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Automated lifetime-based screening and characterization  of fluorescent proteins

Automated lifetime-based screening and characterization of fluorescent proteins

Lambert Instruments has provided a versatile library of interface functions (API) that allows direct communication with the LIFA software via MATLAB, in order to control the LIFA camera and the microscope. With the aid of this API, a custom made MATLAB graphical user interface (GUI) was developed that allows multi-position acquisition.

Revealing Cancer's Infrastructure

Lambert Instruments has been shipping the LIFA to cancer research facilities all over the world for years. We visited Dr. Kees Jalink of the Biophysics of Cell Signaling group at the Netherlands Cancer Institute. His research group purchased the first ever LIFA to leave the labs of Lambert Instruments. Ten years later, the LIFA is still their fluorescence lifetime imaging method of choice for studying signal transduction pathways in living cells.

High-speed in vivo imaging of a zebrafish heart

At the Max Planck Institute for Heart and Lung Research in Bad Nauheim (Germany), the cardiovascular system of the zebrafish is studied. The transparency of the zebrafish and its experimental advantages make it an ideal scale model of the human cardiovascular system.

Ion imaging

For ion imaging, several fluorescent indicators (sensor, construct, tracer, etc) are available that have a change in quantum yield upon ion binding. This means that they emit photons with different energy, thus have different emission wavelength. Their fluorescence lifetime could also change. Therefore, there are two methods in which ion imaging can be done by use of indicators: the ratiometric method and the FLIM method.

Another method ion imaging is by the use of Forster Resonance Energy Transfer based (FRET-based) indicators that change their conformation upon ion binding. Upon the conformational change of a FRET-based indicator, its FRET efficiency changes, which is used as indicator of ion concentration. Examples of these indicators are cameleons. Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM).

Reference: Miyawaki A, Griesbeck O, Heim R, Tsien RY. "Dynamic and quantitative Ca2+ measurements using improved cameleons". Proc Natl Acad Sci USA (PNAS) 96(5):2135-40 (1999)

Demonstration of the Lambert Instruments Toggel camera for single-image FLIM (siFLIM) detection of histamine-induced alterations in Ca2+ concentration. Tiny oscillations in Ca2+ levels (~2.5 s periods) are observed after addition of histamine. Such small and rapid transients would go completely unnoticed when recorded by conventional FLIM.  Video courtesy of the Netherlands Cancer Institute.

Demonstration of the Lambert Instruments Toggel camera for single-image FLIM (siFLIM) detection of histamine-induced alterations in Ca2+ concentration. Tiny oscillations in Ca2+ levels (~2.5 s periods) are observed after addition of histamine. Such small and rapid transients would go completely unnoticed when recorded by conventional FLIM.

Video courtesy of the Netherlands Cancer Institute.

Calcium Imaging

Calcium (Ca2+) is important for signal transduction pathways.

Proton (pH) Imaging

The intracellular proton (H+) concentration (pH), as well as intracellular calcium, is important in the regulation of cellular functions including growth, differentiation, motility, exocytosis and endocytosis. To study this in more detail, measurements of the intracellular pH of resting cells can be done and the pH fluctuations inside cells after environmental perturbations can be followed.

Reference: Hai-Jui Lin, Petr Herman, and Joseph R. Lakowicz. "Fluorescence Lifetime-Resolved pH Imaging of Living Cells". Cytometry Part A 52A:77–89 (2003).

Zinc Imaging

Zinc (Zn2+) is involved in enzyme catalysis, protein structure, protein-protein interactions, and protein-oligonucleotide interactions. Zinc interacts with extracellular binding sites, which are likely to include binding sites involved in the subsequent translocation of this ion to the cell interior. Inside the cell, Zinc binds to cytosolic and organelle binding sites or is taken up by intracellular organelles.

Sodium Imaging

Sodium (Na+) is important in the signal transduction in the central nerve system.

Magnesium Imaging

Many enzymes (like kinases) require the presence of magnesium Mg2+ ions for their catalytic action, especially enzymes utilising ATP.

Chloride Imaging

Chloride (Cl-) plays a role in the central nervous system.

Potassium

Potassium (K+) plays a role in cell growth and cell viability.

Indicators for Ion Imaging by FLIM

  • BCECF (pH)

  • Bis-BTC (heavy metals)

  • Calcium-crimson (Calcium, orange excitation)

  • Calcium-green (Calcium, blue excitation)

  • Carboxyfluorescein (pH)

  • Carboxy-SNAFL-1 (pH)

  • Carboxy-SNAFL-2 (cytosol pH)

  • DM-NERF dextrans (lysosoml pH)

  • Fluo-3 (Calcium)

  • Fura-2 (Calcium)

  • LysoSensor DND-160 (lysosomal pH)

  • LysoSensor probe (pH)

  • Magnesium-green (Magnesium)

  • Mag-quin-1 (Magnesium)

  • Mag-quin-2 (Magnesium)

  • MQAE (Chloride)

  • Newport Green DCF (Zinc)

  • OG-514 carboxylic acid dextrans (lysosoml pH)

  • PBFI (Potassium)

  • Quin-2 (Calcium, blue excitation)

  • SPQ (Chloride)

Medical diagnosis

Biological tissues show intrinsic fluorescence lifetime imaging microscopy contrast because of the presence of autofluorescent structures like riboflavins and NADH. While autofluorescence is disadvantageous for observing tagged proteins of interest with e.g. fluorescence lifetime imaging microscopy, the exploitation of differences in the autofluorescent properties of biological tissue will increase the throughput and reliability of histopathological screening.

Autofluorescence can be used to detect molecular changes arising from diseases such as cancer and to differentiate normal cells/tissues from cancerous cells/ tissues. Namely, between normal and tumour cells differences in lifetime of normal autofluorescence have been detected. One approach is the combination of fluorescence lifetime imaging microscopy to endoscopy, whereby the endoscope is equipped with a laser. During endoscopic research, the tumour cells can be recognised and immediately burned away with the high-power laser.


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