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ibidi FAQs

Find answers to frequently asked questions on LIGHT MICROSCOPY

Compatibility of Surfaces with Microscopy Techniques

Phase Contrast

Phase contrast is a technique that converts small phase shifts in cells into amplitude or intensity contrast. This label-free technique is strongly dependent on having the right alignment of the phase plate and the annular ring in the optical pathway. It is by far the most frequently used method in biological light microscopy. A common problem in phase contrast is the meniscus formation at the air-liquid interfaces, thus making this technique nearly non-applicable to small culture wells, like 96 well plates.

ibidi’s recommendation:

Phase contrast is rarely dependent on the material. What is crucial is the meniscus, which is formed at the air-water interface in small open wells. Therefore, channel μ-Slides are great tools for phase contrast.

For detailed information, please refer to "Phase Contrast in Channel Slides" or read our Application Note 03.


DIC – Differential Interference Contrast

DIC is a label-free microscopy technique with a high sensitivity to thin cellular material, even when it is located within thick tissue. It is less sensitive to meniscus formation than phase contrast. DIC needs low birefringence and is, therefore, not compatible with standard culture ware made out of polystyrene.

ibidi’s recommendation:

DIC is 100 % compatible with the ibidi Standard Bottom and Glass Bottom.  There are drawbacks in using DIC with regular channel slides and plastic lids. Therefore, ibidi offers special DIC lids. Upon request, we will also produce DIC-compatible channel slides.


Wide-Field Fluorescence

Wide-field fluorescence microscopy is a form of light microscopy. The specimen is illuminated with filtered light at wavelengths that excite fluorophores. It requires labeling that uses special antibody reactions, or tagged proteins (e.g., green fluorescent protein). Fluorescence is used to detect structures, molecules, or proteins within the cell, up to highest magnifications.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, making wide-field fluorescence possible without restrictions.


Confocal Microscopy

Confocal microscopy is based on conventional wide-field microscopy. Laser light is focused into the sample, exciting only a small spatial area. Pinholes inside the optical pathway cut off signals that are out of focus, thus creating images of one, single optical plane. With this technique, it is possible to create 3D images from data that was generated from several optical planes.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, so that confocal microscopy is possible without restrictions.


TIRF – Total Internal Reflection Fluorescence

TIRF utilizes the evanescent field, which is created when a beam of light strikes an interface between two media, exciting the fluorescent dyes in the specimen. Although TIRF cannot image deep into a specimen, it allows imaging of the specimen near the interface with a high signal-to-noise ratio. This technique requires two optical media with different refractive indices, such as glass (nD=1.52) and water (nD=1.33).

ibidi’s recommendation:

TIRF is possible with the ibidi Standard Bottom, but we recommend using a glass bottom.


FRET – Förster Resonance Energy Transfer

FRET is a fluorescence technique that determines the precise location and nature of the interactions between fluorophores within living cells. A donor fluorophore in its excited state can transfer its excitation energy to an acceptor fluorophore in a non-radiative fashion. Typically, this happens through dipole-dipole coupling in a distance of less than 10 nm. Beyond that distance (Förster radius), the two fluorophores show normal fluorescence behavior.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, making FRET microscopy possible without restrictions.


FRAP – Fluorescence Recovery After Photobleaching

FRAP is a fluorescence microscopy method to study the mobility of fluorescently-labeled molecules. A typical FRAP experiment involves three distinct phases. After the registration of the initial fluorescence, the fluorescent molecules are photobleached within a selected area using the laser beam. Next, the fluorescence recovery is recorded when it arises from the immediate surroundings, by diffusional or active transport, from the exchange between photobleached molecules and intact ones. Then it is possible to obtain the diffusion coefficient and a local (im)mobile fraction, using modeling.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, making FRAP possible without restrictions.


FLIM – Fluorescence Lifetime Imaging Microscopy

In contrast to normal fluorescence microscopy, where the intensity is used to create an image of the specimen, FLIM uses the lifetime of the signal by analyzing the fluorophore’s exponential decay rate. By detecting differences in lifetime, it is possible to identify fluorophores that have the same excitation and emission spectrum. The fluorescence lifetime is dependent on ion intensity, oxygen concentration, molecular binding, and molecular interaction. However, FLIM signals are independent of dye concentration, excitation light intensity, and photobleaching.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, making FLIM microscopy possible without restrictions.


2-Photon Microscopy

2-Photon microscopy is based on the fact that a fluorophore can be excited when it is hit by two photons at the same time (typically within several femtoseconds). Typically, the wavelength of the two photons doubles the normal excitation wavelength, so that the excitation energy values are combined, causing a fluorescence signal. The probability of finding two photons, at the same time and at the same spot, is only likely in the focal plane of high numerical aperture objective lenses. The high excitation wavelength is less phototoxic, and it enhances the penetration depth when imaging a thick tissue material.

ibidi’s recommendation:

The ibidi Standard Bottom is optimized for fluorescence microscopy, making 2-Photon microscopy possible without restrictions.


Super-Resolution

New optical microscopy methods such as STED, SIM, (F)PALM and (d)STORM bypass the diffraction barrier and enable super-resolution imaging or “Nanoscopy” with substantially improved optical resolution. Nowadays they can provide a spatial resolution in three dimensions that is well below the diffraction limit and close to a near-molecular resolution. They can be applied to biological samples, and provide new views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales.

ibidi’s recommendation:

Super-Resolution using dSTORM is possible with the ibidi Standard Bottom. For other applications, we recommend using a glass bottom.

(Example provided by Markus Sauer, Würzburg)