한국어

KNOWLEDGE · MICROSCOPE IMAGING

How does a phase contrast microscope make transparent, invisible cells visible?

A phase contrast microscope uses two rings to send the light that passes straight through a cell and the light the cell spreads along different paths, then advances only the straight light by a quarter wavelength and dims it, so the two cancel in the image and the transparent cell looks dark.

In a brightfield microscope, colorless, transparent things such as bacteria and cells are hard to tell from the background. Brightfield forms its image from how much light the specimen absorbs, that is, from differences in color and brightness, and such specimens hardly absorb light; they only delay it a little (phase). Staining is often unsuitable for living specimens, and darkfield illumination easily leads to misreading fine structure. Closing the condenser aperture or defocusing brings a little contrast but blurs the detail. In the 1930s Frits Zernike devised a way to turn this delay into differences in brightness by placing a phase plate in the path of the light that goes straight through, and the 1953 Nobel Prize in Physics was awarded for this phase contrast method, especially for his invention of the phase contrast microscope.

Sources: colorless, transparent specimens, the limits of staining and darkfield, the phase plate, the prize citation: NobelPrize.org (award ceremony speech, Facts) · specimens that only delay light: Nikon MicroscopyU · the condenser aperture: Evident, Köhler Illumination · focus in brightfield: Schöll et al. 2014

  1. 01 StructureAdd two rings to brightfield
  2. 02 PracticeSet the number, aperture, centering and focus

01 Structure

Swap the condenser aperture for an annulus, and add a phase plate to the objective

Two places change from a brightfield microscope. The round iris of ⑤ the condenser becomes an annulus, and ⑧ the objective becomes a phase contrast objective with a phase plate at its back focal plane. Switch between the two with the buttons and the path of light and the camera image change together.

⑩ Camera image (computed)

Cell 0.02–1.80×visible

Straight light SLight spread by the specimen DPlanes where light gathers (①⑤⑧)Planes where images form (③⑦⑩)

Phase contrast: the ⑤ annulus lets light in only through a ring, and that ring is imaged onto the ring of the phase plate at the ⑧ objective's back focal plane. The other parts are the same as in brightfield.

⑤ Condenser aperture → annulusswap

Brightfield
A round opening. It lights the specimen with a full cone. Closing it raises the contrast but lowers the resolution.
Phase contrast
A ring-shaped opening. It lights the specimen with a hollow cone, so the straight light S gathers into a ring at ⑧ the back focal plane. The round iris is opened all the way.

⑧ Objective → phase contrast objectiveadd

Brightfield
Collects the light from the specimen to form the image. Nothing sits at its back focal plane.
Phase contrast
A phase plate (a thin ring of film) sits at that ring's place in the back focal plane and advances only S by a quarter wave while dimming it to 25%. The plate is not fitted separately; the Ph number on the barrel is the number of the annulus to use.

How it makes cells visible

  1. Split the light into two paths. ⑤ and the back focal plane of ⑧ are conjugate planes, so the ring of the annulus is imaged onto the ring of the phase plate. Light that goes straight through the specimen (S) gathers in this ring, and light spread by the cell (D) passes outside it.
  2. Advance and dim S alone. The ring of film on the phase plate advances only S by a quarter wave and cuts its intensity to 25%. D is left as it is.
  3. Cancel in the image. D already lags S by a quarter wave, so it is now half a wave out of step with the advanced S and the two cancel. The cell turns dark, and the camera only measures that brightness.
The other eight parts are the same as in brightfield
PartRoleHow it works
① Light sourceGives off the light that illuminates the specimen (an LED or a halogen lamp)Set the brightness with the voltage or ND filters, not with the irises
② Collector lensGathers the light from the sourceImages the light source at ⑤ and fills that place with light
③ Field diaphragmSets how large an area is lit, which cuts glareThe condenser images it onto the specimen plane. Open it to just outside the field of view
④ FilterPicks the color of the lightGreen (550 nm) for phase contrast. The phase plate is made to give a quarter wave at this wavelength
⑥ Condenser lensLights the specimenTurns the light from each point at ⑤ into parallel light going one way, and images ③ onto the specimen plane. Its height sets up Köhler illumination
⑦ SpecimenAbsorbs no light; it only delays itMost of the light goes straight on (S), and the structure spreads some of it in many directions (D)
⑨ Tube lensForms the imageCollects the parallel light from the objective. Magnification = tube lens ÷ objective focal length (Nikon and Leica 200 mm, Olympus 180 mm, Zeiss 165 mm)
⑩ Camera (or eye)Measures only the brightness of the imageIt does no computing for phase contrast. It records, as it is, the result of S and D interfering on the sensor

Sources: the annulus and the phase plate, conjugate planes, S and D, the quarter wave and 25%, the green filter, opening the condenser aperture fully: Nikon MicroscopyU · the roles of the other parts: Evident, Köhler Illumination (Abramowitz & Davidson), Infinity Optical Systems (Spring & Davidson) · the images are computed by this page (a virtual cell, 10×/0.25, green 550 nm) · the drawing is a simplified diagram of the light path

02 Practice

Set the number, the aperture, the centering and the focus

Phase contrast works only when the image of the annulus lies on the ring of the phase plate. The microscope below is still set up for brightfield. Change one thing at a time and watch how the objective's back focal plane, seen through a phase telescope, and the camera image change.

Objective
Condenser turret
Vessel and position

Back focal plane (phase telescope)

Bright ring: annulusDark ring: phase plate

Drag the bright ring, or select the picture and move it with the arrow keys (the annulus's centering screws).

⑩ Camera image (computed)

    The brightfield 4×/0.10 has no version with a phase plate, so 4× is shown with a 4×/0.13 (PhL). 10×/0.25 and 20×/0.40 share one Ph1 annulus. The images are exposed so the background is gray, and the background number counts an empty field without a phase plate as 100%.

    Sources: an annulus with the same number as the objective (PhL 4×, Ph1 10·20×, Ph2 40·60×), overlapping the rings through a phase telescope, opening the condenser aperture fully: Nikon MicroscopyU · specifications of objectives with a phase plate: Nikon, CFI Achromat Series for Phase Contrast · the curved liquid surface in wells: Nienhaus et al. 2023 · brightfield has the least contrast in focus: Schöll et al. 2014 · the images and the ring picture are computed by this page (a virtual cell, green 550 nm). The NA of the phase rings (PhL 0.048, Ph1 0.114, Ph2 0.266) is counted from Webb 2015's setups and Kurata et al. 2023's measurement, and the ring widths, the shift that halves the contrast and the tilt of the liquid surface in a well (contact angle 74°) are this page's model hypothesis

    Summary

    Two overlapping rings get past seeing brightness only

    1. Brightfield sees only brightness. A transparent cell only delays light, so in focus it is nearly as bright as the background.
    2. Add two rings. The condenser annulus and the phase plate at the objective's back focal plane sit in conjugate planes. The phase plate advances only the straight light by a quarter wave and dims it, so it cancels the spread light in the image and the cell looks dark.
    3. Keep the two rings on top of each other. Fit the annulus with the same number as the objective, open the condenser aperture fully, overlap the bright ring with the dark ring through a phase telescope, and look in focus. In wells, image at the center.

    MythA phase contrast image is computed by the camera or by software.

    In factThe phase plate (a glass plate) inside the objective and the interference of light make it. The camera is the same ordinary camera as in brightfield, and looking through the eyepieces gives the same. Computing phase-contrast-like images in software is a separate method.

    MythIn a phase contrast image, darker means thicker.

    In factHowever thick, the inside of a large object returns to the background brightness (shade-off), and a very large delay even makes an object brighter than the background.

    Sources: MicroscopyU · Evident · Nienhaus et al. 2023 (wells, computing it in software) · Kurata et al. 2023

    Questions

    Frequently asked questions

    What is a phase contrast microscope?

    An optical microscope that shows differences in how much a transparent specimen delays light (its phase) as differences in brightness. It is used to look at living, unstained cells. Frits Zernike devised it in the 1930s and received the 1953 Nobel Prize in Physics for it.

    How does phase contrast microscopy work?

    It takes two rings. The condenser annulus lets light in only through a ring, and the light that passes straight through the specimen (S) gathers on a ring of the same size on the phase plate inside the objective. The light spread by the cell (D) passes outside that ring. When the ring of film on the plate advances only S by a quarter wavelength and dims it, S ends up half a wave apart from D, which was already a quarter wave behind, and the two cancel in the image, so the cell looks dark.

    How does a phase contrast microscope differ in structure from a brightfield microscope?

    Two parts differ. Instead of the round iris at the condenser's front focal plane it uses an annulus with a ring-shaped opening, and it uses a phase contrast objective with a ring of film (the phase plate) at its back focal plane. The two rings sit on conjugate planes, so the image of the annulus lands on the ring of the phase plate. The light source, collector lens, field diaphragm, condenser, tube lens and camera are the same, and a green filter matched to the phase plate is recommended.

    What causes the halo and shade-off in phase contrast microscopy?

    The ring of the phase plate has a width, so part of the light spread by large, smooth structures passes through the ring with the straight light and is advanced and dimmed too. That light can no longer cancel, so the inside of a large object returns to the background brightness (shade-off) and a bright rim appears outside its edge (halo). Apodized phase plates, with neutral density rings beside the phase ring, reduce the halo.

    What do Ph1, Ph2 and Ph3 mean on a phase contrast microscope, and how do you match them?

    They are the codes of condenser annuli sized to match the phase rings of objectives. Usually PhL goes with 4× and 5×, Ph1 with 10× and 20×, Ph2 with 40× and 60×, and Ph3 with 100× objectives. Put in the annulus with the same code as the objective, lay the two rings on each other while watching through a phase telescope or Bertrand lens, and open the condenser aperture fully.

    Can you build a phase contrast microscope yourself?

    The phase plate is fixed inside a phase contrast objective, at its back focal plane, so you usually buy a phase contrast objective. The rest is an ordinary brightfield microscope. Place an annulus matched to the NA of that objective's phase ring at the condenser's front focal plane (radius = NA × condenser focal length), or, without a condenser, an LED ring above the specimen (Webb 2015), and match the size and the center while watching through a phase telescope. To use an objective without a phase ring, put the phase plate on a plane conjugate to the back focal plane formed by relay lenses (external phase contrast).

    Why does phase contrast work poorly in 96-well plates?

    Surface tension curves the medium in the well up toward the wall, so it bends the light like a lens and the image of the annulus slides off the ring of the phase plate (the meniscus effect). In one measurement with standard phase contrast, the area where phase contrast worked was 2.3% of the well in a 96-well plate (10×) and 25% in a 6-well plate (4×). Imaging at the center of the well and using larger wells or labware with a flat liquid surface helps.

    Sources: MicroscopyU (principle, configuration, halo, alignment) · Evident (Köhler illumination) · NobelPrize.org · Webb 2015 · Nienhaus et al. 2023, Table 1

    Further reading

    The images are virtual specimens computed by this page (Fourier optics). The same computation was written separately in Python and the numbers checked against each other. No real micrographs are used.