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Advanced Optical Microscopy Centre


Equipment & expertise

Advanced Optical Microscopy Centre

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EXPERTISE OF THE AOMC

Select the different tabs to learn more about the expertise present in the AOMC.


At the AOMC, all microscopes are capable of imaging both live and fixed samples. However, each system (technique) has its own set of advantages and disadvantages, making them more or less suited for different applications. Every microscopy is equipped with an enclosing incubator and stage inserts that allow precise temperature and pH control for optimal sample conditions.

widefield (epi-fluorescence) microscopy

Ideally suited for fast multicolor imaging of live samples. Due to the nature of widefield fluorescence imaging, it is best suited for thin samples such as monolayers of cultured cells to reduce the amount of out-of-focus light that can blur the images. Learn more about the Elyra PS.1 widefield fluorescence microscope here.

total internal reflection microscopy

Total internal reflection fluorescence (TIRF) microscopy and imaging using a highly inclined and laminated optical (HILO) sheet reduces the amount of out-of-focus blur on a widefield fluorescence microscope by limiting the part of the sample that is excitated by the laser beam. While greatly increasing the contrast of images, the sample region that can be images is also reduced (e.g. limited to a few 100nm depth for TIRF). Learn more about the Elyra PS.1 widefield fluorescence microscope here

confocal microscopy

Known as the workhorse microscopy technique in biological and biomedical research. Confocal microscopy allows imaging in thicker samples and while maintaing very sharp focus. The method is extremely well suited for multidimensional (xyztc) acquisitions, but is generally slower than widefield microscopy. Learn more about the LSM510 confocal microscope here and about the LSM880 confocal microscope here.

two-photon excitation microscopy

Two-photon excitation microscopy allows to image even deeper into tissue and is thus ideally suited for imaging thicker tissue samples. The nature of the excitation inherently results in good optical sectioning making the method ideally suited for multidimensional acquisitions. Learn more about the LSM510 confocal microscope here and about the LSM880 confocal microscope here.

The diffraction-limited nature of light microscopy does not always provide sufficient resolution to see all desired details. At the AOMC, multiple techniques are available to record images with improved (super) resolution. Each of these methods has its own set of advantages and disadvantages, so they can be matched to almost any research question.

structured illumination microscopy (SIM)

SIM is capable of providing a doubled spatial resolution by using a patterned (structured) illumination, usually stripes, to excite the sample. The interaction between the illumination pattern and the sample-structure moves high frequency information, which would normally not be resolvable, into a resolvable range. In practice, a series of 9 - 15 images is acquired while rotating and shifting the illumination pattern and the super-resolved image is subsequently calculated.

The low number of input images makes SIM a fast super-resolution microscopy method. Additionally, no special fluorophores (no "blinking") are required, which means it can be used for many samples and fluorophore combinations. The method does, however, only offer a twofold resolution improvement and can be prone to artifacts when out-of-focus light or refraction index mismatch is present.

advantages disadvantages
fast (< 1s) "only" a twofold resolution improvement
no special fluorophores required limited penetration depth (widefield system)
suitable for live samples (prone to artefacts)
gentle  

single molecule localization microscopy (SMLM)

SMLM groups a collection of microscopy techniques that rely on separating and fitting the emission of individual emitters (single molecules) to determine their exact location. Type examples include photo-activatable localization microscopy (PALM), direct stochastic optical reconstruction microscopy (dSTORM) and point accumulation for imaging in nanoscale topography (PAINT). The first two methods use sequenctial photo-activation or -conversion between two distinguishable states of the employed fluorophores to only have a small subset of fluorophores emitting in the detection channel at any single point in time. The last method uses dyes that are minimally fluorescent when freely diffusing, but brightly fluorescent when bound or immobilized by the structure of interest. In all cases, large timeseries of widefield fluorescence images of sparse emitters are acquired and post-processed to obtain very high resolution images.

SMLM is capable of achieving very good spatial resolution, close to 10-fold better than the resolution limit, but often sacrifices time resolution to achieve this. In many cases, the best results are obtained for fixed samples.

advantages disadvantages
very high resolution obtainable (tenfold improvement) slow (> 5min)
  requires specific fluorophores (and buffer conditions)
  limited penetration depth (best results with TIRF)
  mostly fixed samples

super-resolution optical fluctuation imaging (SOFI)

SOFI is a post-processing method to extract super-resolution information in three dimensions from a timeseries of widefield fluorescence images. The method analyzes temporal fluctuations in the intensity of fluorophores (aka "blinking"). While SOFI provides a theoretically unlimited resolution improvement, in practice it is more commonly used to provide a two- to threefold resolution enhancement. This resolution improvement can be achieved with a relatively short timeseries (a few hundred to a few thousand frames) and a relatively poor signal-to-noise ratio. Additionally, because the method does not rely on the identification of single emitters like the SMLM methods described above, a higher fluorophore density can be used. The combination of these factors makes SOFI a robust method for the acquisition of medium-high resolution images (80 - 180 nm resolution).

advantages disadvantages
medium fast (s - min) medium fast (s - min)
medium resolution gain (twofold - fourfold) medium resolution gain (twofold - fourfold)
suitable for live samples requires specific fluorophores
suitable for low SNR samples  

airyscan microscopy

A commercial implementation of the Image Scanning Microscopy (ISM) technique to improve the spatial resolution of confocal laser scanning microscopy by up to a factor of two. The method employs a grid of detectors, each of which acting as a tiny confocal pinhole, to detect the entire emission spot. The detected fluorescence is reassigned in a post-processing step to a more narrow (higher resolution) spot resulting in the improved resolution image. (see an example here)

Airyscan imaging allows the use of a confocal microscope as if it had a very small pinhole, thus unlocking its true resolution potential, whitout compromise to the speed or sensitivity. Moreover, the microscope can be configured in such a way to target an improved resolution, an increased signal-to-noise ratio or a very fast imaging speed ("Fast Airyscan").

advantages disadvantages
very versatile (balance speed, resolution and SNR) "only" up to a twofold resolution improvement
no drawbacks compared to confocal microscopy medium speed (confocal speed)
compatible with two-photon excitation  
suitable for live samples  

   

It is not always required to label a sample with fluorophores in order to visualize it. Several structures and components can be imaged using label-free imaging techniques.

second harmonic generation microscopy

Second harmonic generation (SHG) occurs when high-intensity light hits non-centro-symmetric structures (e.g. microtubules, enamel, collagen, myosin, ...). The energy conserving scattering process that follows 'combines two photons into one with double the frequency (half the wavelength) of the original photons'. Contrast is created because only the highly ordered non-centro-symmetric structures can cause SHG while most biological structures are not highly ordered and thus produce no SHG signal. Because the SHG signal is created through scattering and doesn't require an excited state, there is no bleaching and no time delay. Additionally, the process itself causes no photodamage or phototoxicity, although absorption of the highly intense laserbeam can still cause damage in the sample. SHG microscopy can be performed relatively straightforward on a confocal microscope equipped with non-linear optics such as the LSM510 and LSM880.

label-free imaging of pollutants

Pollutants present in samples can potentially produce a specific and detectable signal when irradiated with high intensity light. At the AOMC we can investigate the spectral fingerprint of the signal and attempt to reliably separate pollutant signal from other sample components.

The AOMC offers a collection of methods that allow the investigation of molecular and (sub-)cellular dynamics and interactions. These methods can be used to study how, when and where different proteins interact with eachother, with other proteins and with the environment. 

Förster resonance energy transfer (FRET) 

FRET allows to measure the distance between a donor and an acceptor fluorophore and can be used to study conformational changes of molecules as well as the interaction between multiple molecules. FRET experiments can be performed at the ensemble and single-molecule level.

time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM)

The fluorescence lifetime of a fluorophore is a key photophysical parameter that can be used to investigate the direct molecular environment of this fluorophore. FLIM is often employed to detect changes in FRET which in turn reports on changes in the distance between the donor and acceptor fluorophore.

fluorescence fluctuation spectroscopy (FFS)

A family of methods that allow quantifying molecular properties such as concentrations, mobility, stoichiometry, interaction affinities of diffusing molecules at equilibrium. The simplest implementation is fluorescence correlation spectroscopy (FCS), where fluorescence signals from freely diffusing molecules is recorded on a confocal microscope (without image scanning). The resulting time trace is analyzed via autocorrelation, which then informs on diffusion rates and concentration.

 

 Illustration of FCS. Adapted from Schrimpf et al., 2018.

Illustration of FCS. Adapted from Schrimpf et al., 2018.

Image correlation spectroscopy extends FFS analysis to imaging data. With Raster ICS (RICS), for example, mobility and concentration information is obtained from confocal laser scanning microscopy images.  

 

Illustration of RICS. Adapted from Schrimpf et al., 2018.

Arbitrary-region ICS (ARICS) even allows mapping molecular properties in e.g. a biological structure, providing detailed insights in the workings of complex biological machineries.

Illustration of ARICS. Adapted from Hendrix et al., 2016.