PSFj is a software tool that automatically analyses the full field-of-view (FOV) performance of a given fluorescence microscope/objective lens combination with respect to its optical resolution and chromatic aberrations. PSFj provides reporting functions to document the momentary performance of a system and it allows for the export of the obtained data, e.g. for image restoration purposes. PSFj is based on ImageJ and JAVA, and runs on Windows, Mac, and Linux PCs as a stand-alone application.
PSFj was developed with a number of applications in mind in order to satisfy the needs of
- Microscope users
- Microscope facility managers
- Microscope developers
as an easy-to-use tool to quantify the performance of fluorescence light microscopes and their optical components for
- Routine checks of microscopes and objective lens performance
- Comparison and selection of best-performing objectives/microscopes
- Calibration of microscopes for quantitative applications
Principle, scope, and deliverables
PSFj is based on the analysis of image stacks of a large number (many hundreds) of fluorescent beads. The quantitative analysis of the fluorescence signals from the beads then provides information about the used microscope with respect to the resolution, the flatness of the focal plane and the fidelity of the chromatic correction along the lateral and axial dimensions. All information is retrieved as a function of position in the FOV. Using 2D fitting algorithms, PSFj provides for each fluorescence channel
- the PSFs lateral and axial full-width-at-half-maximum (FWHM). This provides a measure for the resolution.
- the center-of-mass co-ordinates. This provides measures of planarity and chromatic aberration correction. It furthermore detects misaligned/tilted stages and sample-holders or cameras.
Upon analysis of the data, the following is delivered: The data is visualized using heatmaps to represent the information across the FOV. The data is compared to the diffraction limited (theoretical) performance of the system. A pdf file containing all microscope information and the analysis results is generated to document the measurements. Data can be exported using .csv files. Interactive inspection and export of the results for specific beads in the image stack is possible.
In particular: PSFj optionally outputs an .xls file (Landmark file) to be used with bUnwarpJ, an ImageJ plugin for image registration (Ignacio Arganda-Carreras, Carlos O. S. Sorzano,Roberto Marabini, Jose M. Carazo, Carlos Ortiz de Solorzano, andJan Kybic, “Consistentand Elastic Registration of Histological Sections using Vector-Spline Regularization”, Lecture Notes in Computer Science, Springer Berlin / Heidelberg, volume 4241/2006, CVAMIA: Computer Vision Approaches to Medical Image Analysis, pages 85-95, 2006. ; http://biocomp.cnb.csic.es/~iarganda/bUnwarpJ/) for lateral non-linear (elastic) alignments of images of different channels (“unwarping” chromatic aberrations).
For what types of microscopes can I use PSFj?
Since the analysis is based on image-stacks of fluorescent beads, PSFj can be used to investigate any microscope that can be used to image beads that are mounted on a flat surface, e.g., Confocal-, Widefield-, Spinning Disc-, TIRF-microscopes, etc.
What type of results do I get?
All results are provided as function of position in the FOV and include (see Figure 4.7):
- minimum and maximum lateral FWHM (resolution in the xy-plane:)
- the resolution asymmetry (A), i.e., the ratio of the minimum lateral FWMH over the maximum lateral FWHM
- the orientation of the maximum lateral FWHM (θ).
- the axial FWHM (resolution along the z-axis)
- the z-position of the best focus of each bead. From this the planarity of the focal plane can be measured.
and in the case of dual-color analysis
- the lateral and axial chromatic shifts throughout the FOV, for different color channels (emission wavelengths). This serves as a measure of lateral and axial chromatic aberrations.