Imaging Techniques

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Imaging Techniques

A large selection of microscopy techniques and methods are available to either improve contrast or to only detect discrete structures. A list of common microscopy techniques is available below.

In brightfield microscopy, the sample gets illuminated by white light. The interaction with the sample (absorption, reflection, scattering) attenuates the white light. Only a fraction of the light gets transmitted through the sample. It then gets collected by the objective and forms the brightfield image. Immunohistochemical staining like H&E or DAB is commonly used to prepare slides for brightfield microscopy as thin tissue samples or single cell layers barely show any contrast.

https://en.wikipedia.org/wiki/Bright-field_microscopy

List of microscopes that can image with these techniques:

Olympus VS200
Olympus IX81 spinning disk (DSU)

In fluorescence microscopy, fluorophores are used to selectively label and image properties of samples. The sample is illuminated by light of specific wavelengths that is absorbed by the fluorophores and causes them to emit light of longer wavelengths. Different kinds of fluorophores can be used to image multiple properties in the same sample. Most fluorescence microscopes use the epifluorescence design in which the sample is illuminated through the objective lens and the fluorescence is collected through the same objective lens. Epifluorescence microscopy will generate a crisp image of one focus plane. Fluorescence from above and below the focus is collected as well and will generate out of focus haze which is particularly noticeable in thick samples.

https://en.wikipedia.org/wiki/Fluorescence_microscope

List of microscopes that can image with these techniques:

Olympus VS200
Olympus IX81 spinning disk (DSU)
Zeiss Elyra P.1
Zeiss LSM980
3I Marianas

Differential Interference Contrast is a microscopy technique used to enhance the contrast in unstained, transparent samples.

https://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy

List of microscopes that can image with these techniques:

Olympus FV1000
Zeiss LSM780
Zeiss LSM980

In laser scanning confocal microscopy, the sample is raster scanned with a diffraction limited laser spot that causes fluorescence or reflected light, which is collected by the objective, filtered through a pinhole in a conjugate plane, and recorded by a photodetector. Filtering through the pinhole rejects out of focus light which results in optical sectioning capability that allows for imaging samples in 3D. Laser scanning confocal microscopes can typically accommodate a broad range of fluorescent imaging channels yielding crisp multi-channel images. They are, however, limited in their scan speed as each pixel (voxel) in the image gets sampled sequentially.

https://en.wikipedia.org/wiki/Confocal_microscopy

List of microscopes that can image with these techniques:

Abberior Infinity Line
Abberior STEDYCON
Olympus FV1000 MPE/DIVER
Olympus FV1000
Zeiss LSM780
Zeiss LSM980

Spinning disk confocal microscope parallelizes the laser scanning confocal principle by illuminating the sample in many separate spots, that rapidly scan across the sample, and then filter the fluorescence from these spots through many pinholes and detecting it with a camera. This is facilitated by a pinhole decorated spinning disk that upon laser illumination generates the illumination spots in the imaging plane. The emitted fluorescence gets filtered through the same pinholes before being imaged onto the camera. Spinning disk confocal microscopes can image at higher frame rates and causes less photobleaching than laser scanning confocal microscopes which makes them well suited for live cell or volumetric imaging.

https://en.wikipedia.org/wiki/Confocal_microscopy

List of microscopes that can image with these techniques:

Olympus IX81 spinning disk (DSU)
3I Marianas
3I Vivo

In two-photon excitation, a fluorophore gets excited by absorbing the energy of two simultaneously impinging photons. The energy of a single photon would not be large enough to excite the fluorophore as the excitation light has a longer wavelength (lower energy) than the fluorescence. The two-photon excitation is typically in the near-infrared for fluorophores that get excited and emit in the visible range. Two-photon microscopy requires the use of high-power pulsed femtosecond lasers that raster scan the sample to generate a photon flux in the excitation spot that is high enough to generate two-photon fluorescence. Two-photon microscopy is inherently capable of optical sectioning. It also penetrates deeper into tissue and reduces photobleaching compared to laser scanning confocal microscopy.

https://en.wikipedia.org/wiki/Two-photon_excitation_microscopy

List of microscopes that can image with these techniques:

Olympus FV1000 MPE/DIVER
Olympus FV1000
Zeiss LSM780
Zeiss LSM980

SHG is a process where two photons of the same wavelength interact with non-centrosymmetric crystalline structures and get converted into one photon of twice the energy and half the wavelength of the initial photons. In biological samples, this nonlinear imaging technique can be used for label-free imaging of collagen (collagen I and II) and myosin.

https://en.wikipedia.org/wiki/Second-harmonic_imaging_microscopy

List of microscopes that can image with these techniques:

Olympus FV1000 MPE/DIVER
Zeiss LSM780
Zeiss LSM980

Live cell/tissue imaging requires that the environment (temperature, humidity, gas) of the sample can be controlled. This can be achieved by mounting the sample on the microscope in an environmental enclosure or stage insert chamber.

List of microscopes that can image with these techniques:

Abberior Infinity Line
Olympus FV1000
Zeiss Elyra P.1
Zeiss LSM780
Zeiss LSM980
3I Marianas

Single molecule localization microscopy is the overarching term for a set of techniques (PALM, dSTORM, DNA-PAINT) that all rely on the fact that the position of individually imaged fluorophores can be determined at high accuracy (typically 30nm or better) whereas the resolution in normal fluorescence images is limited by the diffraction limit to about 250nm. SMLM techniques manipulate the fluorophores in the sample in such a way that only a small subset of them fluoresce at a given time. This results in sparse images from which the fluorophore positions can be accurately determined. By imaging a timelapse over which the active fluorophores randomly vary, all fluorophore positions in the sample are eventually recorded and can be used to generate a super-resolution image of the sample.

https://en.wikipedia.org/wiki/Photoactivated_localization_microscopy

List of microscopes that can image with these techniques:

Zeiss Elyra P.1

TIRF microscopy allows to only excite fluorophores in a thin layer (about 150nm) right above the coverslip in an aqueous sample. Fluorophores further away from the coverslip do not get excited and do not cause any out of focus fluorescence.

https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence_microscope

List of microscopes that can image with these techniques:

Zeiss Elyra P.1

FRAP is a fluorescence imaging technique that gives insight into the dynamics of a live sample by first photo-bleaching an area of interest and then recording the fluorescence recovery in this area.

https://en.wikipedia.org/wiki/Fluorescence_recovery_after_photobleaching

List of microscopes that can image with these techniques:

Olympus FV1000
Zeiss LSM780
Zeiss LSM980
3I Marianas
3I VIVO

CARS microscopy is a label-free laser scanning microscopy method in which the strength of a Raman mode (vibration of a chemical bond) is probed in a sample. CARS microscopy can for example highlight lipids and be used for imaging myelin due to the strong CARS signal from the CH2 vibrational mode.

https://en.wikipedia.org/wiki/Coherent_anti-Stokes_Raman_spectroscopy

List of microscopes that can image with these techniques:

Olympus FV1000

FCS is a method of analyzing molecular dynamics and interactions in vivo by observing the correlation of the intensity fluctuations of fluorescent signal via molecules diffusing through the focal volume. FCS can measure concentrations, diffusion coefficients, and interactions of molecules.

https://en.wikipedia.org/wiki/Fluorescence_correlation_spectroscopy

List of microscopes that can image with these techniques:

Zeiss LSM780
Zeiss LSM980

The absorption and emission spectra of commonly used fluorophores are so broad that in multi-channel acquisitions, when one fluorophore is excited, other fluorophores get excited as well (cross excitation), and the emission of a fluorophore also gets registered in other detection channels (bleed-through). If each fluorophores’ relative emission strengths in each
detection channel are known, the contribution of other fluorophores bleeding into a detection channel can be calculated out, which is referred to as spectral unmixing.

https://www.microscopyu.com/techniques/confocal/spectral-imaging-and-linear-unmixing

List of microscopes that can image with these techniques:

Zeiss LSM780
Zeiss LSM980

For many fluorophores, the return from the excited state into the ground state follows a probability distribution function that is mono exponential (~𝑒−𝑡𝜏⁄) and which is characterized by the fluorescence lifetime τ. FLIM detects not only the brightness of the fluorescence signal but also the lifetime of the fluorescence. This additional lifetime information can be used to separate spectrally similar fluorophores, detect FRET (FLIM-FRET), or to monitor changes in mammalian metabolism (NAD(P)H and FAD imaging).

https://en.wikipedia.org/wiki/Fluorescence-lifetime_imaging_microscopy

List of microscopes that can image with these techniques:

Abberior Infinity Line
Olympus FV1000 MPE/DIVER
Zeiss LSM780
Zeiss LSM980

STED microscopy is a super resolution technique that allows to acquire images with lateral resolutions down to 30nm (axially 600nm) in 2D STED and isotropic resolution down to 80nm in 3D STED when using photostable fluorophores. Just like in laser scanning confocal microscopy, the sample is raster scanned with a diffraction limited laser spot that causes fluorescence which is collected by the objective, filtered through a pinhole in a conjugate plane, and recorded by a photodetector. In addition, the STED microscope overlays the excitation spot with a second laser whose intensity profile is zero in the center. This second laser deexcites the excited fluorophores in the periphery of the excitation spot by a process called stimulated emission, thus shrinking the spot from which fluorescence is detected, which increases the resolution in the image.

https://en.wikipedia.org/wiki/STED_microscopy

List of microscopes that can image with these techniques:

Abberior Infinity Line
Abberior STEDYCON

FRET is a distance dependent, non-radiative transfer of energy from a donor fluorophore to an acceptor fluorophore. This transfer of energy typically only occurs if donor and acceptor are less than 10nm apart. Thus, FRET can be used to probe the distance between two labeled properties on the nanometer scale. FRET can either be detected by synchronously imaging the detector and acceptor channel, or by fluorescence lifetime imaging of the donor channel (FLIM-FRET).

https://en.wikipedia.org/wiki/Förster_resonance_energy_transfer

List of microscopes that can image with these techniques:

All Systems can do FRET
Systems which can do FLIM-FRET:
- Abberior Infinity Line
- Zeiss LSM780
- Zeiss LSM980

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