IntroductionPhase Contrast Microscopy (PCM)

The easiest and most common way to image biological samples is using phase contrast, which is a special contrast-enhancing imaging method for transmitted-light microscopes invented by Frits Zernike (1888-1966) in 1932 [1] and introduced into microscopic practice by August Köhler (1866-1948) and Loos in 1941 [2, 3]. Soon it revolutionized biological and medical research and earned its inventor the Nobel Prize in Physics in 1953.

Amplitude and Phase Objects
 
 

	Illustration of the different impact of amplitude and phase objects on transmitted light. (Figure taken from diploma thesis of Steve Pawlizak, 2009.)

Using an ordinary (bright-field) transmitted-light microscope, the produced images of very thin and transparent objects, like living cells or biological tissues, usually suffer from lacking contrast, which makes it very difficult and in many cases even impossible to recognize and distinguish delicate sample structures or image details. This is because light absorption of such objects is too low to provide sufficient amplitude changes of the transmitted light (bright-dark-contrast). However, individual sample structures (e.g. cell nucleus, cytoplasm, organelles) show little density differences, leading to small differences of the refractive indices and, for this reason, to different optical path lengths. If light waves pass those structures, they experience a certain phase shift that corresponds to the respective optical path lengths. The phase contrast technique is intended to convert such phase shifts into amplitude differences that are detectable by the human eye (bright-dark-contrasts).

Principle

When light passes through an object that is more optically dense than its environment (background), the wavefronts are retarded with respect to the unaffected, bypassing background light. It may be assumed that the phase shift of the so-called object light is ≤ 90° (corresponding to an optical path difference of ?/4), as it is the case for most biological samples. The idea behind visualization of phase shifts in the object light is to change the phase of the background light in such a manner that background and object light weaken or even cancel each other out when interfering in the primary image plane. Consequently, the object would appear dark against the background.

Schematic simplified assembly of a phase contrast microscope containing an annular aperture (light ring) in the condenser and an accordant phase ring in the objective. The background light is colored orange and the object light is rose. (Figure taken from diploma thesis of Steve Pawlizak, 2009.)

In order to do that, the background light must be influenced without affecting object light (which is already phase shifted due to the object). This is achieved by placing a annular aperture (light ring) in the front focal plane of the condenser and a matching phase ring in the back focal plane of the objective (see figure on the left). Due to this alignment, the parallel light fronts leaving the annular aperture are focused by the objective directly onto the phase ring, i.e. nearly all background light has to pass through the phase ring, which serves in two ways: On the one hand, the phase ring reduces the intensity of the background light by 70 to 90% like a neutral density filter and, on the other hand, it adds a constant phase shift of 90° (?/4) like a quarter-wave plate. The reduction of the amplitude is necessary, because the object light is much less intense than the bright direct light.
If an object is placed in the light path, the object light gets deflected, only slightly passing through the phase ring. Most object light passes by and remains unaffected as desired. Recombining the background and object light in the primary image plane results in an effective phase shift of about 180° (?/2), which means destructive interference. In fact, for best contrast, both phase shifts should complement one another as well as possible to 180°. For this reason, the properties of the phase ring must take into account the most frequent refractive index and the thickness of the samples.
Starting at a certain thickness, phase contrast objects show light or dark "halos" along their edges and simulate a kind of 3D effect. This is due to the fact that a small part of the diffracted object light passes through the phase ring as well and interferes at the image plane. Thus, these halos may not necessarily represent the actual structure of the sample.

References:
 

[1]		F. Zernike: Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung, Zeitschrift für technische Physik 16:454-457 (1935)
[2]		A. Köhler, W. Loos: Das Phasenkontrastverfahren und seine Anwendungen in der Mikroskopie, Die Naturwissenschaften 29:49-61 (1941)
[3]		W. Loos: Das Phasenkontrastverfahren nach Zernike als biologisches Forschungsmittel, Klinische Wochenschrift (Journal of Molecular Medicine) 20(34):849-853 (1941)
[4]		A. Köhler: Ein neues Beleuchtungsverfahren für mikrophotographische Zwecke, Zeitschrift für wissenschaftliche Mikroskopie und mikroskopische Technik 10:433-440 (1893)