Phase contrast imaging in JPK Nanowizard 3 Bioscience AFM

Phase contrast imaging is an optical imaging mode where very transparent objects can be made more visible. The inverted optical microscope with the JPK Nanowizard 3 Bioscience AFM has this capability. Here is a brief description of how to set this up.

Detailed info about optical phase contrast microscopy

In short the illumination is shaped in a ring and this should match a phase shift ring in the objective. On the JPK system there are two phase contrast objectives, 20× (used with Ph1 aperture) and 40× (used with Ph2 aperture), they have different diameters of their respective phase rings, and therefore the ring-shaped aperture in the condenser have to be switched to fit the chosen objective.

 

Here is an overview of the JPK Nanowizard 3 Bioscience AFM with its most important parts indicated.

The motorized stage is controlled by the stage control box to the left, the stage can be manually controlled by the joystick, but the software tracks all movements of the stage. This makes it possible to stitch AFM-scans and optical images to larger areas.

The AFM scan head, i.e. the cantilever tip can be moved relative to the stage and sample with the two manual screws to the right (Scan head translation X-Y).

 

The phase contrast imaging cannot be done with the AFM scan head in place. The condenser cannot be moved down to its correct position. Here the microscope stage is shown without the scan head mounted.

The condenser with its aperture selector is shown with the focus knobs necessary to set up the phase contrast imaging.

Then what is the use of phase contrast imaging if it cannot be used together with the AFM scanning?
You can collect a phase contrast image before scanning and use as a background map for the AFM-scans.

 

Phase contrast images of human epithelial cells from the inner side of my mouth, simply collected by gently scraping the inside of the chin wall with a plastic spoon and transfer a blob of saliva and cells onto a cover slip. The 20× objective is used together with the Ph1 aperture.
Please note! There is NO contrast enhancement after collecting the image from the camera.

Here is the 20× objective with the Ph2 aperture. which is the wrong combination. Please notice the decreased contrast (not same area as in previous image).
Please note! There is NO contrast enhancement after collecting the image from the camera.

Here is the same area and objective with the A aperture. This is for normal imaging, i.e. no ring shaped illumination at all. Please notice the decreased contrast.
Please note! There is NO contrast enhancement after collecting the image from the camera.

 

Here the 40× objective is used together with the Ph2 aperture.
The 40× objective MUST be used together with cover slips, NOT standard microscope object slides. The shorter working distance (about 0.2 - 0.3 mm) of this objective makes it impossible to focus through an object slide (about 2 mm thick) onto the sample. Remember that this is an inverted microscope, the sample is on the top side of the glass plate and the microscope objective is on the underside.

And here the 40× objective is used together with the A aperture, which also gives a bad contrast.

How to adjust the phase contrast imaging

The illumination ring must be aligned to the phase ring in the objective. There are special optics on the microscope to make this alignment possible.

 

There is a small lever directly under the eye-pieces. This can be switched so that the phase ring of the objective and the illumination ring can be seen in the eye-pieces.

To see these sharply it is necessary to use the special small focus knob on the right side of the eye-piece body.

Please note that this can only be seen in the eye-pieces, never in the CCD camera.

 

Here is what you see in the eye piece when the lever is switched to see the ring shaped phase contrast aperture. This is showing the 20× objective with the Ph1 aperture, the correct combination. The ring shaped illumination is NOT aligned to the phase ring of the objective.
It is difficult to display this in a photo due to multiple reflections of the illumination ring. The phase contrast ring of the objective is barely visible as a weaker ring of the roughly the same diameter as the illumination ring.

 

Here the illumination ring and the phase ring of the objective are properly aligned. This is showing the 20× objective with the Ph1 aperture, the correct combination. Here is the wrong combination of 20× objective with Ph2 aperture. The dark outer ring is the phase ring of the objective, and the smaller bright ring is the illumination from the Ph2 aperture.

 

Wrong combination of 40× objective and Ph1 aperture. The phase ring of the 40× objective is the smaller dark ring, and the outer, brighter ring is the illumination from Ph1 aperture. Here is the also wrong combination of 40× objective with the A aperture. The dark ring is the phase ring of the objective, and the bright circular area is the illumination from the A aperture, used for bright field (normal) imaging.

 

Here is the condenser shown with three aperture settings. Rotate the wheel with the red aperture labels to switch the apertures.

The adjustment knobs for aligning the Ph-apertures to the phase ring are partly visible behind the two horizontal knobs at the upper left and right in the pictures.

Here the knobs are more visible. Please note there is a small locking knob on top of each adjustment knob, that might need loosening before adjusting.

The "A" aperture has a small lever to adjust the size of the aperture.

 

As mentioned earlier, all these adjustments as well as acquisition of a phase contrast image, has to be done with the AFM scan head removed.

When mounting the AFM scan head, the condenser has to be raised, using the condenser focus knobs. Otherwise, the lowest part of the condenser will collide with the scan head and disturb scanning. See to it that there is a clear gap between condenser and scan head.

 

Here is an example of AFM scan with an optical image as background. The mismatch between AFM scan and optical image is a few µm.

 

The CCD camera image during scanning. This is not a phase contrast image, but the contours of the cells are quite visible due to them being dried.

 

To make things a bit simpler I let the saliva sample with epithelial cells dry out. After a while I added a drop of water to practice AFM scanning in liquid. The cells became partly hydrated and much softer. It was then not possible to get a AFM scan of the cells, they were just pushed around by the tip, as can be seen in this short movie.

The movie is not a complete loop of a scan, therefore the sudden jump of the cantilever.

Anders Liljeborg Albanova Nanolab, KTH, SU.