Bright field microscopy

Introduction to Bright field microscopy

Bright-field microscopy is a type of optical microscope or light microscope that uses a system of lenses and visible light to generate magnified images.

In a Bright field microscope, the microscopic field is brightly lit while the microorganisms appear dark as they are only able to absorb some of the light. The visualization of microorganisms can be improved by staining them with a dye which greatly improves their light-absorbing ability. As a result, greater contrast and color differentiation is achieved when the dye is used.

Generally, a Bright field microscope is able to produce a magnification of about X1,000 to X2,000. If the object is magnified over the range, images become fuzzy.

Determinants of Bright field microscopy

The determinants of the functionality of Bright field microscope include:

1. Resolving power

2. Numerical Aperture

3. Limit of Resolution

4. Magnification

Resolving power

• Resolving powers the ability to distinguish two adjacent points as distinct and separate. In other words, the ability to distinctly see separate lines or dots and the greater the ability is, the higher the resolution power of a microscope.

• The largest magnification of the microscope may not be the best for visualization as the images might be fuzzy i.e. greater magnification without greater resolution does not provide benefits.

• The resolving power of the microscope is based on the wavelength of light used and the numerical aperture (NA) of the lens.

Numerical Aperture

• The numerical aperture (NA) is the measure of the aperture of the objective (half-aperture angle) – the angle θ subtended by the optical axis and the outermost rays still covered by the objective.

• The formula for the numerical aperture of Bright field microscopy is:

  NA = n sin θ,

  Where n is the refractive index and θ is the angle subtended by the optical axis

• Limitation includes the degree to which microscope objectives can be altered to increase and the wavelength of light used – between 400nm (blue light) and 700nm (red light).

Limit of Resolution

• The limit of resolution is the smallest distance at which two objects can be placed separately and still be distinguishable as two separate objects.

• Resolution in light microscopy can be maximized by the use of the shortest wavelength of visible light and an objective with the maximum NA.

• The formulae for the limit of resolution are:

  d = λ / 2NA

  Where d is resolution, λ is the wavelength of the light, and NA is the numerical aperture.

Magnification

• If magnification is more than the resolving value, the produced larger images will be fuzzy with less distinction in details.

• In most laboratories, three objectives are present – oil immersion, high-dry, and low-power objectives.

• The total magnification of the system is calculated by multiplying the power of the objective by that of an eyepiece.

Components of Bright field microscopy

Like most optical microscopes, Bright field microscopes also have similar components. These components are as follows:

1. Eyepiece (ocular lens)

2. Revolving nose piece

3. Objective lenses

4. Focus knobs (coarse adjustment, fine adjustment)

5. Stage

6. Light source

7. Diaphragm and condenser

8. Mechanical stage

Eyepiece (ocular lens)

• Eyepiece, also known as the ocular lens, is a cylinder containing two or more lenses.

• It functions by bringing the image into focus for the eye.

• The interchangeable eyepieces, which can be inserted with different degrees of magnification, are inserted into the top end of the body tube.

• In high-performance microscopes (commonly equipped with apochromatic objectives), the optical configuration of the objective lens and eyepiece are matched to give the best possible optical visualization.

Revolving nose piece

• It is part of the microscope whose function is to hold the set of objective lenses and also allow switching between objective lenses

• The revolving nose piece is also known as an Objective turret or revolver.

Objective lens

• The objective lens (one or more) present in the Bright field microscope collects light from the sample.

• Typically, three objective lenses are screwed into a circular nose piece which can be rotated to select the one with a desired objective lens.

• They are designed to be parfocal i.e. if the objective lens is changed, the focus on the sample remains the same.

• The objective lens has two parameters – magnification (5× to 100×) and numerical aperture (0.14 to 0.7) – with focal lengths of about 40 to 2 mm respectively.

• If the objective lenses have higher magnifications, a higher numerical aperture is present, which results in a shorter depth of field.

Focus knobs

• Focus knobs on the Bright field microscope are adjustment knobs that move the stage up and down.

• Two knobs are present - coarse adjustment and fine adjustment.

• It also enables the microscope to adjust to specimens of different thicknesses.

Stage

• The stage is where the specimen being viewed is placed and is below the objective lens.

• It has arms to hold rectangular slides typically measuring 25×75 mm – in which specimens are placed for observation.

• Since moving the slide by hand at 100× magnification is not practical, the stage has control knobs. These control knobs allow tiny horizontal movements of the slide.

• Stage, which is able to move up and down, has a hole at the center through which light passes to illuminate the specimen.

• In order to center the specimen, the focus is done at lower magnification and if higher magnification is required, the stage is moved vertically up and re-focused at higher magnification.

Light Source

• Numerous sources of light can be used for Bright field microscopy – the simplest being daylight which is directed via a mirror

• Other light sources, which can be adjusted and controlled, includes halogen lamp, LEDs, and lasers.

• In some expensive microscopes, Köhler illumination may be installed

Condenser

• The condenser is part of a Bright field microscope that functions by focusing the illumination source onto the sample.

• It includes other features such as a diaphragm and/or filters which manages the quality and intensity of the illumination.

• darkfield condenser lens, which directs a cone of light away from the objective lens, must be used

Significance

• Bright-field microscopy is used extensively in microbiology, biotechnology, mineralogy, microelectronics, nanophysics, and pharmaceutic research.

• In medicine, it is used for medical diagnosis - histopathology, and smear tests.

Limitations

• At high magnifications, objects can be seen fuzzy with airy disks (diffraction rings).

• Resolving power limits the ability to resolve fine details.

• extent and magnitude of the diffraction patterns are affected by the wavelength of light used, refractive materials used, and the numerical aperture (NA).