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The bh FLIM Technique – More than Lifetime Imaging

bh FLIM systems record FLIM images of unprecedented temporal and spatial resolution at an
accuracy level close to the theoretical limit given by photon statistics. But bh FLIM systems do more
that that: The bh FLIM technique is based on a new understanding of FLIM in general. FLIM is not
just considered a way to add additional contrast to microscopy images. It is considered and designed
as a molecular imaging technique. bh FLIM exploits the fact that the fluorescence decay function of a
fluorophore is an indicator of its molecular environment, and that multi-exponential decay analysis
delivers molecular information, such as the metabolic state of live cells and tissues, protein
conformation and protein interaction, reaction of cells to drugs and environment parameters, or
mechanisms of cancer development and cancer progression. To reach this target, bh FLIM systems
have features not available in other systems: Compatibility with live-cell imaging, extraordinarily
high time resolution and photon efficiency, capability to split decay functions into several
components, excitation-wavelength multiplexing in combination with parallel-channel detection,
recording of dynamic lifetime effects caused by fast physiological effects, and simultaneous
FLIM/PLIM.

Keywords: FLIM, PLIM, Molecular Imaging, FRET, Protein Structure, Metabolic Imaging, Live Cell Imaging, Physiological Effects

 

bh FLIM: More than Fluorescence-Lifetime Imaging

bh FLIM systems record FLIM images of unprecedented temporal and spatial resolution at an accuracy level close to the theoretical limit given by photon statistics. But bh FLIM systems do more that that: The bh FLIM technique is based on a new understanding of FLIM in general. FLIM is not just considered a way to add additional contrast to microscopy images. It is considered and designed as a molecular imaging technique. bh FLIM exploits the fact that the fluorescence decay function of a fluorophore is an indicator of its molecular environment, and that multi-exponential decay analysis delivers molecular information, such as the metabolic state of live cells and tissues, protein conformation and protein interaction, reaction of cells to drugs and environment parameters, or mechanisms of cancer development and cancer progression. To reach this target, bh FLIM systems have features not available in other systems: Compatibility with live-cell imaging, extraordinarily high time resolution and photon efficiency, capability to split decay functions into several components, excitation-wavelength multiplexing in combination with parallel-channel detection, recording of dynamic lifetime effects caused by fast physiological effects, and simultaneous FLIM/PLIM.

 

Precision Megapixel FLIM Images

Pixel numbers in the megapixel range and precision decay data. Enjoy the beauty of the images!



The Ultimate in FLIM Time Resolution and Timing Stability

Electrical time resolution 3.5ps fwhm. Timing stability better than 0.4ps rms. System IRF <19 ps fwhm including detector and laser. No need to record an IRF!

        

 

High Resolution Decay Data - The Basis of Metabolic FLIM

NADH FLIM: Metabolic Ratio, lifetimes of unbound and bound NADH

      

 

Simultaneous FLIM of NADH and FAD - Tumor Detection by Metabolic FLIM

Metabolic FLIM, excitation-wavelength multiplexing, simultaneous imaging of NADH and FAD. Perfect Separation of NADH and FAD.

Protein Interaction - Quantitative FRET Results

Precision FLIM data, double-exponential FRET analysis: No need to record free-donor lifetime from reference sample.

Left to right: Classic FRET efficiency, FRET efficiency of interacting donor, amount of interacting donor, donor-acceptor distance.

   

 

Ultra-Fast Decay Processes

Discover fluorescence-decay processes which have never been seen before. Below: Mushroom spores, fast decay component of 11 ps.

   

Below: Melanoma sample, decay curves of healthy tissue and tumor tissue. The tumor has a fast decay component of 13 ps.

Autofluorescence Imaging of Small Organisms

Study environment effects on small organisms

   

 

Triggered Accumulation of Time Series - Recording of Fast Physiological Effects

Left: Calcium transient in cultured neurons. Right: Chlorophyll transient.

   

 

Online FLIM - Minimum Acquisition Time for Given Photon Rate

Track tho object of Interest. FLIM with 200 ms Acquisition Time.

   

Temporal Mosaic FLIM: Precision Lifetime Analysis of Moving Objects

Metabolic FLIM on the moving leg of a water flee. bh Temporal Mosaic FLIM with subsequent Image Segmentation. Precision decay curve shown lower right.

 

Simultaneous FLIM / PLIM

Record the metabolic state of cells in dependence of oxygen concentration.

 

Multi-Wavelength Detection

Explore the unexplored: Simultaneous Detection in 16 Wavelength Channels

 

 

 

For more information please see:

1.      W. Becker, The bh TCSPC handbook, 9th edition. Becker & Hickl GmbH (2021), available online on www.becker-hickl.com. Please contact bh for printed copies.

2.      Becker & Hickl GmbH, FLIM Systems for Laser Scanning Microscopes. Overview brochure, available on www.becker-hickl.com

3.      W. Becker (ed.), Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)

4.      Becker Wolfgang, Suarez-Ibarrola Rodrigo, Miernik Arkadiusz, Braun Lukas, Metabolic Imaging by Simultaneous FLIM of NAD(P)H and FAD. Current Directions in Biomedical Engineering 5(1), 1-3 (2019)

5.      W. Becker, Bigger and Better Photons: The Road to Great FLIM Results. Education brochure, available on www.becker-hickl.com.

6.      Becker & Hickl GmbH, SPCImage next generation FLIM data analysis software. Overview brochure, available on www.becker-hickl.com

7.      Becker & Hickl GmbH, Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation. Application note, available on www.becker-hickl.com

8.      S. Kalinina, V. Shcheslavskiy, W. Becker, J. Breymayer, P. Schäfer, A. Rück, Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping. J. Biophotonics 9(8):800-811 (2016)

 

 

Becker & Hickl GmbH

Nunsdorfer Ring 7-9

12277 Berlin, Germany

Tel.  +49 30 212 800 20, Fax. +49 30 212 800 213

email: info@becker-hickl.com, www.becker-hickl.com

 

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