LM - Light Microscopy Definitions

Abbreviations

• APO = apochromatic
• CCD = charge-coupled device
• CMOS = Complementary metal-oxide-semiconductor
• LWD = long working distance
• NA - numerical aperture
• P = plan
• Ph = Phase contrast
• S = spring loaded (sometimes called R for retractable)
• UW = ultra wide field (also UWF, or SWF for super wide field)
• WD = working distance
• WF = wide field

Binocular vs. trinocular vs. stereo microscopes

A binocular compound microscope has a single optical channel split into two paths brought to the eyes. A trinocular microscope has one optical channel split into 3 paths, two for the eyes and one for a camera. Usually the image for the camera eyepiece is diverted from one of the binocular eyepieces.

A stereo or dissecting microscope has a separate optical channel for each eye which allows three-dimensional viewing. Photos taken with a stereo microscope use only one eyepiece or one binocular eyepiece image is diverted to the phototube; photos are not in stereo. To produce stereo-pair photos requires two images taken at about 10° to each other with the same spot in the centre of the image or two images taken from the both the normal binoc eyepieces.

If a video camera is fitted and the image is observed on a monitor, a single eyepiece microscope may be sufficient, however, most professional microscopes are at least binocular and, for more versatile photo capability, trinocular.

Objectives

A microscope has two sets of lenses, the eyepiece (or ocular) and the objective. Eyepiece lenses are simple; they merely focus the light coming up the barrel of the microscope, whereas the objectives do the serious magnification.

Descriptions of objective lenses for compound microscopes can seem complex at first. Objectives have different levels of correction for chromatic (colour) and spherical aberration, these are explained below. It should be noted that the outer 35%, 20%, or 5% of the field of view will not be out of focus or blurred - but if aberrations occur, they will be found there.

Achromatic

Achromatic objectives have been corrected for colour aberration and have a flat field of focus in the middle 65% of the field of view.

Semi-plan

Semi-plan objectives give a flatter field of view, with 80% of the field of view in focus; edges would require slight refocusing. These lenses also correct for chromatic aberration.

Plan

Plan objectives have about 95% of the field of view in flat focus. These lenses also correct for chromatic aberration. Plan lenses are superior, but more expensive.

Apochromatic

Achromatic lenses are corrected for chromatic (colour) aberrations by focusing two wavelengths of light onto the same plane. Apochromatic (APO) lenses focus three wavelengths on the same plane. These lenses are much more expensive and are only necessary for the most demanding applications.

Immersion Oil

Only used at very high magnifications, an objective lens described with the suffix (Oil) can be used at a very short working distance with a layer of immersion oil between the specimen and the lens. The oil is optically similar to glass, reducing refraction. For more immersion oil details see our online page i1.

S

(S) in an objective description denotes a spring loaded retraction that saves slides and objectives from collision damage. Sometimes called R for retractable.

Explanation of colour codes and numbers on objectives

Example objective appearance:

• Coloured ring
• 10/0.25
• 160/0.17

Coloured ring:

• red = 4x
• yellow = 10x
• blue = 40x
• white = 100x

10/0.25:

• 10 = magnification (10x)
• 0.25 = numerical aperture

160/0.17:

• 160 is a DIN (German Standard) measurement in mm of the tube length required for this lens
• 0.17 is the thickness in mm of the required coverglass (0.17 is No. 1 coverglass)

Understanding an infinity microscope

A basic microscope is a long empty tube with a lens system at each end. Typically, the objective lens produces parallel beams of light within the tube and the eyepieces focus these for our eyes. With an infinity-corrected system, an additional lens system is contained within the tube itself. This allows the microscope objective lens to be positioned further away from the specimen, which results in greater working distance and a safer position for the lens. Infinity-corrected objectives must be used with an infinity-corrected tube which means that such objectives are not interchangeable with those from ordinary microscopes.

Phase contrast microscopy

A phase contrast microscope is one that does not require stained specimens, but instead enhances the contrast of near-transparent specimens. This makes it possible to view living cells and tissues and a variety of low-contrast specimens such as protozoans, bacteria and sperm tails. As light travels through a transparent medium, its amplitude and phase are altered. Amplitude gives rise to colour, but the human eye cannot discern changes in phase. In bright-field microscopes the information carried by phase is lost.

Frits Zernike realised that it was necessary to induce a phase shift in relation to a reference beam. He etched concentric circles on a glass plate and inserted this into the optical path of the microscope. This allows the phase of the light passing through the specimen to be inferred from the intensity of the image produced. The phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in 1953.
Some compound microscopes are equipped as, and some may be converted to, phase contrast instruments. Changes required are special strain-free objectives and matching condenser phase plates for each objective power. Phase objectives are identified with 'PH'.

Fluorescence illuminator - Wave Length