IMAGING OPTICS FUNDAMENTALS FOR LIFE SCIENCES Gregory Hollows Edmund Optics IMAGE QUALITY Resolution Contrast HOW DOES DIFFRACTION AND F/# AFFECT PERFORMANCE? Not even a perfectly designed and manufactured lens can accurately reproduce an object’s detail and contrast. Diffraction will limit the performance of an ideal lens. The size of the aperture will affect the diffraction limit of a lens. The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#) F/# describes the light gathering ability of an imaging lens (lower F/# lenses collect more light). As lens aperture decreases, F/# increases. HOW DOES DIFFRACTION AND F/# AFFECT PERFORMANCE? The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#) HOW DOES DIFFRACTION AND F/# AFFECT PERFORMANCE? The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#) 9 micron pixels 4.5 micron pixels ~f/8 ~f/4 ~f/2 2.2 micron pixels HOW DOES DIFFRACTION AND F/# AFFECT PERFORMANCE? Not even a perfectly designed and manufactured lens can accurately reproduce an object’s detail and contrast. Diffraction will limit the performance of an ideal lens. The size of the aperture will affect the diffraction limit of a lens. The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#) F/# describes the light gathering ability of an imaging lens (lower F/# lenses collect more light). As lens aperture decreases, F/# increases. HOW DOES CONTRAST DEPEND ON FREQUENCY? Suppose two dots are placed close to each other and imaged through a lens. The two spots will blur slightly. Moving the spots closer causes the blur to overlap and contrast is decreased. When the spots are close enough that the contrast becomes limiting, the spacing is our resolution. At each spacing of the spots we obtain a specific contrast. We can plot this information in the form of a Modulation Transfer Function (MTF). HOW DOES DIFFRACTION AND F/# AFFECT PERFORMANCE? Not even a perfectly designed and manufactured lens can accurately reproduce an object’s detail and contrast. Diffraction will limit the performance of an ideal lens. The size of the aperture will affect the diffraction limit of a lens. The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#) F/# describes the light gathering ability of an imaging lens (lower F/# lenses collect more light). As lens aperture decreases, F/# increases. MODULATION TRANSFER FUNCTION (MTF) CURVE DOES INCREASING THE F/#, DECREASING NAHURT PERFORMANCE? HOW IS MTF AFFECTED BY WAVELENGTH? 660nm Light 470nm Light 3b HOW WAVELENGTH AFFECTS RESOLUTION HOW WAVELENGTH AFFECTS RESOLUTION HOW WAVELENGTH AFFECTS RESOLUTION HOW WAVELENGTH AFFECTS RESOLUTION HOW WAVELENGTH AFFECTS RESOLUTION HOW IS MTF AFFECTED BY WAVELENGTH? Chromatic Aberration Chromatic aberrations can be both on axis and off axis Lateral Color Axial Color COLOR BALANCING IN LENSES Achromatic Design v. Apochromatic Design IMAGE QUALITY Depth of Field Depth of Focus HOW CAN APERTURES BE USED TO IMPROVE DEPTH OF FIELD? HOW CAN APERTURES BE USED TO IMPROVE DEPTH OF FIELD? DOF TO RESOLUTION COMPARISON TIP AND TILT IN SENSOR OR OBJECT GLOSSARY & IMPORTANT TERMS • Parfocal Length Distance between the surface of the specimen and the objective mounting position when in focus • Infinity Corrected Optical System An optical system in which the image is formed by an objective and a tube lens with an Infinity Space between them, into which optical accessories can be inserted • Finite Conjugate Optical System An optical system in which the image is formed only by an objective 24 FINITE CONJUGATE DESIGN • Light from a source is focused (not from infinity) • Characterized by DIN or JIS standards • Utilized when cost and ease of design are concerns • Offer little to no filtering or in-line illumination • No tube lens required for focus • Account for majority of basic microscope systems where only simple magnification and lighting is required 25 INFINITE CONJUGATE DESIGN • Offer longer working distances • Allows for larger samples, elaborate mechanics, and room to operate (dyes, reagents, catalysts) • Allow for addition of in-line components • Filters, beamsplitters, and mechanics • Light rays focused with assistance of secondary/tube lenses –Set at specific, long distance from objective (~160-200mm) • Enable in-line illumination –Improved lighting and convenient for space constraints 26 BUILDING A CUSTOM SYSTEM FROM OFF THE SHELF COMPONENTS • Simplified view of an infinity corrected system • The more advanced design offers some distinct advantanges BUILDING A CUSTOM SYSTEM FROM OFF THE SHELF COMPONENTS • The final system can end up looking something like this • The beamsplitter allows for inline illumination to be introduced into the system • Notice that theoretically there has been no change to the imaging capabilities of the system BUILDING A CUSTOM SYSTEM FROM OFF THE SHELF COMPONENTS • Ultimately the system can be come even more complex by following the same logic • In this systems a second beamsplitter is introduced leading to another tube (focusing) lens and second camera • Now with two tube lenses (with different focal lengths) in the system it is possible to get two different magnifications simaltanously from one system BUILDING A CUSTOM SYSTEM FROM OFF THE SHELF COMPONENTS • The results can be seen in the images to the right • This can allow for image processing on two images at the same time • Additionally this saves time when compared to a system that requires you to zoom BUILDING A CUSTOM SYSTEM FROM OFF THE SHELF COMPONENTS • Since in this design there is collimated space between the lenses it is possibly to add components between them to enhance the systems abilities • This includes: • Beamsplitters • Filters • Colored Glass • Thin Film • Interference Filters • Prisms • Other optical components Gregory Hollows Director, Imaging Business Unit Edmund Optics Barrington, New Jersey USA Phone: (856) 547-3488 Email: [email protected] www.edmundoptics.com GLOSSARY & IMPORTANT TERMS • Focal length (F) Distance between a principal point and a focal point. F1 is the focal length of an objective. F2 is the focal length of a tube lens. For infinity-corrected systems, magnification is determined by the ratio of the focal length of the tube lens to that of the objective. Magnification of Objective = Focal length of tube lens / Focal length of objective • Field Number and Field of View (FOV) The field number of an eyepiece (expressed in mm) is determined by the field stop diameter of the eyepiece. FOV is the area of the specimen that is observable, and is determined by the field number of the eyepiece and magnification of the objective. FOV = Field number of eyepiece / magnification of objective • Depth of Field (DOF) Vertical distance in the specimen, measured from above and below the exact plane of focus, which still yields an acceptable image. The larger the NA, the smaller the depth of field. ± DOF = λ / (2*(NA)2) - standard wavelength of 550nm • Aperture Diaphragm Adjusts the amount of light passing through, and is related to the brightness and resolving power of an optical system. This diaphragm is especially useful in width dimension measurement of cylindrical objects with contour illumination, and provides the highest degree of correct measurement/observation by suppressing diffraction in an optimal aperture. 33 GLOSSARY & IMPORTANT TERMS • Oil Immersion Medium used on objectives with an NA high than 0.95 Examples: air, water, glycerin, paraffin oil, synthetic oil, anisole (Refraction index between 1.01-1.65) • Field Stop Used for blocking out unwanted light and preventing it from degrading the image • Vignetting This unwanted effect is the reduction of an image’s brightness or saturation at the periphery compared to the image center. May be caused by external (lens hood) or internal features (dimensions of a multi-element objective). • Double Image An image degrading phenomenon in which an image appears as if it is a double image due to redundant light projection and optical interference within the optical system. • Flare Lens flare is typically seen as several starbursts, rings, or circles in a row across the image or view, and is caused by unwanted image formation mechanisms, such as internal reflection and scattering of light. 34 SPECIFYING AND CHOOSING OBJECTIVES • Objective Specifications • Objective Designs • Finite Conjugate • Infinite Conjugate • Lens Configurations • Industry Standards • Mounting Threads • Tube Lengths • Choosing the Right Objective 35 OBJECTIVE SPECIFICATIONS 36 OBJECTIVE LENS CONFIGURATIONS • Achromatic ~ 3-5 lens elements • Fluorite ~ 5-9 lens elements • Apochromatic ~ 9-18 lens elements Achromatic – corrected for chromatic aberration at the red and blue wavelengths only Apochromatic – corrected for chromatic aberration at the red, blue, and yellow wavelengths Fluorite – to be used in low light level detection, specifically fluorescence emission Plan – objective lens that produces a flat (planar) image by correcting the spherical 37 aberration/curvature of the field of an achromatic/apochromatic lens INDUSTRY STANDARDS Royal Microscopy Society (RMS) - 0.8” x 36TPI, Whitworth • Society Thread • ~200mm tube length Deutsches Insititut fur Normung (DIN) - 0.7965”, 36TPI, 55` Whitworth • 45mm standard objectives • 160mm tube length • Object to image distance 195mm, fix object distance at 45mm, and remaining 150mm for internal real image position (10mm from end of tube) Japanese Industrial Standards (JIS) - 0.7965”, 36TPI, 55` Whitworth