Hyperspectral microscopy correlates spectra presented by the field of view (FOV) with spectra in a reference spectral library. An unlimited FOV is acquired by placing a sample on a slide mounted on a computer controlled translation stage on a microscope. The sample is illuminated with white light for darkfield scatter, %transmission or %reflection, or with a laser for fluorescence or other excited states. To see a video that describes how the PARISS hyperspectral microscope works click here.
Each object in the field of view will present a spectrum. If there are tens of thousands of objects, then PARISS will acquire tens of thousands of spectra many of which are common. The challenge associated with handling thousands of unique and often common spectra is handled with custom software that sorts and classifies all spectra in the FOV.
All or some classes of spectra can then be entered into a reference spectral library. Each library spectrum can be pseudo-colored and linked to a unique target “spectral object”. The presence of one or more pseudo-colors then confirms the location and presence of a target object.
Future samples can then be scanned, and the spectra presented by all objects in the FOV will be correlated with those in the library. Those that meet a minimum correlation coefficient will then be “painted” onto a gray scale image. Various data processing options including counting correlated objects and various mathematical functions.
To see a video that describes how the PARISS microscope works in darkfield click here.
Creating a reference spectral library
The PARISS hyperspectral microscope acquires many thousands of spectra over an unlimited field of view (FOV). The PARISS software evaluates all spectra and sorts them into “classes.” One class will likely be “background spectra” the remaining spectra will be target objects.
Some, or all, classes of spectra can be added into a reference spectral library. Each library spectrum will be pseudo colored either by the software or the instrument user.
All, or selected, spectra from the FOV that correlate with a library spectrum will then be “painted” with pixel-perfect accuracy onto a grayscale image of the FOV.
PARISS hyperspectral microscope model: PHSI includes
Mounting interface: “C” mount to most research upright and inverted microscopes