Patozone

Microscope objectives

 

Important for determining the magnification of a specimen and the resolution with which fine detail can be observed

- named "objectives" because by proximity they are the closest component to the object (specimen) being imaged

 

Achromatic objectives are the most common, and are corrected for axial chromic aberration in the blue (486 nm) and red (656 nm) wavelengths, which are brought to a single common focal point

- also corrected for spherical aberration in the green color (546 nm)

-- if focus is chosen in the green region of the spectrum, images have a redish-magenta halo (termed residual color)

-- achromatic objectives work best when light is passed through a green filter

--- in the past few years, most manufacturers have began providing flat-field corrections for achromat objectives and have given these corrected objectives the name "plan achromats"

 

- apochromats that are the most highly corrected objectives available

 

 objective positioned on the far left in Figure 2 is a 10x achromat, which contains two internal lens doublets and a front lens element. Illustrated in the center of Figure 2 is a 10x fluorite objective having several lens groups including two doublets and a triplet, in addition to a hemispherical front lens and a secondary meniscus lens. On the right in Figure 2 is a 10x apochromat objective that also contains multiple lens groups and single elements.

 

Apochromats represent the most highly corrected microscope lenses currently available, and their high price reflects the sophisticated design and careful assembly required in their manufacture

 

All three types of objectives suffer from pronounced field curvature and project images that are curved rather than flat, an artifact that increases in severity with higher magnification

- Objectives that have flat-field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats

 

In general, plan objectives corrected for field curvature sacrifice a considerable amount of free working distance, and many of the high-magnification versions have a concave front lens, which can be extremely difficult to clean and maintain.

 

The cover glass acts to converge the light cones originating from each point in the specimen, but also introduces chromatic and spherical aberration (and consequent loss of contrast) that must be corrected by the objective. The degree to which light rays are converged is determined by the refractive index, dispersion, and thickness of the cover glass.

 

many high-performance apochromat dry objectives are fitted with correction collars, which allow adjustment to correct for spherical aberration by correcting for variations in cover glass thickness

 

Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective.

- advantages of oil immersion objectives are severely compromised if the wrong immersion fluid is utilized

Properties of Microscope Objectives

 

3 things critical to the resolution:

1. the wavelength of light used to illuminate the specimen

2. the angular aperture of the light cone captured by the objective

3. the refractive index in the object space between the objective front lens and the specimen

 

In a simple two-lens Abbe condenser. Light passing through the condenser is organized into a cone of illumination that emanates onto the specimen and is then transmitted into the objective front lens element as a reversed cone.

- the size and shape of the illumination cone is a function of the combined numerical apertures of the objective and condenser.

 

Resolution in a diffraction-limited optical microscope is minimum detectable distance bwt 2 closely spaced specimen points:

 

R = λ/2n(sin(θ))

 

where R is the separation distance, λ is the illumination wavelength, n is the imaging medium refractive index, and θ is one-half of the objective angular aperture.

- most important, factor in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95).

 

 dry objectives all have a numerical aperture value of less than 1.0 and only objectives designed for liquid immersion media have a numerical aperture that exceeds this value.

 

When a manufacturer's set of matched objectives, e.g. all achromatic objectives of various magnifications are mounted on the nosepiece, they are usually designed to project an image to approximately the same plane in the body tube. Thus, changing objectives by rotating the nosepiece usually requires only minimal use of the fine adjustment knob to re-establish sharp focus. Such a set of objectives is described as being parfocal, a useful convenience and safety feature.

 

The field diameter in an optical microscope is expressed by the field-of-view number or simply field number, which is the diameter of the viewfield expressed in millimeters and measured at the intermediate image plane.

- The field diameter in the object (specimen) plane becomes the field number divided by the magnification of the objective.

 

The axial range through which an objective can be focused without any appreciable change in image sharpness is referred to as the depth of field.

Numerical Aperture

 

Numeical aperture is critical because it limits resolution and image brightness

 

Resolution in epi-fluorescence microscopy:

 

d = (0.61 * L ) / NA

 

L (lambda) = emission wavelength of light

NA = Numerical aperture

 

 

NA = n sin (O)

 

O (theta) = maximum angle of acceptance

 

n = refractive index of medium between the objective front lens and the specimen

 

 

Difficult to get NA > 0.95 with dry objective

- need to increase the refractive index with water (refractive index = 1.33) or immersion oil (refractive index = 1.51)

- the refractive index of oil and coverslips are similar