-Z
To empirically measure the point spread function (PSF), you must optically section a fluorescent bead. GE Healthcare uses a PSF scan that includes images from approximately 4 mm above and below the plane of best focus, at 0.1 mm intervals.
The properties of the objective lens are the primary determinant of the PSF, so it is only necessary to measure PSFs when new lenses are added to the microscope. Since the deconvolution program can adapt to the PSF (actually the OTF) wavelength, it is unnecessary to measure PSFs at more than one wavelength.
As stated before, you should only need to measure the PSF once. For this reason be careful to thoroughly check all imaging conditions; take the extra time necessary to get a good signal-to-noise ratio in the images and to completely scan the bead. An accurately measured PSF is essential to successful deconvolution.
For this procedure you will need:
a clean and aligned DeltaVision system
a bead slide with 0.1mm, or smaller, fluorescent beads
a DeltaVision grid slide, or other microscope ruler
an immersion oil set, if appropriate
Use the following procedure to measure the PSF and obtain the corresponding OTF:
Obtain the lens ID number.
Measure the XY pixel size.
Add the new lens ID number, pixel size, and other relevant information to the system configuration (that is, modify the Resolve3D.SYS file).
Mount the slide on the microscope and focus on the beads to obtain the maximum intensity.
Center a single bead in X and Y directions. Collecting large images, such as 1024x1024, may help. Use the Center Object function when working with DeltaVision's that have XYZ stages.
Adjust the CCD exposure time so that the maximum intensity at the plane of best focus is at least 2000 counts. Make sure that the camera does not saturate at the plane of best focus.
Now use a 256x256 image.
Verify that Resolve3D is accurately configured for lens and auxiliary magnification.
Run the Standard PSF Measurement Macro, shown below.
Use the MakeOTF program to convert the optical sections into an OTF.
First of all, finding beads is easiest with a very dark room.
Bead slides from GE Healthcare include 1mm beads that fluoresce brightly at 617nm. Coarsely focus on the slide by positioning the lens near the slide. Then scan the slide while looking for fluorescence haze from the 1mm beads. When you focus on fluorescence haze from the 1mm beads you should also find the 0.1 mm beads.
In the case where you do not have a slide with large or bright beads, you can also use reflection mode to find beads. Set up reflection mode such that you can focus on the field aperture. With the slide in focus, switch back to fluorescence mode and make small focus adjustments to find the beads. It may be necessary to repeat this procedure several times before you are able to find beads.
# PSF Acquisition Macro for 60X/1.40 and 100X/1.40 Lenses OPF REMOTE
FOCUS 26.4 FOCUS -20.0
DO 128 AVG 4 WRT FOCUS -0.1 ENDDO
FOCUS 6.4 CLF LOCAL
Experience has shown that an index of refraction equal to 1.518 is ideal for measuring beads that are mounted in glycerol on a 1-1/2 coverslip. Use of an inappropriate immersion oil will yield asymmetric PSF measurements as a result of spherical aberration. GE Healthcare strives to measure PSFs with a minimal amount of spherical aberration. The Lens Information program can help you select the proper oil.
To confirm that the immersion oil is a good choice, rotate the 3D image to get a view of the XZ or YZ plane. For example, rotate the image 90 degrees about the X axis, or 90 degrees about the Y axis. "Flip" the data to view XZ sections, rather than XY sections. (To better see the shape of the PSF, it is helpful to do an exponential scaling -- an exponent of .5 usually works well.) Alternatively, use the Volume Viewer to generate a 3D rendering of the bead scan.
Look for symmetric flare in the resulting image. Symmetry indicates that the oil is correct, and in virtually all situations, the most symmetric PSF along the Z axis is also the smallest and has the highest intensity. That is, symmetry corresponds with the highest resolution.
The
following figures demonstrate the effect of using the correct versus incorrect
oil.
+Z
-Z
Immersion Oil is Too Low:
Flare points down, in the direction of negative Z.
+Z
-Z
Flare points up, in the direction of positive Z.
+Z
-Z
The quick PSF scan, below, will facilitate PSF measurement for the purpose of evaluating your choice of immersion oil. The scan acquires only thirty-two 128 x 128 optical sections at 0.2 um intervals. This coarse scan is not sufficient to determine the resolution of the microscope, but is adequate for choosing oils.
# Quick PSF Scan Macro OPF REMOTE FOCUS 23.2 FOCUS -20.0 DO 32 CCD WRT FOCUS -0.2 ENDDO FOCUS 3.2 CLF LOCAL
Repeat the acquisition with a different oil and viewing process until you are satisfied that the optimum immersion oil that generates the most symmetric PSF has been found.