Imaging for Quantative Analysis

Image quality, required sensitivity, magnification and resolution depend on the particular application and so it is the application that dictates the technology. Good quality, high resolution images call for the use of high numerical aperture objectives and high quality thin bottom optical plates.

Confocal imaging enables optical sectioning and the suppression of background light originating from out-of focus sample layers, such as layers within a cell, multi-layered cells or fluorescent probes in the supernatant in homogeneous assays.

The big advantage of confocality is that only a small vertical section of the sample, namely the one in the true focal plane, will be imaged. This gives rise to data acquired from the z-section of real interest only without any out of focus light contribution from other areas of the biological sample. Unlike most of the deconvolution based image restoration methods, quantification of fluorescence in the imaging plane is possible.

Widefield fluorescence image taken with the Operetta using a 60x high NA objective

Confocal image taken with the Operetta using a 60x high NA objective

While most of the HCS applications are being applied in two dimensions only (x, y), taking more than one reading from a cell sample can also be done vertically, i.e. the z direction of the sample. This requires optical sectioning / confocal measurement and is important for situations where the sample has a complex 3-dimensional shape or where the signal distributes vertically over different layers even within one image, as in multi-layered cell samples, tissues and whole organisms.

Visualization and analysis can then be completed with respective 3D representation and analysis software such as our high performance 3D-4D imaging software, Volocity®.