Optical Microscopy Application

Optical Microscopy Application: Brightfield Illumination

Brightfield illumination, which yields dark objects on a bright background, is the simplest technique for optical microscopy. In brightfield illumination, the light source is positioned below the sample. Light then propagates through the sample, and is observed by the objective lens and sensor, which are positioned above the sample. The darker the sample, the denser the specimen, as denser samples absorb more light. The simplicity of brightfield illumination is the main reason this technique is so popular in optical microscopy.

Image Appearance

A typical brightfield illumination image has a dark sample with a white background. The darker the regions on a sample, the more absorption of light that has occurred. For example, plant cells would appear darkest at the nucleus and central region where cellular matter is most dense, and lighter in the cytoplasm void of ribosomes, the endoplasmic reticulum, and other intracellular components. Animal cells are more difficult to image with this technique without staining of the sample, which ultimately kills live cells.

Figure 1: Brightfield Illumination Image of Tissue Paper

Technical Details

There are four key components in the light path for brightfield illumination.

1、Light Source: trans-illumination below sample that propagates through to condenser and objective lens. Typically a broadband source such as a quartz halogen bulb.

2、Condenser Lens: collects trans-illuminated light and focuses to sample.

3、Objective Lens: collects light which propagates through sample and enhances details by a factor of magnification.

4、Eyepiece/Camera: views or records the image.

There are some limitations to the brightfield illumination technique, which include very low contrast for cellular or biological samples, low optical resolution due to limitations of light, and the requirement for stained samples prior to imaging or viewing. However, simplicity of the brightfield technique is a huge benefit when first imaging an unknown sample. Additional optical microscopy applications include darkfield illumination, phase contrast, fluorescence, and differential interference contrast.

Optical Microscopy Application: Darkfield Illumination

Darkfield illumination is a technique in optical microscopy that eliminates scattered light from the sample image. This yields an image with a dark background around the specimen, and is essentially the complete opposite of the brightfield illumination technique. The primary imaging goal of the darkfield illumination technique is to enhance the contrast of an unstained sample, which is incredibly powerful, yet simple, for live cellular analysis or samples that have not gone through the staining process.

Image Appearance

A typical darkfield illumination image has a white/bright specimen with a dark background and environment filling the image. This is the exact opposite of a brightfield illumination image, and is useful for unstained specimens or images that require increased contrast. The advantage with using darkfield illumination is that unstained specimens can remain alive and vital, whereas their brightfield counterparts must be treated and are no longer active. Also, it is possible to acquire more qualitative results with this technique through live cellular analysis. For additional information on the brightfield technique, please read Optical Microscopy Application: Brightfield Illumination.

Figure 1: Darkfield Illumination Image of Tissue Paper

Figure 2: Optical Path for Darkfield Illumination

Technical Details

The light path of the darkfield illumination technique is typically applied to an upright microscope, as seen in Figure 2. The light path consists of three key components.

1 Light Source: enters the microscope and hits the dark field patch stop, which is a disc used to block light from entering the condenser and leaves a circular ring of illumination.

2 Condenser Lens: collects outer ring of illumination and focuses it on the sample.

3 Objective Lens: light hits the sample, and is transmitted or scattered from it. Scattered light enters the objective lens, whereas transmitted light is not collected by the lens. The direct illumination block assists in this step.

Additional optical microscopy applications include brightfield illumination, phase contrast, fluorescence, and differential interference contrast.

Optical Microscopy Application: Differential Interference Contrast

Differential Interference Contrast (DIC) is a polarization technique in optical microscopy that uses a polarizer, analyzer, and additional polarization optics such as a Nomarski or Wollaston prism. In simple DIC setups, the only required components are a polarizer and an analyzer. The polarizer is typically positioned below the specimen, and the analyzer above the objective lens. The polarization axis of each can be rotated to adjust contrast and throughput, but the two components typically act with respect to one another.

Introducing a Nomarski or Wollaston prism into a DIC optical microscopy setup separates polarized light into two rays that are polarized at 90° to one another. The focal point of the prisms rests outside the glass element, and allows active focusing in the upright DIC microscope system. A second prism is required to join the two light rays back together at a polarization of 135°.

Image Appearance

Figure 1 is a darkfield illumination image with specialized polarized techniques applied to it. The tissue fibers exhibit added complexity, detail, and texture as the light scattered by it is controlled and captured differently by its state of polarization. The image shows details about absorption color, optical path boundaries, and refractive indices, along with whether or not a sample is isotropic and anisotropic. Polarization techniques in optical microscopy such as DIC are invaluable in the identification of unknown samples that exhibit birefringence.

Figure 1: Polarization-Contrast Image of Tissue Paper

Technical Details

Figure 1 was captured using a simple DIC microscope. From bottom to top, there are five key assemblies and components.

Light source emits unpolarized light into microscope and is immediately oriented to 45° by the first polarizing filter.

Polarized light enters Wollaston prism and is separated into two rays at 90° to one another.

Both rays are focused by condenser lens to sample at specific points.

Rays propagate through sample at a separation point which mimics the resolution of the microscope (0.5μm resolution, rays focused at 0.5μm separation). Light is collected by objective lens and focused to second Wollaston prism.

Second Wollaston prism combines two rays into one polarized ray at 135°, which results in improved contrast in the sample image: improvements in bright spots, dark spots, and overall focus.

Figure 2: Optical Path of Differential Interference Contrast Microscope

Additional optical microscopy applications include brightfield illumination, darkfield illumination, phase contrast, and fluorescence.

Optical Microscopy Application: Fluorescence

Fluorescence microscopy is an optical microscopy technique that utilizes fluorescence, which is induced using fluorophores, as opposed to absorption, scatter, or reflection. A fluorophore is a type of fluorescent dye used to mark proteins, tissues, and cells with a fluorescent label for examination by fluorescence microscopy. A fluorophore works by absorbing energy of a specific wavelength region, commonly referred to as the excitation range, and re-emitting that energy in another specific wavelength region, commonly referred to as the emission range.

Fluorescence microscope systems can range from very simple, such as an epifluorescent microscope, to extremely complex, such as confocal or multiphoton systems. Whether simple or complex, fluorescence microscopes share the same basic concept: excitation energy is used to illuminate a sample which then emits a type of wavelength energy, albeit weak, that is quantifiable. The excitation and emission wavelengths do not share the same center wavelength, and this allows specialized optical filters to increase overall contrast and signal.

Image Appearance

Figure 1 shows a real-world fluorescence sample. The sample is excited by a shorter wavelength source (350 - 500nm), and then emits a fluorescent wavelength that is longer than the excitation wavelength (500nm +). Images such as this cannot be captured if not for the advanced optical filtering techniques in fluorescence microscopy which allow narrow bandwidths of light to propagate through to the sensor resulting in very crisp, high contrast images. Fluorescent proteins in specimens cause the unique emission colors. These proteins, such as GFP, are often derived from marine life.

Figure 1: Fluorescence Image of Microspheres

Technical Details

In general, the technical details of a fluorescence microscope mirror any standard brightfield illumination microscope or darkfield illumination microscope. The innovation comes from the intricate filtering techniques that are used to selectively utilize narrow bands of light. The three critical filters needed for a precision fluorescence microscope are the excitation, dichroic, and emission filters. For more in-depth information, please read Fluorophores and Filters in Fluorescence Microscopy.

1 Excitation Filter: placed within the illumination path of a fluorescence microscope. It filters out all wavelengths of the light source except for the excitation range of the fluorophore or specimen under inspection.

2 Dichroic Filter: placed between the excitation filter and emission filter at a 45° angle. It reflects the excitation signal towards the fluorophore under inspection and transmits the emission signal towards the detector.

3 Emission Filter: placed within the imaging path of a fluorescence microscope. It filters out the entire excitation range of the fluorophore under inspection and transmits the emission range of the fluorophore.

Figure 2: Basic Optical Filtering Arrangement for Fluorescence Microscopy

Additional optical microscopy applications include brightfield illumination, darkfield illumination, phase contrast, and differential interference contrast.

Optical Microscopy Application: Phase Contrast

Phase contrast was first utilized and described in 1934 by Frits Zernike. This optical microscopy technique enhances the contrast of transparent specimens, yielding high-contrast images of living cells, microorganisms, and other samples. The main advantage of the phase contrast technique is that living cells and tissues do not need to be killed, fixed, stained, or prepared in any way and can, in turn, be examined in their natural state. Analyzing and recording the dynamics of intricate biological processes becomes very easy with phase contrast optical microscopy.

Image Appearance

A typical phase contrast image has a neutral background and surrounding with varying contrast where light is altered by the specimen (Figure 1). Two very common effects seen in a phase contrast image are halo and shade-off patterns. These occur when the infinite-conjugate focal point does not match for the specimen and background. Although these are common and expected in phase contrast images, they diminish the appearances of details. In general, a bright phase contrast halo is typically visible at a boundary between strong and weak specimen features. These halos are evident due to the circular phase-retarding rings. Specialized objectives, known as apodizing phase contrast objectives, are manufactured to reduce this phenomenon.

Figure 1: Phase Contrast Image of Tissue Paper

Technical Details

The phase contrast technique translates extremely tiny variations in phase into a noticeable and corresponding amplitude change, and is evident in the difference of contrast in Figure 1. The most important concept of the phase contrast microscope design is the isolation of wavefronts, both surround (undiffracted) and diffracted, that arise from the specimen. To differentiate intensity profiles between a specimen and its surroundings, the undeviated light must be reduced and the phase retarded by a quarter-wave retardance. A brightfield illumination microscope can be upgraded to a brightfield-phase microscope with the introduction of two components to the optical train. For additional information on the brightfield technique, please read Optical Microscopy Application: Brightfield Illumination.

1 Phase Plate: mounted on the rear of the objective, this plate selectively alters the phase and amplitude of the light passing through the specimen. Often, microscope objectives have a small ring etched into one of their glass elements to eliminate the need for additional elements.

2 Condenser Annulus: opaque flat-black plate with an annular ring that is transparent, and positioned in the front focal plane. This results in the specimen being illuminated by a defocused, parallel light ray.

Figure 2: Optical Path of Phase Contrast Microscope

Additional optical microscopy applications include brightfield illumination, darkfield illumination, fluorescence, and differential interference contrast.