Table of Contents:

• Applications of Fluorescence Lifetime Imaging Microscopy
• FLIM-Based In Vivo Imaging
• Advanced FLIM Data Analysis
• Leading Technology for Applications You Haven’t Dreamed of

Applications of Fluorescence Lifetime Imaging Microscopy

From the beginning, bh Fluorescence Lifetime Imaging Microscopy (FLIM) systems have been designed with application in Life Sciences in mind. High sensitivity, high photon efficiency, high time resolution, fast recording, and the ability to resolve multi-exponential decay functions are the basic requirements in these applications. Moreover, bh systems address the complexity of live systems by their  ability to record several biological parameters simultanously and in their mutual depencence. With functions like simultaneous FLIM and PLIM, parameter-controlled mosaic FLIM, multi-wavelength FLIM, or triggered recording of fast physiological effects bh’s multi-dimensional TCSPC technique addresses these needs almost perfectly. These features make bh FLIM systems superiour to other systems on the market.

Basis of all these applications is the fact that the fluorescence lifetime of most fluorophores is depending on their molecular environment. Accurate measurement of the fluorescence lifetimes or, more exactly, the fluorescence decay functions in the pixels of a FLIM image is therefore to obtain reliable information on biological systems. An inherent advantage of FLIM in this respect is that the fluorescence lifetime, within reasonable limits, does not depend of the concentration of the fluorophore, the laser power, the detector gain, or other experimental or instrumental details. An example of a Calcium-concentration measurement is shown in the figure on the right.

Although the bh FLIM technique is perfectly suited for entry-level applications it develops its full power in advanced applications.

Molecular Imaging

‘Molecular Imaging’ includes a wide range of applications, such as pH measurement in tissues, cells, and subcellular compartments, concentration of physiologically relevant ions, such as Ca⁺⁺, Mg⁺⁺, Na⁺, K⁺, Cl^–, or Oxygen concentration, or Glucose concentration. Also membrane-potentil-measurements fall into this catagory.

Metabolic Imaging

Metabolic Imaging uses the decay functions of NAD(P)H and/or FAD to determine the metabolic state of cells and tissues. Here, the information is not primarily in the fluorescence lifetimes but in the multi-exponential composition of the decay.

FRET Imaging

FRET (Förster Resonance Energy Transfer) measurements use the energy transfer from a donor to an acceptor molecule to probe protein conformation and interaction between different proteins. Also here, the fluoresence decay functions are multi-exponential, and the FLIM technique has to resolve the parameters of the complex decay to extract full FRET information from the data.

Simultaneous NAD(P)H, FAD and pO2 Imaging

The bh technique can combine metabolic imaging with simultaneous pO2 imaging. With this combination, the metabolic state of a cell can be investigated in dependence of the oxygen concentration.

Ultra-Fast Decay in Biological Material

bh FLIM systems open the path to the use of ultra-fast fluorescence-decay effects in biological and medical imaging. Lifetimes of carotenods and  melanins and their dependence on the cell or tissue status remain undetected in other FLIM systems but are easiliy recorded by bh FLIM systems.

FLIM-Based In Vivo Imaging

The methods of Fluorescence Lifetime Imaging Microscopy (FLIM) are particularly suited for in-vivo diagnostics as they are noninvasive and non-destructive. Thus, they are widely used for clinical research and medical examination on live subjects. Examples are given below.

Pre-Clinical FLIM

Tumor progression in small animals can be observed with the DC-120 MACRO FLIM system. The image beow shows an open tumor in a mouse. Tumor tissue is clearly discriminated from normal tissue.

In-vivo Diagnostics of Skin

Multiphoton-lifetime tomography of human skin uses two-photons excitation in combination with non-descanned detection. Multi-photon FLIM delivers optically sectioned images of tissue layers as deep as 100 µm. In-vivo two-photon imaging of human skin cells is possible without impairing the viability of the tissue. From z-stacks of FLIM images three-dimensional structures can be reconstructed at sub-cellular resolution.

Fluorescence-Lifetime Ophthalmoscopy (FLIO)

Ophthalmic FLIM uses a combination of fast beam scanning and excitation by a picosecond diode laser. This method is so sensitive that it is able to record lifetime images of the fundus (background) of the human eye. By this, the early discovery of eye diseases is possible, as these are often accompanied by metabolic changes of the fundus. In turn, these cause changes in the fluorescence decay parameters of endogenous fluorophores.

Personalized Chemotherapy

To target the special type of cancer a patient is suffering from, it is crucial to find the most efficient anti-cancer drug. The response of cancer cells to the different types of drugs is not entirely predictable though. Therefore a biopsy is taken, the cells are cultured and treated with different drugs. At the same time they are repeatedly imaged by FLIM. Fluorescence lifetimes indicate early shifts in the metabolic state of a cell after the treatment. Thus, with these measurements the most efficient medication can be determined within only a few days.

Advanced FLIM Data Analysis

All Becker & Hickl FLIM systems use SPCImage NG FLIM data analysis. SPCImage NG fits the decay data in the pixels if the image by a single, double, or triple-exponential model. The fit procedure uses a Maximum Likelihood Estimation (MLE) algorithem. Compared to the traditional Weighted Least Square (WLS) fit MLE is free of lifetime bias at low photon numbers per pixel and delivers a far better resolution of multi-exponential decay parameters. The calculation runs on a Graphics Processing Unit (GPU) The GPU runs the data analysis in a large number of pixels in parallel. Calculation therefore runs at unprecendented speed. Large FLIM images which formerly required calculation times of 10 minutes and more are now analysed within a few seconds.

SPCImage NG combines Time-Domain analysis with Phasor Analysis. The ‘Phasor Plot’ allows the user to combine the temporal data of pixels of similar decay signature into a single fluorescence decay curve. This way, eccurate multi-exponential decay data are obtained from data of low photon number. In particular, moving objects in large FLIM Mosaic data can be identified, the corresponding pixel data combined, and the results be analysed at unprecendented precision.

SPCImage also does away with an old problem of FLIM data anylysis analysis. Decay analysis requires the Instrument-Response Function (IRF) of the recording system to be known. Measurement of the IRF in a FLIM system is difficult or even impossible. SPCImage NG therefore determines the IRF form a multi-parameter analysis of the FLIM data themselves. This not only makes the use of the FLIM system more user friendly, it also avoids artefacts from an incorrectly measured IRF.

Leading Technology for Applications your Haven’t Dreamed of

Choose the ideal equipment for your laboratory or clinic from our complete high-grade FLIM systems or our versatile upgrades for your existing microscope and scanner. You need further information on which solution would be best for your desired application? Send us an inquiry via our contact form or call us at +49 (30) 212 80 02-0!