Application Archives | Photonscore

Light-sheet lifetime imaging

stefanrichter — Fri, 12 Nov 2021 10:36:55 +0000

Light-sheet lifetime imaging

Rat embryo acquired with light-sheet microscope

One of the well-known drawbacks of widefield microscopy imaging its low axial resolution compared to confocal microscopy. Several methods are known to overcome those limitations introducing confocality. Here we demonstrate FLIM images acquired with optical sectioning with light-sheet illumination.

The recorded slices of the specimen can be combined into a real 3D representation of the FLIM data, allowing it to get a vivid visualization of the sample

Drosophila larvae

A commercially available light-sheet system equipped with a pulsed laser source and a CCD with c-mount. For the user it is just a drop-in replacement of the CCD by LINCam to start imaging. An image below shows 128 FLIM sections acquired with 10 seconds per frame.

Again the recorded data can be fused into a 3D representation.

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Measurement induced entanglement of stored ions

stefanrichter — Mon, 11 Oct 2021 16:37:28 +0000

Measurement induced entanglement of stored ions

Typically single photon emitters show an emission behaviour that is called anti-bunching. This means that the probability to detect a second photon after detection a first one is suppressed. By using two of this single photon emitters in form of trapped calcium ions, it is possible to create a ligh source consisting of two single calcium ions.

If the scattered light does not contain information of its creating ion (achieved by measuring the far-field), some interesting properties of this light source can be observed. As described in fig. 1, the two ion system can be described by the Dicke-Basis [1]. Interestingly the anti symmetric state |a> does not couple to the laser field. The system now shows behaviour of typical single photon emitters, an anti-bunched photon statistics, if it driven in the symmetric decay channel. If a photon is emitted in a way that the system ends up in the anti-symmetric case, it becomes invisible for the driving laser and a second photon has to be emitted to bring the system back t0 the ground state. Such behaviour of the emission of two photons in a short period of time is called bunching and can be found normally only for chaotic or thermal light sources.

Interestingly the decay channel can be chosen by the angle of investigation of the trapped ion crystal, deliviring a light source whose emission statistics can be tuned from non-classical anti-bunching to classical bunching and everything in between by just changing the angle under which it is observed.

In fig. 2 the used setup is illustrated. Two calcium ions are stored in a segmented Paul trap and cooled to the Doppler-Limit. The emitted fluorescence light is collected by a lense that images the ion crystal on a amplified CCD camera. Only 10% of the light is used for this, the other 90% of the light are reflected by a 90/10 beam splitter to a Hanbury-Brown and Twiss (HBT) setup in which the detectors are replaced by two spatially resolving LINCam systems. The CCD camera is used to monitor the status of the ion crystal, so that the measurement can be paused upon ion loss and restarted once the crystals is ready again. The HBT setup is used to record all incomingdouble photon events with a coincidence window of +-40ns and a precision of 50ps at 1000*1000 spatial pixels.

From this event stream, the second order auto and cross-correlation function can be calculated on the fly. The resulting histogram is displayed live to monitor the success of the experiment.

In [1] the measurement was conducted by using avalanche photo diodes and a TDC (Time-To-Digital-Converter). The angle of observation was chosen by a slit. With this method the majority of the light is discarded and thus the measurement takes 2-3 days per point to gather sufficient statistics. In fig 3. the results of this measurement campaign can be seen. The acquisition of the 8 measured angles took roughly 30 days.

In [2] and fig.4 the LINCam was used to redo the experiment with the aim of measuring more than 8 angles. By using two synchronized LINCams it was possible to record a two-photon-event stream and correlating those events on the fly. The measurement campaign took 30 days again but delivered not only 8 observation angles, but 96. With the APD setup the campaing would have last one year.

Publications

[1] Sebastian Wolf, Stefan Richter, Joachim von Zanthier, and Ferdinand Schmidt-Kaler, Light of Two Atoms in Free Space: Bunching or Antibunching?, Phys. Rev. Lett. 124, 063603, https://doi.org/10.1103/PhysRevLett.124.063603

[2] Stefan Richter, Sebastian Wolf, Joachim von Zanthier, and Ferdinand Schmidt-Kaler, Imaging Trapped Ion Structures via Fluorescence Cross-Correlation Detection, Phys. Rev. Lett. 126, 173602, https://doi.org/10.1103/PhysRevLett.126.173602

References

S. Wolf, S. Richter, J. von Zanthier, and F. Schmidt-Kaler, "Light of Two Atoms in Free Space: Bunching or Antibunching?", Physical Review Letters, vol. 124, 2020. http://dx.doi.org/10.1103/PhysRevLett.124.063603

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Spinning Disc

stefanrichter — Mon, 11 Oct 2021 16:36:39 +0000

Confocal superresolution FLIM microscopy

Photonscore LINCam used here as a drop-in replacement of the Hamamatsu CCD camera. The detector housing has a c-mount female thread that allows a direct attachment to the CSU. Acquisition electronic module controls the camera and transfers acquired data to the computer with a standard USB interface. A pulsed laser is required to perform time-resolved single photon counting. Here, Omicron QuixX 488 diode laser was coupled into a light input of CSU using a single mode fibre.

Primary hippocampal neurons from rats. To visualize excitatory synaptic contacts, neurons were stained with rat anti-homer, guinea pig anti-MAP2, rat anti-Ctip2 and mouse anti- Prox1 antibodies. Subsequently, samples were incubated with anti-rat Alexa 488-, anti-guinea pig Cy5-, and anti-mouse Alexa 350- conjugated donkey secondary antibodies.

Lymphocytes (Jurakt T-cells) were transfected with a monomeric CFP-YFP Lck-biosensor and stimulated by CD3. After fixation cells were immune stained by an anti-GFP antibody, labelled with Atto 647N. The optical sections clearly show the shuttling of Lck positive vesicle between the plasma membrane and an inner compartment (most likely associated with the Golgi complex). Intensity weighted 3D stack of FLIM images of T-cells, 400 × 400 bins, 20 seconds per slice.

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Glycolytic oscillations in eukaryotic cells followed by NADH imaging

stefanrichter — Mon, 11 Oct 2021 16:36:22 +0000

Glycolytic oscillations in eukaryotic cells followed by NADH imaging

Monitoring intrinsic energy metabolism over long periods of time allows the study of cellular communication between cell populations. By using the metabolite NADH as an intrinsic marker for glycolysis, the dynamics of individual cells can be monitored and their interactions studied. Glucose consumption by glycolysis and alcoholic fermentation leads to the production of metabolites, some of which are released. Coupling between yeast cells depends on the release and sensing of the messenger acetaldehyde, which diffuses through the extracellular medium. Yeast cells are well known for the oscillatory behavior of the glycolysis and their metabolic organization. The exchange of messenger molecules can result in waves and synchronized patterns in which all cells oscillate in concert. Essential to this study is an ultrasensitive detection system that allows excitation of the weak fluorescence of NADH by low-intensity UV light.

(a) The time-series of the collective NADH fluorescence signal for a yeast population of cell density ρ=0.1%. Partial synchronisation of intracellular oscillations occurs at 760 s ≤t≤1100 s. (b) Development of the relative amplitudes of oscillations of each cell, and (c) of their phases. In (b) and (c) the cells are sorted according to their phases at time t=900 s. (d) Evolution of the distribution of instantaneous frequencies fi of the cells, and (e) of the distribution of the phase difference Δϕi between the phase ϕi of each individual cell to that of the average phase Φ of all cells of the population. (f) Time dependence of the order parameter R. The field of view had a diameter of 169 μm, and hosted 232 cells. Glucose was added to the cell suspension at t=−158 s.

Publications

[1] Weber, A., Zuschratter, W. & Hauser, M.J.B. Partial synchronisation of glycolytic oscillations in yeast cell populations. Sci Rep 10, 19714 (2020). https://doi.org/10.1038/s41598-020-76242-8

[2] Weber, A., Prokazov, Y., Zuschratter, W., Hauser, M.J.B. (2012) Desynchronisation of Glycolytic Oscillations in Yeast Cell Populations. PLOS ONE 7(9): e43276. https://doi.org/10.1371/journal.pone.0043276

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MIET (Metal induced energy transfer)

stefanrichter — Sun, 18 Jul 2021 16:36:02 +0000

Metal induced energy transfer with wide-field microscopy

Introduction

In Förster resonance energy transfer (FRET) an excited donor molecule transfers energy to an acceptor molecule. In MIET technique a metal plasmon surface acts like an acceptor allowing to measure distances with nanometer accuracy ¹. On the figure a general MIET setup is depicted. A gold film coating of a coverslip acts as an energy acceptor by efficiently quench- ing the fluorescence of chromophores of the sample. The efficiency of the energy transfer depends on the distance between the molecule and the surface: the closer the chromophore is located to the gold layer, the higher the probability of the transfer. This efficiency is observable as a change of the lifetime of the excited state allowing one to measure the distances h1 and h2. The reported efficient depth range of this technique is up to 100 nm matching to the typical working light penetration depth in total internal reflection fluorescence (TIRF) microscopy.

Fixed cell measurement

T-lymphocytes transfected with a GFP marked protein kinase (GFP-Lck) were fixed on gold coated coverslips. First, measurements were made under standard epifluorescence illumination conditions with 488 nm excitation wavelength. The resulting intensity weighted mean lifetime (Fig. 2a) and the intensity (Fig. 2c) images show the typical lifetime and grey value range for GFP with a slight quenching on the contact sites. The second measurement of the same cells was performed with TIRF illumination revealing much higher lifetime contrast (Fig. 2b) related to MIET effect.

To discriminate fluorescence lifetime components, we performed a maximum entropy method (MEM) analysis (Fig. 4). The results of per pixel amplitude fitting on basis of the MEM analysis are shown in figure 3.

The amplitudes corresponding to the lifetime components of 0,38 ns and 0,82 ns were summed into the intensity image (Fig. 3c) and the amplitude weighted lifetime map (Fig. 3a). The results for two other components of 2,29 ns and 5,58 ns are shown as well (Fig. 3b, 3d). The short components images demonstrate typical clustering of the protein kinase along the plasma membrane after T-cell receptor stimulation.

Publications

[1] A. Chizhik, J. Rother, I. Gregor, A. Janshoff and J. Enderlein, Metal-induced energy transfer for live cell nanoscopy, Nat. Photonics, Vol. 8, 2014
DOI: 10.1038/nphoton.2013.345

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Widefield single molecule acquisition

stefanrichter — Fri, 26 Mar 2021 10:27:49 +0000

Widefield single molecule acquisition

Cy5, Atto 655 and Atto 647N are nearly unresolvable spectrally however they are characterized by significantly different lifetimes. LINCam is capable of imaging Surface-immobilized single molecules in the most challenging for its (QY ~2%) far-red spectral region. In the top figure the

three fluorophores are imaged separately. The precision in lifetime determination is less than 0.2 ns.

Same three types (bottom figure) of fluorophores on the surface measured in a mix. Lifetime- based identification of the fluorophore type is straight- forward.

Read full story: https://doi.org/10.1021/acs.jpca.0c01513

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FLIM (Fluorescence Lifetime Imaging)

stefanrichter — Sun, 08 Nov 2020 13:55:08 +0000

Fluorescence Lifetime Imaging (FLIM)

Example of lifetime imaging of a lily of the valley slice sample. The intensity image (a) is a histogram of the positions of acquired photons. Lifetime analysis reveals four lifetime components: τ1 = 0,19; τ2 = 0,67; τ3 = 1,95 and τ4 = 3,75 ns. The resulting overlay image (b) of the intensity image and average lifetime is shown.

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Application Archives | Photonscore

Light-sheet lifetime imaging

Light-sheet lifetime imaging

Rat embryo acquired with light-sheet microscope

Drosophila larvae

COMPANY

LEGAL

CONTACT

Measurement induced entanglement of stored ions

Measurement induced entanglement of stored ions

Publications

References

Spinning Disc

Confocal superresolution FLIM microscopy

Glycolytic oscillations in eukaryotic cells followed by NADH imaging

Glycolytic oscillations in eukaryotic cells followed by NADH imaging

Publications

MIET (Metal induced energy transfer)

Metal induced energy transfer with wide-field microscopy

Introduction

Fixed cell measurement

Publications

Widefield single molecule acquisition

Widefield single molecule acquisition

FLIM (Fluorescence Lifetime Imaging)

Fluorescence Lifetime Imaging (FLIM)