Darkfield Microscopy

Dark-field microscopy is a technique that can be used for the observation of living, unstained cells and microorganisms. In this microscopy, the specimen is brightly illuminated while the background is dark. It is one type of light microscope, others being bright-field, phase-contrast, differential interface contrast, and fluorescence (Figure 1).

Principle of the Darkfield Microscope

A dark field microscope is arranged so that the light source is blocked off, causing light to scatter as it hits the specimen [1].

This is ideal for making objects with refractive values similar to the background appear bright against a dark background.

When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing the individual to view a specimen in dark field.

Important Features

The dark-ground microscopy makes use of the dark-ground microscope, a special type of compound light microscope [2].                

Figure 1: Principle of the Darkfield Microscope

The dark-field condenser with a central circular stop, which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope.

This microscope uses reflected light instead of transmitted light used in the ordinary light microscope.

It prevents light from falling directly on the objective lens.

Light rays falling on the object are reflected or scattered onto the objective lens with the result that the microorganisms appear brightly stained against a dark background.

Uses of Darkfield Microscope

The dark ground microscopy has the following uses:

• It is useful for the demonstration of very thin bacteria not visible under ordinary illumination since the reflection of the light makes them appear larger.

• This is a frequently used method for rapid demonstration of Treponema pallidum in clinical specimens.

• It is also useful for the demonstration of the motility of flagellated bacteria and protozoa.

• Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics.

• Darkfield is used to study mounted cells and tissues.

• It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

Advantages of Darkfield Microscope:

Dark-field microscopy is a very simple yet effective technique [3].

It is well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms.

Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.

Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature of the process.

A researcher can achieve a dark field by making modifications to his/her microscope.

Limitations of Darkfield Microscope

The main limitation of dark-field microscopy is the low light levels seen in the final image. The sample must be very strongly illuminated, which can cause damage to the sample. 

Phase-contrast Microscopy

Unstained living cells absorb practically no light. Poor light absorption results in extremely small differences in the intensity distribution in the image. This makes the cells barely, or not at all, visible in a brightfield microscope. Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in the  light passing through a transparent specimen to brightness changes in the image. It was first described in 1934 by Dutch physicist Frits Zernike [4].

Principle of Phase contrast Microscopy

When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast (Figure 2).

The Working of Phase contrast Microscopy

Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.

Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen.

Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase-contrast image observed in the eyepieces [5].

Figure 2: Principle of Phase contrast Microscopy

Parts of Phase contrast Microscopy

Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted.

The Annular Diaphragm:

• It is situated below the condenser

• It is made up of a circular disc having a circular annular groove

• The light rays are allowed to pass through the annular groove

• Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied

• At the back focal plane of the objective develops an image

• The annular phase plate is placed at this back focal plane

The Phase Plate

It is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular groove [6].

This thick or thin area in the phase plate is called the conjugate area. The phase plate is a transparent disc [7].

With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this microscope. This is obtained by separating the direct rays from the diffracted rays.

The direct light rays pass through the annular groove whereas the diffracted light rays pass through the region outside the groove. Depending upon the different refractive indices of different cell components, the object to be studied shows a different degree of contrast in this micro­scope.

Applications of Phase Contrast Microscopy

To produce high-contrast images of transparent specimens, such as:

• Living cells (usually in culture),

• Microorganisms,

• Thin tissue slices,

• Lithographic patterns,

• Fibers,

• Latex dispersions,

• Glass fragments, and

• Subcellular particles (including nuclei and other organelles).

Applications of phase-contrast microscopy in biological research are numerous.

Advantages of Phase Contrast Microscopy

Living cells can be observed in their natural state without previous fixation or labeling. It makes a highly transparent object more visible. No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast microscope which saves a lot of time.

Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles. It made it possible for biologists to study living cells and how they proliferate through cell division [8-10].

Phase-contrast optical components can be added to virtually any brightfield microscope, provided the specialized phase objectives conform to the tube length parameters, and the condenser will accept an annular phase ring of the correct size. 

Limitations of Phase contrast Microscopy

Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions.

To use phase-contrast the light path must be aligned. Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the technique is based on the diminishment of the brightness of most objects.

Figure 3: Principle of Fluorescence Microscope

Fluorescence Microscope

A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study the properties of organic or inorganic substances. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation while phosphorescence is a specific type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. The fluorescence microscope was devised in the early part of the twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann, among others (Figure 3).

Principle of Fluorescence Microscope

Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes [11].

Fluorescent dyes, also known as fluorophores or fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds.

The emission light can then be filtered from the excitation light to reveal the location of the fluorophores. Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emits light of a longer wavelength.

The image produced is based on the second light source or the emission wavelength of the fluorescent species rather than from the light originally used to illuminate, and excite, the sample.

Working

Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused on the detector by the objective. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light.

Forms

The “fluorescence microscope” refers to any microscope that uses fluorescence to generate an image, whether it is a simpler set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image.

Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective).

Parts of Fluorescence Microscope

Typical components of a fluorescence microscope are:

Fluorescent dyes (Fluorophore): A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds.

Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules that are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains like DAPI and Hoechst, phalloidin which is used to stain actin fibers in mammalian cells [12].

A light source

Four main types of light sources are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, and high- power LEDs.

Lasers are mostly used for complex fluorescence microscopy techniques, while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used for wide-field epifluorescence microscopes.

Figure 4: Parts of Fluorescence Microscope

The excitation filter

The exciter is typically a bandpass filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing the excitation of other sources of fluorescence and blocking excitation light in the fluorescence emission band [13]. 

The dichroic mirror

A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors [14].

The emission filter

The emitter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light [15,16].

By blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence, optical filters ensure the darkest background. 

Applications of Fluorescence Microscope

To identify structures in fixed and live biological samples. 

Fluorescence microscopy is a common tool for today’s life science research because it allows the use of multicolor staining, labeling of structures within cells, and the measurement of the physiological state of a cell.

Advantages of Fluorescence Microscope

Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live-cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.

Different molecules can now be stained with different colors, allowing multiple types of the molecule to be tracked simultaneously. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.

Limitations of Fluorescence Microscope

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Cells are susceptible to phototoxicity, particularly with short-wavelength light. Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.

Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence. 

1. Dark-field microscopy. Wikipedia, Wikimedia Foundation, 16 Jun. 2022, https://en.wikipedia.org/wiki/Dark-field_microscopy. Accessed 16 June 2022.

2. Caprette, David R. "Dark Field Viewing." Experimental Biosciences: Resources for Introductory and Intermediate Level Laboratory Courses, Rice University, 10 Aug. 2012, https://www.ruf.rice.edu/~bioslabs/methods/microscopy/dfield.html. Accessed 17 June 2022.

3. De Grand, Alec. "What Is Darkfield Microscopy?" Olympus Life Science, Olympus Corporation, 17 Dec. 2020, https://www.olympus-lifescience.com/ en/discovery/ what-is-darkfield-microscopy/. Accessed 18 June 2022.

4. Rijal, Nisha. "Dark-field Microscopy: Principle and Uses." Microbe Online, n.d., https://microbeonline.com/dark-field-microscopy/. Accessed 16 June 2022.

5. Aryal, Sagar. "Darkfield Microscope – Definition, Principle, Uses, Diagram." Microbe Notes, 27 May 2022, https://microbenotes.com/darkfield-microscopy/. Accessed 18 June 2022.

6. "Phase-contrast microscopy." Wikipedia, Wikimedia Foundation, 31 May 2025, https://en.wikipedia.org/wiki/Phase-contrast_microscopy. Accessed 19 June 2022.

7. "Introduction to Phase Contrast Microscopy." Nikon’s MicroscopyU, Nikon Instruments Inc., n.d., https://www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-contrast-microscopy. Accessed 17 June 2022.

8. "A guide to Phase Contrast." Scientifica (Learning Zone), Scientifica Ltd., 7 Jan. 2019, https://www.scientifica.uk.com/learning-zone/a-guide-to-phase-contrast. Accessed 16 June 2022.

9. Aryal, Sagar. "Phase Contrast Microscopy – Definition, Principle, Parts, Uses." Microbe Notes, 27 May 2022, https://microbenotes.com/ phase-contrast-microscopy/. Accessed 17 June 2022.

10. Caprette, David R. "Phase-Contrast Microscopy." Teledyne Photometrics, n.d., https://www.photometrics.com/ learn/microscopy-basics/ phase-contrast-microscopy. Accessed 16 June 2022.

11. Phase Contrast Light Microscopes. Leica Microsystems, Leica Microsystems GmbH, n.d., https://www.leica-microsystems.com/products/light-microscopes/ phase-contrast-light-microscopes/. Accessed 18 June 2022.

12. "Fluorescence microscope." Wikipedia, Wikimedia Foundation, 8 Aug. 2025, https://en.wikipedia.org/wiki/Fluorescence_microscope. Accessed 16 June 2022.

13. Spring, Kenneth R., and Michael W. Davidson. "Introduction to Fluorescence Microscopy." Nikon’s MicroscopyU, Nikon Instruments Inc., n.d., https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy. Accessed 19 June 2022.

14. Karki, Prakriti. "Fluorescence Microscope: Principle, Parts, Uses, Examples." Microbe Notes, 16 Apr. 2024, https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-advantages-limitations/. Accessed 16 June 2022.

15. Rice, George. "Fluorescent Microscopy." Microbial Life Educational Resources, Science Education Resource Center, Carleton College, 23 Feb. 2007, https://serc.carleton.edu/microbelife/research_methods/microscopy/fluromic.html. Accessed 17 June 2022.

16. Koenig, Fred. "Fluorescence Microscopy – Explanation and Labelled Images." New York Microscope Company, 16 Dec. 2020, https://microscopeinternational.com/fluorescence-microscopy/. Accessed 18 June 2022.