Microscope Contrast Techniques

When using a microscope with brightfield, some samples will have a natural contrast that is easily viewed, such as bright plants and flowers, metals, and pigments. Some samples can be stained to increase the microscope contrast. However, low contrast samples such as unstained bacteria, thin tissue slices, live cells, or reflective metal parts require the use of additional microscope contrast techniques in order to view the sample. Here we will explore each of these different microscopy contrasting techniques.

Top 7 Microscope Contrast Techniques:

1. Darkfield
2. Phase Contrast
3. Polarization
4. Fluorescence
5. Oblique Illumination
6. Differential Interference Contrast (DIC)
7. Improved Hoffman Modulation Contrast (iHMC)
   

 

Darkfield Microscopy

When using darkfield microscopy, no light from the illuminator will pass into the imaging system, only light diffracted by the structure is captured by the objective. This is what makes the structures appear rich in contrast, and very bright against a dark background. Darkfield microscopy is especially useful to detect tiny and isolated structural details, such as bacteria. When working with reflected light, darkfield illumination is used to identify grain boundaries in polished and etched metal sections, as well as to detect contaminants and flaws in surfaces.

This image of salt was captured using a darkfield slider on the Zeiss Primostar 3 microscope.

 

Phase Contrast Microscopy

When using a brightfield microscope, thin and transparent samples have a very low contrast and can be barely visible. Phase contrast microscopy is the method of choice for viewing thin, unstained samples such as culture cells. Phase contrast uses a special condenser and objective lenses to enhance the contrast seen in the microscope image. Phase contrast is not useful for thick specimens such as plant or animal tissue sections.

This image of rabbit tastebuds was captured using the Zeiss Primsotar 3 phase contrast microscope.

Polarization

Polarizing microscopes must be equipped with two crossed polarizers. In most cases at least one polarizer and one analyzer are used. Many materials, like most crystals and some biological structures such as muscle cells, are birefringent, meaning they have two refractive indices. This phenomenon fulfills an important diagnostic function in mineralogy, pharmacology, forensic microscopy, polymer research, or the quality control of textile fibers. Reflected light is used to visualize the contrasts in the origin of structure of opaque metals such as aluminum, zirconium, and others.

This image of crystals was captured using a polarizing microscope.

 

Fluorescence Microscopy

Fluorescence is a low-energy form of radiation (emission) that results from a previous high energy illumination (excitation). As soon as the excitation stops, the fluorescence emission stops almost immediately. The particular advantages of fluorescence microscopy include a strong image contrast and its specificity; that is, its ability to specifically detect individual structures down to individual molecules. Fluorescence microscope illuminators often use gas discharge lamps such as HBO, XBO, HXP or long-life LED light sources. LEDs make it possible to change the excitation wavelengths extremely quickly. The downside to LED light sources is that they still exhibit rather low excitation intensity in certain spectral ranges.

This image of Convallaria (flower) was captured using a fluorescence microscope.  

Oblique Illumination

Oblique illumination is recommended to contrast objects that are too thick for alternative contrasting techniques. Oblique illumination directs light beams onto the specimen at different angles. As a result of highly directed one-sided illumination, the sample will have one side that appears bright and the other darker, which results in a shadow-casting relief image showing fine structural details. Oblique illumination microscopy is utilized to view thick tissue samples or solid samples that do not allow light to pass through them, such as automotive parts.

This is an automotive part captured under the Zeiss Stemi 305 stereo microscope using oblique illumination.

 

Differential Interference Contrast (DIC) Microscopy

In interference contrast, previously split partial beams are combined as in phase contrast, interfering with each other as a result. Due to the interference of the two twin images in the intermediate image plane, a light-dark contrast is generated at the edges of objects. By laterally shifting the objective-sided DIC prism, also known as the DIC slider, the contrast can be adjusted in such a way that the light and dark object edges appear on a gray background. As the illumination appears darker on one side and brighter on the other side, a pseudo-relief image is perceived by our brain. This can - but does not have to - match the actual topography of the sample.

This image of Navicula Lyra (algae) was captured using DIC on the Zeiss Axioscope 5 microscope.

 

Improved Hoffman Modulation Contrast (iHMC)

Hoffman modulation contrast is a form of oblique transmitted light illumination in combination with a grayscale modulator comprising neutral gray absorbing layers mounted in the objective's back focal plane. The modulator consists of three grayscale strips that run vertically through the pupil of the objective. This results in this method's middle gray image background and modulates a relief contrast. iHMC is used in the live-cell microscopy of sperm and egg cells when conducting artificial insemination in human and veterinary medicine.

This image of an embryo was captured under the Zeiss Axio Vert A1 microscope using iHMC.

References: Koenen and Zölffel, Microscopy for Dummies, Wiley, 2020.

If you have any questions about microscope contrasting techniques or which microscope provides the best contrast for your application, please contact Microscope World.

Related Articles of Interest:

Different Types of Microscopes and Their Uses

Microscopy Polarization Explained

Darkfield Microscopy Explained