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Understanding Fluorescence Lifetime Imaging Microscopy (FLIM)

Fluorescence Lifetime Imaging Microscopy (FLIM) represents a powerful imaging technique that enables researchers to investigate the dynamics of fluorescence signals within biological samples with remarkable precision and specificity. In this article, we’ll delve into the world of FLIM, exploring its techniques, applications, and recent advancements, shedding light on its significance in biomedical research and beyond.

FLIM is a non-invasive imaging technique that measures the fluorescence lifetime of fluorophores within biological samples. Unlike conventional fluorescence microscopy, which relies on intensity measurements, FLIM focuses on the temporal characteristics of fluorescence emission, providing valuable insights into molecular interactions, microenvironment changes, and cellular processes.

Techniques and Principles of FLIM

FLIM relies on the principles of fluorescence spectroscopy and time-correlated single-photon counting (TCSPC) to measure the lifetime of fluorescent molecules. The technique involves exciting fluorophores with a short pulse of light and then detecting the decay of fluorescence emission over time. By analyzing the decay curve, researchers can determine the fluorescence lifetime of the fluorophores, which is influenced by factors such as molecular environment, pH, and molecular interactions.

FLIM Cameras

LIFA FLIM

LIFA FLIM

with SPAD powered vTAU camera

Applications of FLIM in Biomedical Research

Protein-Protein Interactions:

FLIM is widely used to study protein-protein interactions within cells, providing insights into protein dynamics, localization, and complex formation. By labeling proteins of interest with fluorescent tags and performing FLIM analysis, researchers can visualize and quantify protein interactions in real-time, helping to unravel the complexities of cellular signaling pathways and molecular mechanisms.

Metabolic Imaging:

FLIM has emerged as a powerful tool for metabolic imaging, enabling researchers to monitor metabolic processes such as glycolysis, oxidative phosphorylation, and lipid metabolism in living cells and tissues. By measuring the fluorescence lifetime of metabolic fluorophores such as NAD(P)H and flavins, FLIM can provide valuable information about cellular metabolism and energy production, offering potential applications in cancer research, neurobiology, and metabolic diseases.

Drug Discovery and Development:

FLIM is increasingly being utilized in drug discovery and development processes to evaluate the efficacy and toxicity of pharmaceutical compounds. By monitoring changes in cellular fluorescence lifetime in response to drug treatments, FLIM can provide insights into drug-target interactions, cellular uptake, and cytotoxicity, facilitating the screening and optimization of potential therapeutics.

Recent Advancements and Future Directions

Recent advancements in FLIM technology have expanded its capabilities and applications, making it more accessible and versatile for researchers across disciplines. Innovations such as multiphoton FLIM, fluorescence lifetime imaging with phasor analysis (FLIM-Phasor), and time-gated FLIM have enhanced the spatial and temporal resolution of FLIM imaging, enabling new discoveries in areas such as neurobiology, stem cell research, and tissue engineering.

Conclusion

Fluorescence Lifetime Imaging Microscopy (FLIM) represents a sophisticated imaging technique that offers unique insights into the dynamics of fluorescence signals within biological samples. With its ability to visualize molecular interactions, metabolic processes, and cellular dynamics in real-time, FLIM has become an indispensable tool in biomedical research, driving discoveries and advancements in fields such as cell biology, pharmacology, and disease pathology. As technology continues to evolve, the future of FLIM holds immense promise, with the potential to unlock new frontiers in understanding the complexities of life at the molecular level.

Discover More: https://en.wikipedia.org/wiki/Fluorescence-lifetime_imaging_microscopy

Fluorescence Imaging: A Comprehensive Guide

In the realm of modern scientific exploration and medical diagnostics, fluorescence imaging stands out as a powerful technique, offering unparalleled insights into the world of cells, molecules, and tissues. From unraveling the mysteries of biological processes to advancing medical diagnostics, it has revolutionized the way researchers and clinicians visualize and understand the intricate workings of life.

Understanding Fluorescence Imaging
It is a versatile imaging technique that utilizes the natural fluorescence properties of certain molecules or artificially introduced fluorophores to generate detailed, high-resolution images. This technique relies on the principle of fluorescence, wherein certain substances absorb light at a specific wavelength and then emit light at a longer wavelength.

Applications in Scientific Research
In scientific research, it plays a crucial role in elucidating various biological phenomena. Researchers use fluorescent markers or dyes to label specific molecules or structures within cells, allowing them to track molecular interactions, observe cellular processes, and study the localization of proteins with remarkable precision.

From studying the dynamics of gene expression to investigating the behavior of individual cells in complex biological systems, fluorescence imaging provides researchers with a powerful tool to explore the intricacies of life at the molecular level.

Advancements in Medical Diagnostics
In the field of medical diagnostics, it offers a non-invasive and highly sensitive approach for detecting and visualizing diseased tissues. Fluorescent contrast agents can be selectively targeted to specific biomarkers or pathological features, enabling clinicians to identify tumors, visualize blood vessels, and assess tissue viability with exceptional accuracy.

Cutting-Edge Technologies
Recent advancements in fluorescence imaging technologies have further expanded its capabilities and applications. High-speed cameras, advanced microscopy techniques, and sophisticated image analysis algorithms have enhanced the resolution, sensitivity, and speed of fluorescence imaging, enabling researchers and clinicians to delve deeper into the intricacies of biological system

Fluorescence Imaging Cameras

High-Speed Camera

HiCAM Fluo

Cooled High-speed Camera for Fluorescence Imaging

Low-Light Imaging

TRiCAM

Intensified high-speed high-sensitivity imaging camera

TRiCATT

Compact lens-coupled image intensifier

Future Directions

As this type of imaging continues to evolve, researchers are exploring new avenues and pushing the boundaries of what is possible. Emerging technologies such as super-resolution microscopy, multiphoton imaging, and optogenetics promise to further revolutionize our understanding of cellular dynamics and disease mechanisms.

Conclusion
Fluorescence imaging represents a cornerstone of modern scientific research and medical diagnostics, offering unprecedented insights into the inner workings of life. With its versatility, sensitivity, and non-invasive nature, fluorescence imaging continues to drive discovery and innovation across a wide range of disciplines. As we look to the future, the potential of fluorescence imaging to uncover new mysteries and transform healthcare remains boundless.

For more information click here: https://en.wikipedia.org/wiki/Fluorescence_imaging

Frequency-Domain FLIM: Basic Equations

In this article the first principles of frequency domain (FD) fluorescence lifetime imaging microscopy (FLIM) are further explained through use of equations. Although these principles not necessary for the execution of basic lifetime measurements, a thorough understanding provides the groundwork that enables deeper insight into your results and into the possibilities of FD lifetime imaging.

Fluorescence Lifetime 

For an ensemble of fluorescent molecules the rate at which molecules decay from the excited state to the ground state (cf. Fig.1) is proportional to the number of excited state molecules N:

Homodyne Detection

FLIM Papers and Reviews

A selection of papers papers based on Lambert Instruments FLIM systems is maintained here.

The following is a selection of books and papers on FLIM technology:

BOOKS / REVIEWS:

Gadella TW Jr., FRET and FLIM techniques, 33. Elsevier, ISBN-13: 978-0080549583. (Dec 2008) 560 pages. Elsevier link

Periasamy, A & Clegg RM, FLIM Microscopy in Biology and Medicine. Chapman & Hall/CRC, 1st edition, ISBN-13: 978-1420078909. (Jul 2009) 368 pages. Amazon link

Lakowicz JR. Principles of fluorescence spectroscopy, 3rd edition, ISBN-13: 978-0387312781 . Springer, 3rd edition (Sep 2006) 954 pages. Amazon link

Van Munster EB, Gadella TW Jr. Fluorescence lifetime imaging microscopy (FLIM). Review. Adv Biochem Eng Biotechnol. (2005) 95:143-75. Pubmed link

BOOKS; FLUORESCENT PROTEINS / CELL BIOLOGY:

Sullivan KF, Fluorescent Proteins, 2nd Edition Volume 85. Academic Press, ISBN-13: 978-0123725585. (Dec 2007) 660 pages. Elsevier link
Sullivan KF, Kay SA, Wilson L, Matsudaira PT,Green Fluorescent Proteins, Volume 58. Academic Press, ISBN-13: 978-0125441605. (1998) 386 pages. Amazon link


PAPER MULTIFREQUENCY:

Squire A, Verveer PJ, Bastiaens PIH, Multiple frequency Fluorescence lifetime imaging microscopy. Journal of Microscopy, (2000) 197(2):136-149. Pubmed link


PAPER PHASE STEP ORDER:

Van Munster EB, Gadella TW Jr, Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order. Cytometry A. (2004) 58(2):185-94. Pubmed link


PAPERS POLAR PLOT:

Redford GI, Clegg RM, Polar plot representation for frequency-domain analysis of fluorescence lifetimes. Journal of Fluorescence (2005) 15(5):805-815. Pubmed link
Clayton AHA, Hanley QS, Verveer PJ. Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy (2004) 213(1):1-5. Pubmed link


PAPERS TIRF-FLIM:

Valdembri D, Caswell PT, Anderson KI, Schwarz JP, König I, Astanina E, Caccavari F, Norman JC, Humphries MJ, Bussolino F, Serini G,

Fluorescence Lifetime Imaging Microscopy

What is the Fluorescence lifetime?
The fluorescence lifetime – the average decay time of a fluorescence molecule’s excited state – is a quantitative signature which can be used to probe structure and dynamics at micro- and nano scales. FLIM (Fluorescence Lifetime Imaging Microscopy) is used as a routine technique in cell biology to map the lifetime within living cells, tissues and whole organisms. The fluorescence lifetime is affected by a range of biophysical phenomena and hence the applications of FLIM are many: from ion imaging and oxygen imaging to studying cell function and cell disease in quantitative cell biology using FRET.

For fluorescent molecules the temporal decay can be assumed as an exponential decay probability function:

where t is time and τ is the excited state lifetime.

More complex fluorophores can be described using a multiple exponential probability density function:

where t is time, τi is the lifetime of each component and αi is the relative contribution of each component.

Why Measure Fluorescence Lifetime?

A key advantage of the fluorescence lifetime is that it is a basic physical parameter that does not change with variations in local fluorophore concentration and is independent of the fluorescence excitation. Hence the lifetime is a direct quantitative measure, and its measurement – in contrast to e.g. the recorded fluorescence intensity – does not require detailed calibrations. Excited state lifetimes are also independent of the optical path of the microscope, photobleaching (at least to first order), and the local fluorescence detection efficiency.

The fluorescence lifetime does change when the molecules undergo de-excitation through other processes than fluorescence such as dynamic quenching through molecular collisions with small soluble molecules like ions or oxygen (Stern-Volmer quenching) or energy transfer to a nearby molecule through FRET. As a result the fluorophores (in the excited state) lose their energy at a higher rate, causing a distinct decrease in the fluorescence lifetime. The measured rate of fluorescence is actually a summation of all of the rates of de-excitation. In this way the fluorescent lifetime mirrors any process in the micro-environment that quenches the fluorophores; and spatial differences in the amount of quenching reveals itself as contrast in a lifetime image.

Frequency-Domain FLIM for Beginners

Fluorescence lifetime imaging microscopy (FLIM) can be performed in the time domain and in the frequency domain. Scanning single point lifetime detection units on confocal laser scanning microscopes mainly operate in the time domain. Camera-based lifetime detection on widefield, multi-beam confocal and total internal reflection fluorescence (TIRF) microscopes operate both in time domain and frequency domain. The Lambert Instruments LIFA for example is a fast frequency-domain system, whereas the Lambert Instruments TRiCAM can be operated both in the frequency and time domains.

Time Domain

In the time domain the fluorescence decay can be measured by using time-correlated single photon counting (TCSPC) or fast-gated image intensifiers. A measurement requires short excitation pulses of high intensity and fast detection circuits. Each point in the sample is excited sequentially. TCSPC records a histogram of photon arrival times at each spatial location using Photo Multiplier Tubes (PMTs) or comparable single photon counting detectors. Fast-gated image intensifiers measure fluorescence intensity in a series of different time windows. With both time domain techniques lifetimes are derived from exponential fits to the decay data. When sufficient channels (time windows) are used, multi-exponential lifetimes can be extracted.

Fluorescence lifetime imaging microscopy (FLIM) can be performed in the time domain and in the frequency domain. Scanning single point lifetime detection units on confocal laser scanning microscopes mainly operate in the time domain. Camera-based lifetime detection on widefield, multi-beam confocal and total internal reflection fluorescence (TIRF) microscopes operate both in time domain and frequency domain. The Lambert Instruments LIFA for example is a fast frequency-domain system, whereas the Lambert Instruments TRiCAM can be operated both in the frequency and time domains.

Time Domain

In the time domain the fluorescence decay can be measured by using time-correlated single photon counting (TCSPC) or fast-gated image intensifiers. A measurement requires short excitation pulses of high intensity and fast detection circuits. Each point in the sample is excited sequentially. TCSPC records a histogram of photon arrival times at each spatial location using Photo Multiplier Tubes (PMTs) or comparable single photon counting detectors. Fast-gated image intensifiers measure fluorescence intensity in a series of different time windows. With both time domain techniques lifetimes are derived from exponential fits to the decay data. When sufficient channels (time windows) are used, multi-exponential lifetimes can be extracted.

Frequency Domain

The frequency-domain FLIM technique requires a modulated light source and a modulated detector. The excitation light is modulated or pulsed in intensity at a certain radio frequency (the blue curve in the figure below). The induced fluorescence emission will mirror this modulation pattern and show, due to the fluorescence decay, a delay in time in the form of a phase-shift (the red curve). In addition, the modulation depth will decrease with respect to the excitation light, while the average intensity remains the same. The phase-shift and modulation-depth directly depend on the fluorescence lifetime and the known modulation frequency (see figure).

To extract the phase shift and modulation depth from the fluorescence emission signal, a homodyne detection method is often used. In this method the sensitivity of the detector – often an intensified camera – is modulated (or gated) with the same radio frequency as the light source (the green curve in the figure on the right). For a camera detector the result is an intensity image with a fixed brightness. By shifting the phase of the image intensifier with respect to the light source in a series of fixed steps a low-pass signal is generated for each pixel: the output image will be brighter or dimmer depending on whether the detector sensitivity is in or out of phase with the fluorescence emission. The result is a frequency-domain FLIM signal as a function of the phase difference between light source and camera (purple curve in the figure on the right) for each pixel in the image.

The key is that this frequency-domain signal (purple curve) exactly mirrors the phase shift and demodulation in the time domain. The phase and modulation depth can be directly extracted from the measurements and are the fundamental data in a homodyne FD FLIM measurement.

From the acquired modulation depth and phase shifts, two independent determinations of the fluorescence lifetime can be calculated. For an absolute determination the system needs to be calibrated at the pixel level with a reference fluorophore of known lifetime. For this calibration the only requirement is a FLIM acquisition of the reference fluorophore with known lifetime (figure on the right).

Multi-Exponential Decay  
Some fluorophores have a multi-exponential decay, consisting of two or more lifetime components. For example, the decay of CFP is bi-exponential. These multiple lifetime components can be separated and extracted using multiple frequency measurements and the polar (or phasor) plot.

 

 

Advantages of Frequency-domain FLIM 
The key advantage of frequency-domain FLIM is its fast lifetime image acquisition making it suitable for dynamic applications such as live cell research: the entire field of view is excited semi-continously – using relatively broad excitation pulses – and read out simultaneously. Hence frequency domain lifetime imaging can be near instantaneous. Another advantage of a camera-based FLIM setup, such as the Lambert Instruments LIFA, is its ease of use and its low maintenance requirements. For more information about our products, please visit the FLIM product pages and our FLIM software page.

Confocal FLIM

Confocal microscopy is a technique for high-resolution three-dimensional imaging which uses a pinhole to increase resolution in the image plane and eliminate out-of-focus light in thick specimens. The thickness of the focal plane is generally defined mostly by the objective lens and also by the optical properties of the specimen and the ambient conditions. With an imaging confocal only the light within the focal plane is detected, so that the resulting confocal images appear crisper than widefield images [read more at the cell biology wiki]. Typical applications occur within the life sciences, e.g. in cell biology. In essence there are two classes of confocal systems: single beam and multi-beam.

Confocal Scanning with CSU Spinning Disk
A Nipkow spinning disk is a multi-beam confocal scanner. The main advantage of this type of confocal imaging is the relatively fast imaging acquisition making it useful for live cell imaging applications.

The operating principle of the Yokogawa CSU spinning disk is explained here. Briefly, the disk has a spiral pattern of pinholes that is illuminated by an expanded laser beam. This generates a multi-beam illumination pattern which which the sample is illuminated. By rapidly rotating the disk this multi-beam visits all positions in the sample plane near simultaneously. The part of the fluorescence that travels back through the pinholes generates a full field confocal image at the camera detector.

Lifetime images of mixed polled grains, at three different z-positions.

Being a camera-based system, the Lambert Instruments LIFA system for frequency domain FLIM is compatible with multi-beam confocal microscopy techniques, most notably the Yokogawa CSU spinning disk series (based on the Nipkow disk scanner), and the VTInfinity series by Visitech International Ltd.

 

TIRF FLIM

Total Internal Reflection Fluorescence (TIRF) microscopy is a super-resolution technique with high sensitivity of fluorescence near the cover glass. TIRF does not disturb cellular activity, and enables tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. TIRF enables the selective visualisation of processes and structures of the cell membrane and pre-membrane space such as vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The combination of TIRF and frequency domain FLIM makes it possible to measure fluorescence lifetimes of for instance small focal adhesions near the cover glass.

High NA TIRF objectives (up to 1.49) make it possible to introduce illumination at incident angles greater than the critical angle (θ
) resulting in TIR (Total Internal Reflection) accompanied by the formation of an evanescent wave immediately adjacent to the coverglass-specimen interface. The evanescent wave energy drops off exponentially with distance from the coverglass and reaches about a hundred nanometers into the specimen. For TIR to occur, the refractive index of the coverglass should be higher than the refractive index of the specimen (which is e.g. the case when using buffered saline solution).
The white-TIRF as well as the laser-TIRF system utilises this evanescent wave to excite fluorescent molecules in a very thin section in contact with the coverglass (here: green dots). Because the specimen is not excited beyond the evanescent wave (here: white dots), this imaging system can produce fluorescence images with an extremely high signal-to-noise (S/N) ratio and z-resolution.

For more information: WikipediaOlympus FluoView.

Single-Image Fluorescence Lifetime Imaging Microscopy

LIFA Flim features a unique image sensor that was designed and optimized specifically for fluorescence lifetime imaging applications. It enables lifetime imaging at unprecedented frame rates with the single-image fluorescence lifetime imaging microscopy (siFLIM) method [1].

“We used a prototype of the vTAU camera to develop a method for acquiring quantitative lifetime images from a single exposure.” says professor Kees Jalink of the Netherlands Cancer Institute. “siFLIM takes advantage of the technical capabilities of the Toggel camera and simultaneously records two 180°-phase-shifted images. This allows for video-rate lifetime imaging with minimal phototoxicity and bleaching.”

 

Demonstration of the Lambert Instruments Toggel camera for single-image FLIM (siFLIM) detection of histamine-induced alterations in Ca2+ concentration. Tiny …

During a single exposure, the Toggel camera records two images. The electrons in each pixel of the sensor are toggled between two storage areas at the same frequency as the modulation frequency of the light source. This results in two images that are shifted 180° in phase with respect to each other.

Because both images are recorded simultaneously, there are no artifacts of cellular movements and there is a significant improvement in the photon efficiency of the image acquisition.

These two images of convallaria were recorded simultaneously, and because of the 180° phase shift between the two images, the recorded light intensity differs. The difference in light intensity between these two images depends on the phase shift and demodulation of the fluorescence light and can be used to calculate changes in the fluorescence lifetime of a sample [1].

[1] siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data, M. Raspe et al, Nature Methods 13, 501–504 (2016)

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.