So sánh top và phase contrast

Phase contrast microscopy translates small changes in the phase into changes in amplitude (brightness), which are then seen as differences in image contrast.

Unstained specimens that do not absorb light are known as phase objects. This is because they slightly change the phase of light that is diffracted by them; the light is usually phase-shifted by about ¼ wavelength compared to the background light. Our eyes are unable to detect these slight phase differences as they can only detect variations in the frequency and intensity of light.

Phase contrast enables high contrast images to be produced by further increasing the difference of the light phase. It is this characteristic that enables background light to be separated from specimen diffracted light. The difference of the light phase is increased by slowing down (or advancing) the background light by a ¼ wavelength, with a phase plate just before the image plane. When the light is focused on the image plane, the diffracted and background light cause destructive (or constructive) interference which decreases (or increases) the brightness of the areas that contain the sample, in comparison to the background light.

Light from a tungsten-halogen lamp goes through the condenser annulus in the substage condenser before it reaches the specimen. This allows the specimen to be illuminated by parallel light that has been defocused.

Some of the light that passes through the specimen will not be diffracted (bright yellow in the picture). These light waves form a bright image on the rear aperture of the objective. The light waves that are diffracted by the specimen pass the diffracted plane and focus on the image plane only. This allows the background light and the diffracted light to be separated.

The phase plate then changes the background light’s speed by ¼ wavelength. When the light is focused on the image plane, the diffracted and background light will cause destructive or constructive interference, which changes the brightness of the areas that contain the sample in comparison to the background light. Often the background is also dimmed by 60 to 90% by a grey filter ring.

Figure 1: The path of light in an upright microscope set up for phase contrast.

Aligning your microscope for phase contrast

All of the components required for phase contrast need to be aligned and centred. Some phase sliders are pre-centred, we therefore recommend that you check beforehand. Details of how to centre and align phase components are below:

1. If possible, set up Koehler illumination on your microscope. Read Scientifica’s 6 step guide to Koehler illumination to help you set this up.

2. Install the phase rings in the condenser.

3. Remove the eyepieces and replace these with the phase contrast centring telescope.

4. Put the phase contrast telescope into focus, so that the phase plate and phase ring are in focus.

5. Put the lowest magnification phase objective and corresponding phase annulus in place. For example, a 10x Ph1 objective with a Ph1 phase annulus.

6. Look at the phase plate and phase ring through the phase telescope.

7. Using the adjustment screws on the condenser, centre the phase plate and phase ring so the segmented circle of light sits on the black ring.

Figure 2: On the left, the objective phase ring and condenser annulus are mis-aligned. On the right, the components are correctly aligned, as the segmented circle of light created by the condenser annulus sits on the black ring.

8. Repeat steps 4-6 for the rest of the objectives.

9. Once all objectives have been aligned and centred, remove the phase contrast centring telescope and replace this with the eyepieces.

You shouldn’t need to re-centre the phase contrast microscope. It is, however, recommended to regularly check that the set-up is centred using the phase contrast telescope.

Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.

When light waves travel through a medium other than a vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength-dependent and may give rise to colors. Photographic equipment and the human eye are only sensitive to amplitude variations. Without special arrangements, phase changes are therefore invisible. Yet, phase changes often convey important information.

The same cells imaged with traditional bright-field microscopy (left), and with phase-contrast microscopy (right)

Phase-contrast microscopy is particularly important in biology. It reveals many cellular structures that are invisible with a bright-field microscope, as exemplified in the figure. These structures were made visible to earlier microscopists by staining, but this required additional preparation and death of the cells. The phase-contrast microscope made it possible for biologists to study living cells and how they proliferate through cell division. It is one of the few methods available to quantify cellular structure and components that does not use fluorescence. After its invention in the early 1930s, phase-contrast microscopy proved to be such an advancement in microscopy that its inventor Frits Zernike was awarded the Nobel Prize in Physics in 1953.

Working principle[edit]

Dark field and phase contrast microscopies operating principle

The basic principle to make phase changes visible in phase-contrast microscopy is to separate the illuminating (background) light from the specimen-scattered light (which makes up the foreground details) and to manipulate these differently.

The ring-shaped illuminating light (green) that passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The remaining light is unaffected by the specimen and forms the background light (red). When observing an unstained biological specimen, the scattered light is weak and typically phase-shifted by −90° (due to both the typical thickness of specimens and the refractive index difference between biological tissue and the surrounding medium) relative to the background light. This leads to the foreground (blue vector) and background (red vector) having nearly the same intensity, resulting in low image contrast.

In a phase-contrast microscope, image contrast is increased in two ways: by generating constructive interference between scattered and background light rays in regions of the field of view that contain the specimen, and by reducing the amount of background light that reaches the image plane. First, the background light is phase-shifted by −90° by passing it through a phase-shift ring, which eliminates the phase difference between the background and the scattered light rays.

When the light is then focused on the image plane (where a camera or eyepiece is placed), this phase shift causes background and scattered light rays originating from regions of the field of view that contain the sample (i.e., the foreground) to constructively interfere, resulting in an increase in the brightness of these areas compared to regions that do not contain the sample. Finally, the background is dimmed ~70-90% by a gray filter ring; this method maximizes the amount of scattered light generated by the illumination light, while minimizing the amount of illumination light that reaches the image plane. Some of the scattered light that illuminates the entire surface of the filter will be phase-shifted and dimmed by the rings, but to a much lesser extent than the background light, which only illuminates the phase-shift and gray filter rings.

The above describes negative phase contrast. In its positive form, the background light is instead phase-shifted by +90°. The background light will thus be 180° out of phase relative to the scattered light. The scattered light will then be subtracted from the background light to form an image with a darker foreground and a lighter background, as shown in the first figure.

[edit]

S. cerevisiae cells imaged by DIC microscopyA quantitative phase-contrast microscopy image of cells in culture. The height and color of an image point correspond to the optical thickness, which only depends on the object's thickness and the relative refractive index. The volume of an object can thus be determined when the difference in refractive index between the object and the surrounding media is known.

The success of the phase-contrast microscope has led to a number of subsequent phase-imaging methods. In 1952, Georges Nomarski patented what is today known as differential interference contrast (DIC) microscopy. It enhances contrast by creating artificial shadows, as if the object is illuminated from the side. But DIC microscopy is unsuitable when the object or its container alter polarization. With the growing use of polarizing plastic containers in cell biology, DIC microscopy is increasingly replaced by Hoffman modulation contrast microscopy, invented by Robert Hoffman in 1975.

Traditional phase-contrast methods enhance contrast optically, blending brightness and phase information in a single image. Since the introduction of the digital camera in the mid-1990s, several new digital phase-imaging methods have been developed, collectively known as quantitative phase-contrast microscopy. These methods digitally create two separate images, an ordinary bright-field image and a so-called phase-shift image. In each image point, the phase-shift image displays the quantified phase shift induced by the object, which is proportional to the optical thickness of the object. In this way measurement of the associated optical field can remedy the halo artifacts associated with conventional phase contrast by solving an optical inverse problem to computationally reconstruct the scattering potential of the object.

See also[edit]

• Live cell imaging
• Phase-contrast imaging
• Phase-contrast X-ray imaging

References[edit]

• "The phase contrast microscope". Nobel Media AB.
• Zernike, F. (1955). "How I Discovered Phase Contrast". Science. 121 (3141): 345–349. Bibcode:1955Sci...121..345Z. doi:10.1126/science.121.3141.345. PMID 13237991.
• "The Nobel Prize in Physics 1953". Nobel Media AB.
• Mertz, Jerome (2010). Introduction to optical microscopy. Greenwood Village, Colo.: Roberts. pp. 189–190. ISBN 978-0-9815194-8-7.
• Frits Zernike (1942). "Phase contrast, a new method for the microscopic observation of transparent objects part I". Physica. 9 (7): 686–698. Bibcode:1942Phy.....9..686Z. doi:10.1016/S0031-8914(42)80035-X.
• Frits Zernike (1942). "Phase contrast, a new method for the microscopic observation of transparent objects part II". Physica. 9 (10): 974–980. Bibcode:1942Phy.....9..974Z. doi:10.1016/S0031-8914(42)80079-8.
• Oscar Richards (1956). "Phase Microscopy 1954-56". Science. 124 (3226): 810–814. Bibcode:1956Sci...124..810R. doi:10.1126/science.124.3226.810. PMID 13380397.
• US2924142, Georges Nomarski, "INTERFERENTIAL POLARIZING DEVICE FOR STUDY OF PHASE OBJECTS" US4200354, Robert Hoffman, "Microscopy systems with rectangular illumination particularly adapted for viewing transparent object"