No specialized optics
Iatia's QPI generates phase data based on a minimum of two conventional brightfield images captured digitally at slightly different focal planes.
QPI is not bound by the inherent limitations of specialized phase contrast optical systems, allowing:
- use in applications where the addition of specialized optics is impractical and cumbersome (eg. high throughput image analysis)
- simultaneous capture of brightfield and phase data without the need to change optical configurations or employ complicated optical systems which may degrade image quality, allowing for more accurate co-localization between phase and brightfield images (eg. confocal microscopy)
- unpolarized illumination
- use with birefringent plastic cultureware
- generation of phase contrast images at all available magnifications
Case study - co-localization in confocal microscopy
QPI technology is being used by researchers at the Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute, Australia, with confocal laser scanning microscopes (CSLMs) to generate phase contrast images simultaneously with confocal fluorescence images for more accurate co-localization in a simplified workflow1.
Utilizing QPI technology, brightfield images collected with the TLD of a CSLM may be captured at the same time as confocal fluorescence images. The benefits of using QPI to simultaneously capture brightfield and confocal fluorescence images include:
- a simplified image collection process
- phase contrast images such as DIC, optical phase contrast and Hoffman Modulation Contrast can be generated from the archival brightfield data
- no specialized optics which may compromise the intensity or quality of confocal fluorescent images
- no misalignment of image sets for improved co-localization
- no temporal changes in samples between capture of fluorescent and registration image sets
Rat mast cell
An activated rat mast cell stained with antibodies to protein phosphatase 1 (PP1). Fluorescent and TLD brightfield images were collected simultaneously using a Bio-Rad MRC 1024 confocal microscope attached to a Nikon TE-300 inverted microscope at 60x magnification. A z-series was collected at 0.5µm intervals and DIC images were generated utilizing QPI technology.
TLD brightfield
|
Fluorescent
|
QPI DIC
|
QPI DIC with fluorescent overlay
|
Confocal fluorescent images were able to be captured with maximum intensity and overlaid on the QPI DIC image. Note the registration of the fluorescence at the cell margins demonstrates excellent alignment of the confocal fluorescent and QPI DIC images.
References
-
A simple method allowing DIC imaging in conjunction with confocal microscopy
S.H. Cody, S.D. Xiang, M.J. Layton, E. Handman, M.H.C. Lam, J.E. Layton, E.C. Nice, and J.K. Heath, Journal of Microscopy 217, 265-274 (2005).