Lecture 2 The Principles of Microscopy BMS 524

Lecture 2 The Principles of Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine J. Paul Robinson, Ph. D. Professor of Immunopharmacology Director, Purdue University Cytometry Laboratories These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J. Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2 nd Ed. A number of the ideas and figures in these lecture notes are taken from this text. UPDATED October 27, 1998 Slide 1 t: /powerpnt/confoc/lect 2 nu. ppt Purdue University Cytometry Laboratories

Review • Microscope Basics • Magnification • Optical systems Purdue University Cytometry Laboratories Slide 2 t: /powerpnt/confoc/lect 2 nu. ppt

Microscope Components • • • Purdue University Cytometry Laboratories Fluorescence Microscope Numerical Aperture Refractive Index Aberrations Objectives Slide 3 t: /powerpnt/confoc/lect 2 nu. ppt

Definitions • Refractive Index • Aberrations • Fluorescence Purdue University Cytometry Laboratories Slide 4 t: /powerpnt/confoc/lect 2 nu. ppt

Fluorescent Microscope Arc Lamp Excitation Diaphragm Excitation Filter EPI-Illumination Ocular Objective Emission Filter Purdue University Cytometry Laboratories Slide 5 t: /powerpnt/confoc/lect 2 nu. ppt

Construction of Filters Dielectric filter components “glue” Single Optical filter Purdue University Cytometry Laboratories Slide 6 t: /powerpnt/confoc/lect 2 nu. ppt

Anti-Reflection Coatings are often magnesium fluoride Optical Filter Multiple Elements Purdue University Cytometry Laboratories Slide 7 t: /powerpnt/confoc/lect 2 nu. ppt

Standard Band Pass Filters 630 nm Band. Pass Filter White Light Source Transmitted Light 620 -640 nm Light Purdue University Cytometry Laboratories Slide 8 t: /powerpnt/confoc/lect 2 nu. ppt

Standard Long Pass Filters Light Source 520 nm Long Pass Filter Transmitted Light >520 nm Light Standard Short Pass Filters Light Source Purdue University Cytometry Laboratories 575 nm Short Pass Filter Transmitted Light <575 nm Light Slide 9 t: /powerpnt/confoc/lect 2 nu. ppt

Optical Filters 510 LP dichroic Mirror Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light Purdue University Cytometry Laboratories Slide 10 t: /powerpnt/confoc/lect 2 nu. ppt

Filter Properties Light Transmission 100 Notch 50 Bandpass %T 0 Wavelength Purdue University Cytometry Laboratories Slide 11 t: /powerpnt/confoc/lect 2 nu. ppt

Refraction Short wavelengths are “bent” more than long wavelengths dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. Purdue University Cytometry Laboratories Slide 12 t: /powerpnt/confoc/lect 2 nu. ppt

Light striking a surface Incident Beam i Transmitted (refracted)Beam t r Reflected Beam Purdue University Cytometry Laboratories Slide 13 t: /powerpnt/confoc/lect 2 nu. ppt

Properties of thin Lenses f f p 1 p Resolution (R) = 0. 61 x (lateral) (Rayleigh criterion) Purdue University Cytometry Laboratories q + l NA 1 q = 1 f q Magnification = p Slide 14 t: /powerpnt/confoc/lect 2 nu. ppt

Microscope Objectives Purdue University Cytometry Laboratories Slide 15 t: /powerpnt/confoc/lect 2 nu. ppt

Objectives PLAN-APO-40 X 1. 30 N. A. 160/0. 22 Flat field Apochromat Magnification Numerical Tube Coverglass Aperture Length Thickness Factor Purdue University Cytometry Laboratories Slide 16 t: /powerpnt/confoc/lect 2 nu. ppt

Objectives Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): d = 1. 22 This defines a “resel” or “resolution element” Thus high NUMERICAL APERTURE is critical for high magnification In a medium of refractive index n the wavelength gets shorter: n Purdue University Cytometry Laboratories Slide 17 t: /powerpnt/confoc/lect 2 nu. ppt

Numerical Aperture • The wider the angle the lens is capable of receiving light at, the greater its resolving power • The higher the NA, the shorter the working distance Purdue University Cytometry Laboratories Slide 18 t: /powerpnt/confoc/lect 2 nu. ppt

Numerical Aperture • Resolving power is directly related to numerical aperture. • The higher the NA the greater the resolution • Resolving power: The ability of an objective to resolve two distinct lines very close together NA = sin – (n=the lowest refractive index between the object and first objective element) (hopefully 1) – is 1/2 the angular aperture of the objective Purdue University Cytometry Laboratories Slide 19 t: /powerpnt/confoc/lect 2 nu. ppt

Numerical Aperture • For a narrow light beam (i. e. closed illumination aperture diaphragm) the finest resolution is (at the brightest point of the visible spectrum i. e. 530 nm)…. NA = . 00053 1. 00 = 0. 53 m • With a cone of light filling the entire aperture theoretical resolution is…. . 2 x NA Purdue University Cytometry Laboratories = . 00053 = 0. 265 m 2 x 1. 00 Slide 20 t: /powerpnt/confoc/lect 2 nu. ppt

Object Resolution • Example: 40 x 1. 3 N. A. objective 2 x NA . 00053 = 0. 20 m 2 x 1. 3 = 40 x 0. 65 N. A. objective 2 x NA Purdue University Cytometry Laboratories = . 00053 = 0. 405 m 2 x. 65 Slide 21 t: /powerpnt/confoc/lect 2 nu. ppt

Microscope Objectives 60 1. 4 NA Plan. Apo Oil Microscope Objective Stage Coverslip Specimen Purdue University Cytometry Laboratories Slide 22 t: /powerpnt/confoc/lect 2 nu. ppt

Refractive Index Objective n = 1. 52 n=1. 52 Purdue University Cytometry Laboratories Oil n = 1. 5 n = 1. 0 Air n = 1. 52 Coverslip Specimen Wat er n=1. 33 Slide 23 t: /powerpnt/confoc/lect 2 nu. ppt

Sources of Aberrations • Monochromatic Aberrations – Spherical aberration – Coma – Astigmatism – Flatness of field – Distortion • Chromatic Aberrations – Longitudinal aberration – Lateral aberration Purdue University Cytometry Laboratories Slide 24 t: /powerpnt/confoc/lect 2 nu. ppt

Monochromatic Aberration – Spherical aberration F 1 F 2 F 1 Corrected lens Generated by nonspherical wavefronts produced by the objective, and increased tube length, or inserted objects such as coverslips, immersion oil, etc. Essentially, it is desirable only to use the center part of a lens to avoid this problem. Purdue University Cytometry Laboratories Slide 25 t: /powerpnt/confoc/lect 2 nu. ppt

Monochromatic Aberrations – Coma Fig 12 p 117 From: ”Handbook of Biological Confocal Microscopy” J. B. Pawley, Plenum Press, NY, 1995, 2 nd Ed The figure is not reproduced in this presentation because we do not have permission to place this figure onto a public site. Note: For class use Figure is under box Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems. From: Handbook of Biological Confocal Microscopy J. B. Pawley, Plenum Press, NY, 1995, 2 nd Ed Purdue University Cytometry Laboratories Slide 26 t: /powerpnt/confoc/lect 2 nu. ppt

Monochromatic Aberrations –Astigmatism Fig 13 p 118 From: ”Handbook of Biological Confocal Microscopy” J. B. Pawley, Plenum Press, NY, 1995, 2 nd Ed The figure is not reproduced in this presentation because we do not have permission to place this figure onto a public site. Note: For class use Figure is under box If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated. Fig 13 p 118 Purdue University Cytometry Laboratories From: Handbook of Biological Confocal Microscopy J. B. Pawley, Plenum Press, NY, 1995, 2 nd Ed. Slide 27 t: /powerpnt/confoc/lect 2 nu. ppt

• Monochromatic Aberrations – Flatness of Field – Distortion Lenses are spherical and since points of a flat image are focused onto a spherical dish, the central and peripheral zones will not be in focus. Complex Achromat and PLANAPOCHROMAT lenses partially solve this problem but at reduced transmission. DISTORTION occurs for objects components out of axis. Most objectives correct to reduce distortion to less than 2% of the radial distance from the axis. Purdue University Cytometry Laboratories Slide 28 t: /powerpnt/confoc/lect 2 nu. ppt

Useful Facts • The intensity of light collected decreases as the square of the magnification • The intensity of light increases as the square of the numerical aperture Thus when possible, use low magnification and high NA objectives. Purdue University Cytometry Laboratories Slide 29 t: /powerpnt/confoc/lect 2 nu. ppt

Fluorescence Microscopes • Cannot view fluorescence emission in a single optical plane • Generally use light sources of much lower flux than confocal systems • Are much cheaper than confocal systems • Give high quality photographic images (actual photographs) whereas confocal systems are restricted to small resolution images Purdue University Cytometry Laboratories Slide 30 t: /powerpnt/confoc/lect 2 nu. ppt

Summary Lecture 2 • • • Properties of optical filters Objectives Numerical Aperture Refractive Index/Refraction Aberrations Fluorescence Microscope - introduction Purdue University Cytometry Laboratories Slide 31 t: /powerpnt/confoc/lect 2 nu. ppt