CCAM s Selection of. Zeiss Microscope Objectives
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1 CCAM s Selection of Zeiss Microscope Objectives
2 1. Magnification Image scale 2. Resolution The minimum separation distance between two points that are clearly resolved. The resolution of an objective is limited due to diffraction and the nature of light Defined by Abbe s formula d= l /2NA (l = wavelength of light used, NA = the numerical aperture of the objective)
3 3. Numerical Aperture (NA) Objective s ability to collect light and resolve specimen detail at a fixed distance. n = refractive index of medium between front lens element and cover slip. m = ½ the angular aperture (A) (
4 3. Numerical Aperture/Refractive index (cont.) The refractive index is the limiting factor in achieving numerical apertures greater than 1.0. To obtain a higher numerical aperture, a medium with a higher refractive index must be used. Highly corrected lenses are designed with higher numerical apertures.
5 4. Working Distance Distance between the front lens of the objective and the cover glass of the specimen. Note the working distance is reduced with the increase in numerical aperture and magnification.
6 4. Flatness of Field Correction of field curvature Objectives provide a common focus through the field of view. Such objectives are traditionally named as plan Edges in focus Entire field in focus Center in focus
7 6. Chromatic aberration Color correction, focuses different wavelengths of light to the same point Achromatic focuses two wavelengths of light, e.g. red and blue Apochromatic focuses three wavelength of light, e.g. red, green and blue
8 7. Light Transmission The various classes of objectives transmit wavelengths of light, with different efficiencies.
9 8. Contrast Method/Application Objectives are customized to be used for particular imaging techniques; bright-field, fluorescence, differential interference contrast (DIC), phase contrast. Bright-field Phase contrast DIC Fluorescence ( (
10 Objective Class Objective Spherical Aberration Chromatic Aberration Field Curvature Achromat 1 color 2 colors No Plan-Achromat 1 color 2 colors Yes Fluorite 2-3 colors 2-3 colors No Plan-Fluorite 3-4 colors 2-4 colors Yes 3-4 colors 4-5 colors Yes Note: 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.
11 Objective Class (cont.) Objective C-Apochromat Fluar Plan-Neofluar C-Achroplan Features Best chromatic correction, flatness of field and the highest numerical apertures. Adjustable correction collar to correct for differences in refractive indices and preparation thickness, water immersion variety is useful for aqueous specimens. High numerical apertures, high transmission of visible spectrum to near UV wavelengths, objective of choice for weak fluorescent signals. Chromatic correction, high resolving power, flatness of field. Corrected for axial chromatic aberration in two wavelengths, blue and red, flatness of field and adjustable correction collar.
12 Looking at the Objective; A Wealth of Information Information that is labeled on the barrel of the objective: Type Magnification/Numerical Aperture Tube Length/Cover Glass Thickness Color Coded Contrast Method of Contrast Magnification Immersion fluid
13 Preparation Notes The CCAM Zeiss objectives are all designed to be used with a 0.17mm/#1.5 glass cover slip (standard variation of mm). Avoid Spherical Aberration Spherical aberration focuses axial and peripheral rays to different points, it 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. Use 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
14 CCAM Objectives on Zeiss LSM 780 Objective Numerical Aperture Working Distance (mm) Confocal XY resolutio n (mm) (l em = 488 nm Optimal Pixel Size (mm) Confocal Z Resolution (mm) (l exc = 488 nm) Optimal Slice Spacing (mm) C-Apochromat 10x W x Air C-Achroplan 32x W x W x Oil DIC x Oil DIC XY res =.6. l exc /NA (widefield) For widefield PH > 5 A.U. XY res =.4. l exc /NA (confocal) Pixel size = XY res/2 (Nyquist criterion) (Resolution based on Rayleigh criteria) Z resolution = 2. l em. n/na 2 (widefield) Z resolution = 1.4. l em. n/na 2 (confocal) Slice spacing = Z res/2 (Nyquist criterion) (Resolution based on Rayleigh criteria) M = magnification PH = pinhole AU = Airy Units n = ref. index Air = 1.0 Water = Oil = Brightness = ana 4 /M 2
15 CCAM Objectives on Zeiss LSM 880 Objective Numerical Aperture Working Distance (mm) Confocal XY resolutio n (mm) (l em = 488 nm Optimal Pixel Size (mm) Confocal Z Resolution (mm) (l exc = 488 nm) Optimal Slice Spacing (mm) EC Plan-Neofluar 10x Air x Air LD LCI Plan- Apochromat 25x Variable Immersion Water = 1.43 Silicon Oil = 1.50 Glycerin = 1.57 Oil = 1.62 Water =.71 Silicon Oil =.75 Glycerin =.78 Oil =.81 40x W Fluar 40x Oil x Oil DIC XY res =.6. l exc /NA (widefield) For widefield PH > 5 A.U. XY res =.4. l exc /NA (confocal) Pixel size = XY res/2 (Nyquist criterion) (Resolution based on Rayleigh criteria) Z resolution = 2. l em. n/na 2 (widefield) Z resolution = 1.4. l em. n/na 2 (confocal) Slice spacing = Z res/2 (Nyquist criterion) (Resolution based on Rayleigh criteria) M = magnification PH = pinhole AU = Airy Units n = ref. index Air = 1.0 Water = Oil = Silicone Oil = Gylcerin = Brightness = ana 4 /M 2
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