CCAM Microscope Objectives

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Griselda Francis
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1 CCAM Microscope Objectives Things to consider when selecting an objective Magnification Numerical Aperture (NA) resolving power and light intensity of the objective Working Distance distance between the front lens of the objective and the cover glass of the specimen Flatness of Field correction of field curvature Color Correction image different colors at the same point Light Transmission transmission of different wavelengths Use in Techniques brightfield, fluorescence, differential interference contrast (DIC), phase contrast Types Plan-Apochromat best chromatic correction, flatness of field and the highest numerical apertures. Used for brightfield, DIC and fluorescence. C-Apochromat adjustable correction collar to correct for differences in refractive indices and preparation thickness, water immersion variety is useful for aqueous specimens. Fluar high numerical apertures, high transmission of visible spectrum to near UV wavelengths, objective of choice for weak fluorescent signals Plan-Neofluar chromatic correction, high resolving power, flatness of field. Preparation All Zeiss objectives are designed to be used with a 0.17mm glass coverslip.

2 Looking at the Objective Information is labeled on the barrel of the objective Type Magnification Tube Length/Correction Collar Color coded for magnification and immersion fluid

3 Background Information Resolution The smallest distance between two structures at which the structures are imaged separately The resolution of an objective is limited due to diffraction and the nature of light Defined by Abbe s formula d= λ /2NA (λ = wavelength of light used, NA = the numerical aperture of the objective) Numerical aperture The light capturing capability of the objective; this takes into consideration the immersion medium being used. NA = n(sinα) (n = refractive index between cover glass and front lens of the objective, α = one-half angular opening of the objective) Imaging Aberrations Spherical - focuses axial and peripheral rays to different points, blurs the image of a point source of light leading to reduced image contrast and sharpness and eliminates much of the fluorescence of an object. Avoid spherical aberration Using immersion and mounting medias of similar refractive indices; i.e. do not image using an oil immersion objective (n=1.52) for a specimen mounted in a watery solution (n=1..33) Spherical aberration increases with sample depth therefore it is best to position the specimen directly under the cover glass

4 Background Information (cont.) Imaging Aberrations (cont.) Chromatic arises from lenses having different focal distances for different wavelengths of light. Appears as reddish or greenish fringes around specimen structures. Higher resolution lenses are more likely to have chromatic aberration because the higher aperture magnifies the color errors of the imaging system. These high resolution objectives are crafted with lenses to remove such errors. These objectives include achromats, fluorites, and apochromats; the latter having the highest amount of correction. Apochromats are corrected for up to seven wavelengths, ranging from UV to IR. Field Curvature simple lenses focus image points from a flat structure on to a spherical surface. Plan (flat-field) objectives provide a corrected flat field. An uncorrected lens may provide only 10-12mm of flatness while a plan objective can provide a flat field across 18-26mm.

5 CCAM Zeiss Objectives (dry-air) Objective Numerical Aperture Working Distance (mm) XY resolution (μm) (λ em = 515 nm, PH = 1 A.U.) Z Resolution (μm) (λ exc = 488 nm) Optimal Pixel Size (μm) (λ exc = 488 nm) Optimal Slice Spacing (μm) (λ em = 515 nm, PH = 1 A.U.) Plan Neofluar 5x x Fluar 10x Plan Neofluar 10x x x Plan-Neofluar 20x x Achrostigmat (Ph1 LD) 32x x Plan Apochromat 40x corr x Brightness = αna 4 /M 2 Z resolution = λ em.n. PH/NA 2 Pixel size = XY res/2 (Nyquist criterion) XY res =.61. λ exc /NA For widefield PH > 5 A.U. Slice spacing = Z res/2 (Nyquist criterion) M = magnification PH = pinhole AU = Airy Units n = ref. index Air = 1.0, water = 1.337, oil = 1.518

6 CCAM Zeiss Objectives (immersion) Objective Numerical Aperture Working Distance (mm) XY resolution (μm) (λ em = 515 nm, PH = 1 A.U.) Z Resolution (μm) (λ exc = 488 nm) Optimal Pixel Size (μm) (λ exc = 488 nm) Optimal Slice Spacing (μm) (λ em = 515 nm, PH = 1 A.U.) C-Apochromat 40x corr W.28 with cover glass.124x Achrostigmat 40x Oil Fluar 40x Oil x Plan Neofluar 63x Oil Plan Apochromat 63x Oil x x Plan Apochromat 100x Oil x Brightness = αna 4 /M 2 Z resolution = λ em.n. PH/NA 2 Pixel size = XY res/2 (Nyquist criterion) XY res =.61. λ exc /NA For widefield PH > 5 A.U. Slice spacing = Z res/2 (Nyquist criterion) M = magnification PH = pinhole AU = Airy Units n = ref. index Air = 1.0, water = 1.337, oil = 1.518

CCAM s Selection of. Zeiss Microscope Objectives

CCAM s Selection of. Zeiss Microscope Objectives CCAM s Selection of Zeiss Microscope Objectives 1. Magnification Image scale 2. Resolution The minimum separation distance between two points that are clearly resolved. The resolution of an objective is