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Microscope Applications in Life Science

4015 views - 2019-10-28 17:52:14



Life Science


Life science refers to the fields of science that involve the scientific study of processes and structures of living organisms. While biology remains the centerpiece of the life sciences, related fields such as biochemistry, biophysics, or medicine are also included. Life science applications aim at understanding organisms and their place in the ecosystem.





rapidFLIM (Fluorescence Lifetime Imaging)

Fast imaging technique redefining the standards for dynamic FLIM imaging


rapidFLIM measurements enable the imaging of dynamic processes via fluorescence lifetime imaging (FLIM). This new approach allows for fast FLIM acquisition up to several frames per second for imaging of dynamic processes (e.g., protein interaction, chemical reaction, or ion flux), highly mobile species (e.g., mobility of cell organelles or particles, cell migration), and investigating FRET dynamics. More than 10 frames per second can be acquired, depending on sample brightness and image size.




Fluorescence Lifetime Imaging (FLIM)

Imaging technique based on differences in the excited state decay rate


Fluorescence Lifetime Imaging (FLIM) produces an image based on the differences in the excited state decay rate from a fluorescent sample. Thus, FLIM is a fluorescence imaging technique where the contrast is based on the lifetime of individual fluorophores rather than their emission spectra. The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon.

As the fluorescence lifetime does not depend on concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity it is more robust than intensity based methods. At the same time, the fluorescence lifetime depends on a wealth of environmental parameters such as pH, ion or oxygen concentration, molecular binding or the proximity of energy acceptors making it the technique of choice for functional imaging of many kinds.




Phosphorescence Lifetime Imaging (PLIM) 

Imaging technique for long lived samples


Phosphorescence Lifetime Imaging (PLIM) is similar to Fluorescence Lifetime Imaging (FLIM), only that it images the phosphorescence from the sample and consequently covers time ranges up to milliseconds. Analogous to FLIM, the contrast in a PLIM image is based on the lifetime of individual fluorophores rather than their emission spectra. The phosphorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon.

PLIM, or generally the characterization of phosphorescent compounds has been of great importance in the field of materials science namely chemical sensing for many years , and has renewed its interest over the past decade with the booming development of Organic Light Emitting Diode (OLED) technology. Other typical samples include metal ions complexed with organic ligands, which can be used to, e.g., image oxygen consumption in living cells.




Foerster Resonance Energy Transfer (FRET)

Lifetime-based Foerster Resonance Energy Transfer (FLIM-FRET)


FRET is a non-radiative process whereby energy from an excited fluorescent molecule (Donor) is transferred to a second, non-excited fluorophore (Acceptor) in its direct vicinity. FRET is used as a molecular ruler due to its sensitivity in the range of 2-10 nm. The energy transfer results in donor quenching and leads to changes in the fluorescence intensity and the fluorescence lifetimes of the two fluorophores. As a prerequisite, the donor is usually directly excited whereas direct excitation of the acceptor should be avoided.




Pulsed Interleaved Excitation (PIE)

PIE for single-pair Foerster Resonance Energy Transfer (spFRET) and Fluoresence Lifetime Cross-Correlation Spectroscopy (FLCCS)


Pulsed Interleaved Excitation (PIE) is a technique of synchronizing several pulsed lasers. The laser pulses are separated on a nanosecond time scale to allow simultaneous recording of the temporal behaviour of a sample molecule corresponding to each individual laser. For this method usually two lasers are chosen with suitable wavelengths for sequential excitation of both fluorophores. The laser pulses are delayed with respect to each other to yield a pulse sequence with interleaved pulses. To avoid crosstalk, the laser repetition rate is adjusted in that way that the fluorescence of each dye decays completely before excitation of the other fluorophore.

PIE is often used in combination with single-species Foerster Resonance Energy Transfer (spFRET) wherein donor and acceptor are excited alternately. In this way, the acceptor dye is excited independently of the FRET process to prove its existence and photoactivity. Molecules lacking an active donor or acceptor are separated from active FRET complexes. This allows to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescing acceptor.





Fluorescence Correlation Spectroscopy (FCS)

Correlation analysis of fluorescence intensity fluctuations)


Fluorescence Correlation Spectroscopy (FCS) is a correlation analysis of temporal fluctuations of the fluorescence intensity. It offers insights into the photophysics that cause these characteristic fluorescence intensity fluctuations as well as diffusion behaviour and absolute concentrations of detected particles. The most prominent and easily observed cause of fluorescence fluctuations is the fluctuation of the concentration of fluorescent particles (molecules) in the obeservation volume. The method records temporal changes in the fluorescence emission intensity caused by single fluorophores passing the detection volume. These intensity changes can be quantified in their strength and duration by temporally auto-correlating the recorded intensity signal, leading to the average number of fluorescent particles in the detction volume and their average diffusion time through the volume. Eventually, important biochemical parameters as the concentration and size or shape of the particle (molecule) or viscosity of the environment can be determined.




Fluorescence Lifetime Correlation Spectroscopy (FLCS)

Fluorescence Lifetime weighted correlation analysis of fluorescence intensity fluctuations


The fusion of Time-Correlated Single Photon Counting and Fluorescence Correlation Spectroscopy, called Fluorescence Lifetime Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions.

FLCS is of particular advantage when using spectrally inseperable fluorophores that differ in their lifetime for Fluorescence Cross-Correlation Spectroscopy (FCCS) because it offers elimination of spectral cross talk and background. It also offers a way around detector afterpulsing artifacts.

In FLCS, a separate autocorrelation function is calculated for each fluorophore component determined by its decay pattern, emitted, for example, by various species in the sample. The only assumption is that various emissions have different TCSPC histograms (i.e., different fluorescence lifetimes), which is practically always the case. The core of the method is a statistical separation of different intensity contributions with similar lifetimes, performed on a single photon level.




Dual-focus Fluorescence Correlation Spectroscopy (2fFCS)

Reliable, artifact-free measurement of absolute diffusion coefficients


To quantitatively evaluate a FCS experiment, one has to exactly know the shape and the size of the confocal volume. The problem is that the confocal volume sensibly depends on numerous parameters of the optical set-up, refractive index mismatch of the sample solution and the objective’s immersion medium, coverslide thickness variations and especially the dependence on optical saturation of the fluorescent dye, which can occur at even very low excitation powers.

In contrast to conventional FCS dual-focus FCS (2fFCS) is robust against these FCS artifacts thus allowing to measure absolute diffusion coefficients. This is achieved because instead of the size and shape of the confocal volume 2fFCS uses the distance between two confocal volumina as a reference.




Stimulated Emission Depletion Microscopy (STED)

Imaging below the optical diffraction limit


Stimulated emission depletion microscopy (STED) is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes. The resolution enhancement is essentially based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the diffraction limited excitation focus. This intense radiation causes almost all of the excited molecules to return to the ground state. Fluorescence from the remaining excited dye molecules in the center of the excitation focus is then detected and used to form the high resolution images. An even further resolution enhancement  is possible by applying time gates to the collected data (gated STED or gSTED). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS. In that case, collecting data at different observation volume diameters can help to disentangle complex 2D diffusion scenarios in heterogeneous samples such as biological membranes.




Single Molecule Detection

Studying individual emitters


Many systems are either dynamically or stationary inhomogeneous and the inhomogeneities, especially dynamic inhomogeneities, are the key to understand their interactions and functions. Ensemble measurements naturally can only yield the mean value of the ensemble. Single-molecule trajectories on the other hand are direct records of the fluctuation that contain detailed dynamical and statistical information. Since trajectories may vary for different members of the ensemble the average trajectory contains less information, the ensemble might not even show fluctuations at all as they could be averaged out during the timespan of the experiment.

Single molecule detection techniques are also essential in overcoming the diffraction limit in microscopy and have been honored with the 2014 Nobel prize in chemistry. PicoQuant has a long history of supporting the single molecule community both with state of the art instrumentation as well as through hosting single molecule events around the world for many years.




Time-resolved Fluorescence

Measurements of the fluorescence lifetime


The fluorescence (or in general photoluminescence) lifetime is characteristic for each fluorescent or phosphorescent molecule and can thus be used to characterize a sample. It is, however, also influenced by the chemical composition of its environment. Additional processes like Förster Resonance Energy Transfer (FRET), quenching, charge transfer, solvation dynamics, or molecular rotation also have an effect on the decay kinetics. Lifetime changes can therefore be used to gain information about the local chemical environment or to follow reaction mechanisms.




Fluorescence Anisotropy (Polarization)

Study molecular orientation and mobility


Measurement of steady-state and particularly time-resolved fluorescence anisotropy offers fascinating possibilities to study molecular orientation and mobility as well as processes that affect them. Except of special cases, anisotropy does not depend on the concentration of fluorophores, thus on the detected signal intensity. Owing to this similarity with the behavior of fluorescence lifetime, we can regard anisotropy as a yet another dimension of fluorescence information.

Molecules with their absorption transition moment (ATM) aligned with the polarization plane of excitation are preferentially excited. This is called photo-selection, because at the same time molecules with ATM oriented perpendicular to the excitation polarization plane are not excited, they remain in their ground state.




Pattern Matching Analysis

Advanced FLIM / STED analysis feature of the SymPhoTime 64 software


A pattern matching analysis is included in the SymPhoTime 64 software that permits an unambiguous identification and separation of different populations (like fluorophores, background and autofluorescence) in each image pixel. The fluorescence decay acts as fingerprints for specific fluorescence contributions and provides an excellent tool to analytically determine the fluorescence lifetime. This method allows to distinguish between e.g., different cell types and autofluorescence based on their individual overall fluorescence lifetime without the need of time-consuming data fitting. No information about the different contributions is necessary prior to analysis, one simply defines the decay “fingerprints” of the individual molecules (for example of the background or the different fluorescent molecules) within the FLIM or STED image or the scatter plot, which are then saved as “patterns”.The scatter plot (decay diversity map) is very intuitive with meaningful parameters like the average fluorescence lifetime, which is analytically determined and represents the center of mass of the average photon arrival times. The second plot parameter delta_tau indicates the number of exponentials or components that are included in the fluorescence decay. By pattern matching, the intensities of the defined patterns corresponding to the different species are determined in each image pixel. As a result, the pattern matching approach applying the full lifetime information fastens and refines FLIM and STED analysis. Specific regions within the sample which have been identified by this method could further be analyzed via multi-exponential fitting                                                                                                                                              



Diffuse Optical Tomography and Imaging

Studying optical properties of physiological tissue


In Two-Photon Excitation (TPE), a high power pulsed laser with very short pulse width is focused into the sample. The high photon density in the focus leads to a certain probability that a fluorophore absorbs two photons quasi simultaneously. The wavelength of the excitation laser is chosen such that the combined energy of two photons spans the gap between the ground state and first excited electronic state of the molecule. Lasers that are usually used for this application are Ti:Sa lasers with femtosecond pulse width and a tunable wavelength in the range between 690 nm and 1040 nm. At these infrared wavelengths, the excitation light is scattered only little inside the sample, and a good focal spot can be created even millimeters deep into the sample. Since the probability of excitation depends quadratically on the photon density, z-sectioning is provided by the excitation alone, and the pinhole in the detection path can be omitted.




Two-Photon Excitation (TPE)

Deep-tissue imaging with Two-Photon Excitation


Diffuse Optical Tomography (DOT) and Imaging (DOI) are non-invasive techniques that utilize light in the near infrared spectral region to measure the optical properties of physiological tissue. The techniques rely on the object under study being at least partially light-transmitting or translucent, so it works best on soft tissues such as breast and brain tissue. By monitoring spatial-temporal variations in the light absorption and scattering properties of tissue, regional variations in oxy- and deoxy-hemoglobin concentration as well as cellular scattering can be imaged. Based on these measurements, spatial maps of tissue properties such as total hemoglobin concentration, blood oxygen saturation and scattering can be obtained using model-based reconstruction algorithms. DOT and DOI have been applied in various deep-tissue applications including breast cancer imaging, brain functional imaging, stroke detection, muscle functional studies, photodynamic therapy, and radiation therapy monitoring.




Singlet Oxygen

Sensitive measurements in the NIR


Singlet oxygen is the common name of an electronically excited state of molecular oxygen which is less stable than molecular oxygen in the electronic ground state. It is typically generated via energy transfer from the excited state of a photosensitizer to the oxygen molecule. The reactive properties of singlet oxygen are, for example, used to destroy cancer cells in photodynamic therapy. In order to optimize such therapies, current research tries to design photosensitizer molecules that optimize the generation of singlet oxygen. Other studies focus on the emission lifetime of singlet oxygen, which is solvent dependent and can therefore be used to gain information about the environment of the emitting oxygen molecules. Singlet oxygen studies are usually performed by steady-state and time-resolved phosphorescence measurements with emission detection around 1270 nm. Such measurements are usually challenging, because the singlet oxygen emission is very weak compared to, e.g., the fluorescence signal of the photosensitizer.




Laser cutting/ablation

Coupling of pulsed lasers to a microscope for selective photomanipulation of sample


Pulsed lasers enable photomanipulation of various structures within tissues and cells, even down to the molecular level, as well as the interference with cellular processes. The selective laser cutting of cellular structures like microtubules or the removal of whole cells by ablation permit to study cell and developmental biology. This technique provides new insights into the architecture of the mitotic spindle, the process of chromosome segregation as well as cell locomotion. Furthermore, it gives new information about origin, fate, or function of individual cells in the developing organism and can also be used for gene activation.

As a primary advantage, laser-based cutting / ablation is a very flexible method that can be performed at any cellular site, in any cell pattern and at any time in development within living tissues and cells. It reaches submicrometer resolution thus allowing to target specific cell organells. The ablation efficiency depends on the fluorecent label. Efficient ablation requires pulsed laser excitation to reach high peak intensities with moderate average power. In this way, cell damage is minimized in untreated regions. In addition, the heat generation is significantly reduced due to the short pulse duration. Thus, by using pulsed laser light, the necessary ablation illumination times are very short without causing visible collateral damage.





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