides you through your FLIM experiments from start to finish. A live view from the camera makes finding the right FLIM settings easy. the software records the FLIM data and instantly calculates the fluorescence lifetime. A time-lapsed video of the sample can also be recorded to see how the lifetime changes over time. Results can be analysed as statistical data or in several visual representations including histograms, scatter plots or a phasor plot. Lambert hardware is integrated seamlessly, allowing researchers to focus on the experiment.
Configurations
Widefield Configuration
On widefield microscopes, the LIFA vTAU camera in combination with the Multi-LED offers a capable and compact FLIM solution. vTAU connects to the widefield microscope via the camera port, while the Multi-LED connects via the standard epifluorescence port, creating in all-in-one solution.
Spinning-Disk Confocal Configuration
Being a camera-based system, the Lambert Instruments LIFA system for frequency domain FLIM is compatible with multibeam confocal microscopy techniques, most notably the Yokogawa CSU spinning disk series (based on the Nipkow disk scanner) and the VTInfinity series by Visitech International.
TIRF Configuration
Total Internal Reflection Fluorescence (TIRF) microscopy facilitates extremely high contrast visualization and thereby high sensitivity of fluorescence near the cover glass. Typically, the optical section adjacent to the cover glass is about 100nm. The unique combination of TIRF and frequency-domain FLIM makes it possible to measure lifetimes of, for instance small focal adhesions near the cover glass.
Lambert History
Lambert understands the diverse requirements of FLIM applications and offers customisable solutions. Researchers can select from different camera options excitation sources, and detectors to tailor the FLIM system to their specific needs. Known for providing reliable customer support and service, they offer technical assistance, training, and ongoing support to ensure that researchers can make the most of their FLIM systems.
1992 Lambert Instruments was founded by Bert van Geest, a specialist in cooled intensifiers and very high-speed cameras for Astronomy and Scientific Markets.
Early 2000 the first LIFA (Lambert Instruments Fluorescence attachment) FLIM system was developed. The LIFA was then developed into a commercial application, which later became a turnkey product including hardware (intensifier) and software, allowing biologists to upgrade their existing fluorescence microscope, into an advanced fluorescence microscope capable of FLIM imaging.
2014 the first FLIM System with a Toggel camera based on solid-state technology without the image intensifier was developed. Lambert has over 80 units installed Worldwide.
2023 The New LIFA camera is released. The New LIFA camera is a unique product that operates in the frequency domain. The fully integrated solution combines a FLIM system with a light source, high speed detector and software. The LIFA detector has excellent temporal resolution in the sub-nanosecond or even picosecond range. This high time resolution enables accurate measurement of short fluorescence lifetimes.
About the Author
Johan Herz, MSc Biomedical Engineering, Business Development Manager at Lambert Instruments. Johan joined Lambert as an intern during his final year at university in 2011 and later became the Specialist Service Engineer at Lambert for the LIFA Fluorescence Lifetime Imaging Microscopy (FLIM) system.
