IMAGING OPTICS FUNDAMENTALS for LIFE SCIENCES Gregory

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?

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?

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 • 36mm standard objectives • 170mm tube length • Rare cases have slight variation on Parfocal Distance and Tube Length

38 INDUSTRY STANDARDS

Typical Objective Manufacturer Specifications

• Olympus Standards - RMS thread type, 180mm tube lens focal length, 45mm parfocal distance • Nikon Standards - M25 thread type, 200mm tube lens focal length, 60mm parfocal distance • Zeiss Standards - RMS thread type, 165mm tube lens focal length, 45mm parfocal distance • Mitutoyo Standards - 26mm x 0.706mm pitch (36 TPI), 200mm tube lens focal length, 95mm parfocal 39 distance

SELECTING THE RIGHT OBJECTIVE

“For any conventional optical microscope configuration, the objective is the most critical component of the system in determining the information content of the image.” Microscopy Techniques -Olympus Microscopy Resource Center • Brightfield • Darkfield • Phase Contrast • Differential Interference Contrast (DIC) • Fluorescence • Confocal • Multiphoton

40 BRIGHTFIELD & DARKFIELD

Brightfield Illumination • Sample contrast comes from absorbance of light in the sample • The most basic technique in light microscopy • Typical appearance is a bright background with dark objects • Zero sample preparation

Darkfield Illumination • Sample contrast comes from light scattered by the sample • Technique used to enhance contrast in unstained samples • Typical appearance is a dark background with bright objects • Zero sample preparation, but also lacks overall intensity and resolution Images from Wiki – tissue paper, 1.6um/pixel

BRIGHTFIELD & DARKFIELD

42 BRIGHTFIELD OBJECTIVES

• Almost any objective will work for this microscopy technique! • The illumination source, detector, and sample heavily influence objective choice • The lowest cost, most basic objective would work (<$100), but your image and system could suffer

43 DARKFIELD OBJECTIVES

• Depending on the light source and illumination method, a specific objective type might be necessary • Darkfield attachments and darkfield ring lights exist, which allow for the use of standard brightfield objectives • However, specialized reflected light darkfield objectives exist which redirect light in an oblique fashion via reflector lenses working in unison with basic Olympus Resource Center objective design

44 PHASE CONTRAST

• Displays proportional differences in optical density • Shows differences in refractive index as contrast difference • Nucleus appears darker than surrounding ECM • Typically a grey background with light and dark features - these features represent change in optical density

45 PHASE OBJECTIVES

• Specific phase contrast objectives exist

• Almost any objective can be used here as well, but more intricate mechanical designs/systems needed at image and object space

• Mechanical Apertures

• Phase Plates

• Phase plates and apertures control phase shifts and light. Image is made up of a combination of scattered light and background light

• These specialized phase objectives can be intricate in design, difficult to use, and expensive to acquire

46 FLUORESCENCE

• Technique that utilizes fluorescence, as opposed to scatter, dispersion, or reflection • Fluorescence describes light emission that continues only during the absorption of excitation light • Requires the use of filters, to separate excitation energy from emission energy

Green Fluorescent Protein – image courtesy of Nexcelom Bioscience 47 FLUORESCENCE OBJECTIVES

• More intricate objective assemblies required • Low, or weak emission levels are typical • This requires objectives with extremely high transmission and high numerical apertures • Often, wavelength correction is required so an apochromatic objective becomes necessary • High numerical apertures result in short working distances

48 CONFOCAL MICROSCOPY

• Confocal microscopy shares a common optical pathway with fluorescence microscopy • Differences are… • Addition of a pinhole aperture between light source and excitation filter

• Addition of pinhole aperture between detector and emission filter • These apertures block out-of-focus light rays

49 CONFOCAL OBJECTIVES

• Almost always requires planar, apochromatic, immersion objective designs • Correction collar to compensate for aberrations from cover glass changes or depth changes in the sample • Extremely high numerical apertures required (20x/0.75, 40x/0.9, 60x/1.2-1.25)

Olympus Resource Center 50 FILTERING GLOSSARY & IMPORTANT TERMS

• Angle of Incidence (AOI) – the angle at which a filter is tilted relative to the primary ray. For example, an AOI of 0 means the primary ray is perpendicular to the plane of the filter • Bandpass filter – a filter than transmits a band of wavelengths while blocking the wavelengths on either side of the passband • Blocking – the level of rejection of light outside the passband • Center Wavelength (CWL) – center of the passband for a bandpass filter. Another common definition is the average of the 50% of peak of each edge of a bandpass filter • Cone angle – the range of angles of light incident on the filter about the AOI • Cone angle average – average of transmission or reflection across the cone angle • Dichroic – a filter that transmits one band and reflects a different band • Edge filter – a filter that transmits a band of wavelengths and blocks only low side (long wave pass) or high side (shortwave pass) of wavelengths • Fused Silica – substrate material composed of amorphous silicon dioxide • Long Wave Pass (LWP) – an edge-type filter that transmits longer wavelengths and blocks shorter wavelengths • Notch Filter – a filter that blocks a narrow range while transmitting wavelengths on either side of the blocked wavelength range

52 GLOSSARY & IMPORTANT TERMS

• Optical Density (OD) – the log (base 10) of transmittance (T)

• Passband – the range of wavelengths that are desired to be transmitted

• Ripple – variations in transmission in the passband

• Shortwave Pass (SWP) – an edge-type filter that transmits the shorter wavelengths and blocks higher wavelengths

• Spectral Slope – rate of change of the spectrum from a passband to a blocking band

• Transmitted Wavefront Error (TWE) – distortion of the wavefront of a plane wave as it transmits through a filter, generally given in units of the HeNe wavelength (633nm)

53 TYPES OF FILTERS

Colored Glass TYPES OF FILTERS

Shortpass and Longpass TYPES OF FILTERS

Bandpass and Notch TRADITIONAL VS HARD COATED

• Traditional filters – multiple layers, heavier, and larger in size. Decrease in transmission due to number of layers • Hard Coated filters – single layer, advanced coating decreases overall size, weight, and results in improved blocking and transmission

57 MULTILAYER COATINGS

• Coated filers have an angular dependence to them • This can create blue shift in the coating transmission

58 FABRICATION TECHNIQUES

• Optical filters can be broken down into two main categories • Absorptive – light is blocked based on the absorption properties of the glass substrate used • Dichroic – unwanted light is reflected, and specific ranges are transmitted • Absorptive filters are not angularly sensitive, while dichroic filters are heavily depending on angle of incidence

• Dichroic filters are desirable as they can separate light into two sources. Dichroics utilize multiple layers to exploit the interference nature of light waves

59 TYPES OF POPULAR FILTERS

• Bandpass Filters – narrowband to broadband, these filters block the surrounding wavelengths and are typically very sensitive to angles. Hard sputtered filters are ideal to maximize transmission and blocking • Edge filters – longpass or shortpass filters, these filters have a designated cut-on or cut-off wavelength that allows transmission before or after the designated wavelength. A longpass and shortpass set can create custom bandpass filters • Notch Filters – designed to block a pre-selected bandwidth while transmitting all other wavelengths within the design range of the filter. Typically used to remove a single laser wavelength, or narrow band, from an optical system • Dichroic Filters – coated with a thin film that can vary transmission and reflection properties. These filters are typically utilized at 45 degree AOI, but can be varied for a specific wavelength parameter. • Neutral Density Filters – designed to reduce, or attenuate, light in an optical system. Either absorptive or reflective, they are specified by optical density on the logarithmic scale

60 SYSTEM LAYOUTS AND FILTERING COST SENSITIVITY

Attribute Filter Price Impact

Low Medium High

Size (area) X

Flatness X X

TWE X

Wavelength Tolerance X

Spectral Steepness X

Blocking X

Blocking Range X

Substrate Material X

Custom Sizing X

Center Wavelength X

FWHM X

SWP/LWP X 62