Optics and Optical Microscopes

A Basic Primer

Biosciences Edition

Ver. 1.0

April 1, 2010 Nikon Corporation Instruments Company

BS Instruction Committee

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Contents

1. Fundamentals of Light and Lenses 1-1. What is light? 1-2 Speed of light 1-3 Wavelength 1-4 Interference 1-5 Diffraction 1-6 Reflection and refraction 1-7 Concave and convex lenses 1-8 Real and virtual images 1-9 Focal point and focal length 2. The Structure of an Optical Microscope 2-1 Configuration of optical systems 2-2 CFI (Chromatic aberration-Free Infinity) optical systems 2-3 Microscope optical path diagrams 2-4 Microscope part names 2-5 Cover slip standards 3. Optical Performance 3-1 Total magnification 3-2 Actual field of view. 3-3 Numerical aperture (NA) 3-4 Image brightness 3-5 Resolution 3-6 Working distance 3-7 Depth of focus 3-8 Aberration 4. Functions and Usage of the Illumination System 4-1 illumination optics 4-2 Light source 4-3 Color and brightness of illumination 4-4 Condensers 4-5 Functions and usage of diaphragms 4-6 Flare and ghosting 5 Objectives and Eyepieces 5-1 Objectives 5-2 Eyepiece lenses 5-3 Eyepiece tube

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6 Types of Microscope 6-1 Upright microscopes and inverted microscopes 6-2 Transmitted light illumination and reflected light illumination 7 Observation Methods 7-1 Brightfield microscopy 7-2 Phase contrast microscopy 7-3 Differential interference contrast microscopy 7-4 Fluorescence microscopy 7-5 Polarized-light microscopy 7-6 Darkfield microscopy 7-7 Modulation contrast microscopy 7-8 Asbestos dispersion staining microscopy 7-9 Stereoscopic microscopy 7-10 Cell culture observation systems 8. Adjustment Method and Use of Accessories 8-1 Alignment of mercury lamp 8-2 Adjusting the Khler illumination 8-3 Diopter adjustment for the eyepiece lens 8-4 Adjusting the correction ring on the objective 8-5 Checking and adjusting the cross-Nichols for differential interference contrast microscopy 8-6 Centering of the objective 8-7 Adjusting the position of the phase ring 8-8 Adjusting the darkfield condenser 8-9 Adjusting the modulation contrast condenser slider 8-10 How to use an objective micrometer 8-11 How to use an eyepiece micrometer 8-12 How to select an immersion oil 9. Confocal Microscopy 9-1 What is confocal microscopy? 9-2 Structure of a laser-scanning confocal microscope 9-3 Structure of a disk scan confocal microscope 10 Regular care 10-1 Required cleaning implements 10-2 How to wind on the lens cleaning paper 10-3 Cleaning sequence 10-4 Places that should not be cleaned 10-5 Tips on wiping lenses 11. Maintenance of Microscopes

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11-1 Mold on optical systems 11-2 Rust 11-3 Storing a microscope 11-4 Care of mechanical systems 12: Images of Specimens 12-1 Brightfield specimens 12-2 Polarized-light specimens 12-3 Fluorescence specimens 12-4 Differential interference contrast specimens 12-5 Phase contrast specimens 12-6 Apodized phase contrast specimens (APC method) 12-7 Modulation contrast specimens 12-8 Stereoscopic specimen 13. Bibliography

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1. Fundamentals of Light and Lenses

1-1. What is light? Light is a type of electromagnetic wave that has both wave-like and particle-like properties. When the emphasis is on lights wave-like character, it is referred to in terms of light waves; when the emphasis is on its particle-like character, it is referred to in terms of photons. Wavelength, amplitude and phasekey properties of lightcan be understood in terms of wave motion. If the wavelength and amplitude of light are represented in terms of a sine wave, as in the diagram below, one wavelength cycle can be represented as 360 or 2radians.

Wavelength (T): This is a periodic length of a wave propagating through space. Amplitude (H): Represents the magnitude of the wave, which corresponds to its brightness or

light intensity. Phase (P): Expresses (normally in terms of angle) a position within a single period for a

periodic phenomenon such as wave motion. The phase is delayed or advanced in phase contrast observation and polarizing observation

1-2 Speed of light In a vacuum or in the air, C = 3.00 108 m/sec (approximately 300,000 km/sec). This is equivalent to traveling seven and a half times round the Earth in one second. Speed of light (C) = wavelength () frequency (v) C [m/sec] =[m] v [1/sec] [Additional information 1] Speed and wavelength of light in water: The speed of light in water is slowed to 1/1.333 of the speed of light in air, since the index of refraction for water is 1.333.

Traveling direction

Wavelength

Time

Amplitude

Sine vibration

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In other words, since the frequency does not change, the wavelength is reduced. This phenomenon is the reason why the resolution of microscopes using water immersion objectives or oil immersion objectives is improved. 1-3 Wavelength Most observations using microscopes are performed within the visible light spectrum. There are also shorter-wavelength ultra-violet light and longer-wavelength infra-red light, but the human eye is not sensitive to these wavelengths and so cannot perceive these types of light. The visible spectrum is normally regarded as the range 400nm700nm; however, defined in some literature as being from 380nm to 780nm. For a more exact explanation, please refer to [Additional information 2]. The use of ultra-violet light in biological microscopes has been reduced in order to avoid the effects of photo-toxicity. Infra-red light, which has little adverse effect on cells, is now widely used instead. In addition to the lack of photo-toxicity, another advantage of long-wavelength light is that it does not scatter very much; the light thus penetrates deeper into living cells, allowing better observation. [Additional information 2] The definition of visible light: The definition is not set in stone, since the visibility of light depends on whether it has sufficient radiative power to reach the retina and on the degree of responsiveness of the observer. Hence, the visible spectrum is stipulated as running from between 360nm400nm at the lower (shorter-wavelength) end to between 760nm830nm at the upper (longer-wavelength) end. Light with a wavelength that falls within this spectrum is termed visible light.

Specific wavelengths The following is a list of standard wavelengths for microscopy and their corresponding names.

Wavelength (nm) Light color Symbol Light source 768.2 Infra-red A K (potassium) 656.3 Red C H (hydrogen) 589.3 Yellow D* Na (sodium) 587.6 Yellow d He (helium) 546.1 Yellowish-green e Hg (mercury) 486.1 Blue F H (hydrogen) 435.8 Purple g Hg (mercury) 404.7 Purple h Hg (mercury) 365.0 Ultra-violet i Hg (mercury) Note: The wavelength given for D* is the median value of D1 (589.6nm) and D2 (589.0nm).

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1-4 Interference Interference is the formation of a new waveform through the superposition of multiple waves. Interference can easily take place with light of uniform wavelength and phase, such as laser light. When two waves are in-phase with one another (and thus have a phase difference of 0), they intensify one another by means of interferencethat is, their respective peaks and troughs coincide. In this instance the light is intensified by the addition of the respective peaks and troughs. When two waves are in reverse phase with one another (and thus have a phase difference of 180), they cancel one another out by means of interference.

The wavelength is the same but the intensity is doubled.

The waves cancel one another out.

Reference 1

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Example 1 If two lights of the same intensity interfere with one another, with the phase of one light being 90 (/2) behind the other, how will the interference work? If we call the first light and the light whose phase is 90 behind , the mutual interference between them produces a composite light that is stronger . [Additional information 3] What is coherence? Coherence is the ease with which two waves whose respective amplitudes and phases are in fixed relation to one another can interfere with one another. If two waves interfere with one another, they are described as being mutually coherent. There is also the term incoherent, with the opposite meaning. 1-5 Diffraction Diffraction is a phenomenon in which a wave (or wave-like motion) traveling through a medium and encountering an obstacle is diverted into a region (for example, behind the obstacle). As shown in the picture, monochromatic light emerging from a small pinhole diffracts and appears to spread out in concentric fashion, instead of forming a single point. The angle of diffraction varies according to the size and shape of the pinhole. From the center outwards, the light is referred to as zero-order ray, first-order ray, second-order ray and so on. The intensity of each order of light ray as a percentage of the total light is as follows: zero-order ray: 90%, first-order ray: 4.5%, second-order ray: 1.5%, Hence, it can be seen that nearly all the light that enters the pinhole is concentrated into the zero-order ray.

Zero-order ray

First-order ray

Second-order ray

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1-6 Reflection and refraction Reflection is a phenomenon in which light striking the surface of a smooth plane mirror returns from the mirror at the same angle as it entered it. Thus, light which enters the mirror at an angle of incidence returns from it at an angle of reflection . Refraction is a phenomenon in which the direction of travel of a light or sound wave changes at the interface between two different media. Snells law defines the relationship between incident light and refracted light. Snells law If the refractive indices of two media are n1 and n2, and if n2>n1, the angle of incidence and the angle of refraction are related as shown in the following equation. n1 sin () = n2 sin () Sample refractive indices Air: 1 Water: 1.333 Cells: approximately 1.35 (varies depending on the cell contents) Glycerol: 1.473 Oil: 1.515 Microscope slide: 1.515 What is total reflection? Total reflection is the name given to the phenomenon whereby the angle of incidence for light passing from a substance with a high refractive index to a substance with a low refractive index exceeds a certain critical angle, causing the light to be reflected instead of refracted. For example, the critical angle for light passing from glass (1.515) to water (1.333) can be calculated as follows. Since the critical angle is the angle of incidence when the angle of refraction is 90, the critical angle can be calculated by applying Snells equation as follows: 1.515 sin () = 1.333 sin (90). [Additional information 4] Definition and expression of refractive index: The refractive index of a substance is expressed as the speed of light in a vacuum divided by the speed of light in that substance. Thus, if the refractive index of a substance is high, it can be said that it is difficult for light to pass through it, or that light passes through it slowly. It is impossible to simply compare numerical values, since refractive indices vary

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according to temperature and according to the wavelengths used to measure them. When the refraction of immersion oil is measured using the D rays at 23C, the refraction is written as nD. 1-7 Concave and convex lenses A lens that converges parallel light rays that are incident upon it is known as a convex lens, while a lens that does the opposite and diverges parallel light rays is known as a concave lens. The following explanation is based on a thin lens. Light that passes through a convex lens exhibits the following characteristics: Light rays that are parallel to the optical axis will converge on the focal point after passing through the convex lens. Light rays emitted from the focal point will travel parallel to the optical axis after passing through the convex lens. Light ray that passes through the principle point will continue its travel without any change in angle. Light that passes through a concave lens exhibits the following characteristics: After passing through the concave lens, light rays parallel to the axis spread out

(diverge) as if from a single point on the axis (the focal point) on the incident side. Light rays that were heading towards the rear focal point of the lens proceed in

parallel to the axis after passing through the concave lens. Light rays that pass through the principal point proceed in a straight line with no

change in angle. Concave lens

Focal point

Focal point

Principle point

Focal point

Principle point

Focal point

Convex lens

Reference 2

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1-8 Real and virtual images The term real image describes an image formed when refracted or reflected light rays from an object beyond the focal point of a convex lens or concave mirror actually converge. Examples: the image formed by the objective of a microscope; the image on a movie screen. The term virtual image describes an image that appears as if it is formed at a point in the reverse direction to the refraction or reflection when light rays are refracted or reflected by a lens or mirror, and do not actually converge.. Examples: the image seen through the eyepiece lenses of a microscope; the image seen through a magnifying glass. 1-9 Focal point and focal length Focal point The focal point is the point to which light from infinity is focused. It needs to be noted that it is the point at which light from infinity (that is, parallel light rays) convergesnot a point at which divergent or convergent light rays converge. The point at which divergent or convergent light rays converge is known as the imaging point. The focal point can be said to be the imaging point for an object on the optical axis that is at infinity. What are the front focal point and rear focal point? If parallel rays of light that enter the front (or object side) of a lens converge at the rear (or image side) of the lens, the point of convergence is known as the rear focal point. Parallel rays of light that enter the rear (or image side) of a lens converge at the front (or object side) of the lens, and this point of convergence is known as the front focal point. In a microscope the front focal point is closer to the illuminating light source.

Reference 3

Rear focal point

Front focal point

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Focal length With the following parameters for the positional relationship between a lens and an object,

f: focal length of the lens S1: distance from the center of the lens to the object S2: distance from the center of the lens to the image plane

the equation giving the lenss focal length is as follows: 1/f = 1/S1 + 1/S2

Various case studies illustrating this are as follows: Calculating the imaging distance for a convex lens An object situated further away than the focal position produces an inverted real image.

Example 2 Calculate S2 if f = 50mm and S1 = 100mm. Since 1/50 = 1/100 + 1/S2, S2 = 100mm. An inverted real image is produced 100mm from the lens on the image side.

An object situated closer to the lens than the focal position produces an erect virtual image on the object side.

Example 3 Calculate S2 if f = 50mm and S1 = 25mm. Since 1/50 = 1/25 + 1/S2, S2 = 50mm. An erect virtual image is produced 50mm from the lens on the object side.

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Calculating the imaging distance for a concave lens An object situated further away than the focal position always produces an erect virtual image closer to the lens than the focal position.

Example 4 Calculate S2 if f = 50mm and S1 = 100mm. Since 1/50 = 1/100 + 1/S2, S2 = 33mm. An erect virtual image is produced 33mm from the lens on the object side.

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2. The Structure of an Optical Microscope

2-1 Configuration of optical systems An optical microscope is an instrument that combines two convex lens systems in a manner suitable for carrying out enlargement magnification of specimens. The lens closest to the specimen is known as the objective. For a specimen y, this lens produces a magnified real image y that is 1100 the size. The lens closest to the eye is known as the eyepiece lens and magnifies the real image y that was produced by the objective a further 1015 to produce an image at a reference viewing distance (250mm from the eye). Thus, when we use a microscope, we are observing an enlarged virtual image.

Finite optical systems In traditional microscopes, finite optical systems (as shown below) were adopted. In a finite optical system the light from an object passes through the objective and then converges at the primary image plane.

Optical configuration of a microscope

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[Additional information 5] Positioning of the specimen with a finite optical system: The specimen is placed at a distance from the objective that is further than the distance between the lens and the lens focal position. If it is placed at the focal position or closer to the lens than the focal position, no image will be formed. Infinity-corrected optical systems In this kind of optical system, light from the object travels in a parallel optical path after passing through the objective, and is then converged by a tube lens. This is illustrated in the following diagram,.

In general, creating a parallel optical path has the following advantages. [Additional information 6] Positioning of the specimen with an infinity-corrected optical system: The specimen is placed at the focal position for the objective. This causes the light from the object plane to travel in parallel.

The use of the parallel optical path formed by the objective allows an intermediate attachment to be easily inserted in it.

The focal position will not shift, even if an optical component such as a half-mirror is

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The phenomenon of vignetting Vignetting is the phenomenon in which the surrounding area appears dark during observation with a microscope. Although this phenomenon can occur with both finite and infinite optical systems, it will be explained here in the context of an infinite optical system. One feature of infinite optical systems is that they allow the insertion of components such as epi-fluorescence equipment into the parallel system. If the parallel system is too long, however, it cannot supply enough light to the tube lens, since the tube lens is of fixed size. (This is demonstrated by the diagram below.) The insufficient light results in this darkening phenomenon.

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2-2 CFI (Chromatic aberration-Free Infinity) optical systems With the traditional compensation method, the objective is only corrected for axial chromatic aberration, while lateral chromatic aberration of the objective is canceled out by the eyepiece lens. In 1976, Nikon developed the CF optical system, in which the objective and the eyepiece lens both independently correct for axial chromatic aberration and lateral chromatic aberration. Furthermore, Nikon develop the current high-performance CFI optical system that can both exhibit a high optical performance and system expandability both are realized by adopting a new proprietary standard to make the numerical aperture (NA) large and the working distance (WD) longdespite the fact that the WD and NA are complementary to one another. Parfocal distance This refers to the distance from the plane of objective lens mount to the object

plane when the microscope is focused. When a cover slip is used, this distance is measured from the bottom surface of the

cover slip (with upright microscopes). Parfocal distance = 60mm

[Additional information 7] Parfocal distance: This was 45mm for finite optical systems. However, in CFI optical systems, the prolonged parfocal distance allows the working distance to be increased. For all infinite optical systems manufactured by other companies, the parfocal distance is 45mm.

Focal length of tube lens This is the distance from the principal point of the tube lens to the image plane. Focal length = 200mm

[Additional information 8] Focal length of tube lens: This was 160mm for finite optical systems. Infinite optical systems manufactured by Company Z, Company O, and Company L have focal lengths of 160mm, 180mm, and 200mm respectively.

Screw diameter of objective: As well as making the screw diameter larger, changing the objective diameter in

CFI optical systems has greatly increased both the numerical aperture and the working distance.

Screw diameter of objective = 25mm

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[Additional information 9] Screw diameter: This was 20.32mm for finite optical systems. However, in CFI optical systems, the enlarged screw diameter allows the numerical aperture to be increased. Systems manufactured by other companies have screw diameters of 25mm (Company L), 20.32mm (Company O), and 20.32mm or 27mm (Company Z).

2-3 Microscope optical path diagrams Illumination is a key function in a microscope. The Khler illumination method, which uniformly illuminates the entire field of view, is used for general observation. The uniformly illuminated specimen image travels through the parallel optical system and the image is formed by the tube lens mounted under the eyepiece tube.

Episcopic- illumination system

Diascopic- illumination system

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2-4 Microscope part names The names of the parts of a microscope are shown below. (The microscope shown is the ECLIPSE 80i.)

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2-5 Cover slip standards Cover slips A biological specimen placed on a microscope slide is normally protected by a cover slip. Thus, when the specimen is observed through the microscope, the user is always observing it through the cover slip. Japan Industrial Standards (JIS), which meets global standards, specifies the following values for cover slips for use with microscopes.

Cover slips are manufactured to these standards, and objectives for biological microscopy are designed to match a cover slip thickness of 0.17mm. In order to make proper use of a microscope, it is thus vital to use cover slips that meet these standards. Microscope slides Microscope slides are glass plates on which a minute sample is mounted for observations with optical microscopes. These are normally approximately 2.5cm wide, 7.5cm long and 1.2mm thick.

Proper cover slip used Cover slip with a thickness deviation used

Index of refraction ne = 1.5255 0.0015 Abbe number e = 56 2 Degree of flatness 0.010 mm Standard Thickness range No. 1 0.120.17 No. 1-S 0.150.18

Effect of cover slip thickness deviation

Japan Industrial Standards (JIS) standards for cover slips

Reference 4

Reference 5

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3. Optical Performance

3-1 Total magnification Magnification is the ratio of the size of the image to the size of the object. In the case of a microscope, however, the image that has been enlarged by the objective is further enlarged by the eyepiece lens. Thus, calling the magnification by the objective Mo and the magnification by the eyepiece lens Me, the total magnification for observation of an optical microscope (M) is the product of Mo and Me. M = Mo Me (combined magnification by the objective and the eyepiece lens) In an optical microscope a variable magnification lens is sometimes inserted between the objective and the eyepiece lens. For example, in inverted microscopes there is an intermediate lens that can magnify by 1.5. When calling magnification by the intermediate lens Mi, the total magnification M is as follows: M = Mo Mi Me (combined magnification by objective, intermediate lens and eyepiece lens) When calling magnification of the photographic lens Mp, the magnification, which varies according to the type of relay lens or adaptor used, on the film plane or on the CCD (Mf) is expressed as follows: Mf = Mo Mi Mp (combined magnification by objective, intermediate lens and photographic lens) Digital cameras have recently come into widespread use. To find the magnification on the monitor screen of a digital camera, it is necessary to further multiply by the ratio of the monitors screen size to the size of the CCD as monitor magnification. When calling monitor magnification Mc, magnification on a monitor screen (Mm) can be expressed as follows: Mc = monitor size / CCD size. Mm = Mf Mc (product of image plane magnification and monitor magnification)

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[Additional information 10] CCD size: The size of a CCD or monitor is normally expressed in terms of its diagonal size in inches. An inch is 25.4mm. Actual dimensions are as shown below. CCD size (4:3) 2/3 inch 8.8mm 6.6mm 1/1.8 inches 6.9mm 5.2mm 1/3 inch 4.8mm 3.6mm LCD monitor size (4:3) 19 inches 386mm 289mm 24 inches 487mm 365mm CCD size denotes a size equivalent to the imaging area of the pick-up tube. The pick-up tube diameter is used as a shorthand name for the CCD size. Hence, for a 2/3 inch CCD, the CCD diagonal size is designated as 11mm (not 16.9mm), which corresponds to a 2/3 inch pick-up tube's image area. This convention came about because CCD image sensors were initially used in place of the pick-up tubes used in TV cameras, and was thus regarded as convenient when optical elements such as lenses were designed or selected. Example 5 Calculate the magnification on a monitor when an image is shot on an inverted microscope with the following configuration. Objective lens 20, intermediate magnification 1.5 2/3-inch camera, 0.6 C-mount TV adaptor, 19-inch monitor Mm = 20 1.5 0.6 386/8.8 Thus, the image on the monitor is magnified by a factor of 789. 3-2 Actual field of view. The size of the field which can be observed through a microscope eyepiece lens is known as the actual field of view. The actual field of view is calculated as follows, based on the magnification of the objective and the field number* of the eyepiece lens. Actual field of view (mm) = field number of eyepiece lens / magnification of objective For microscopes in which intermediate magnification is carried out, such as inverted microscopes, it is also necessary to divide the above equation for actual field of view by the intermediate magnification.

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* The field number (FN) expresses without units (mm) the diameter of the fixed field stop ring in the eyepiece lens, which limits the size of the image that is produced by the objective. i-Series microscopes have field numbers of 25 or 22. Example 6 Calculate the actual field of view when a 100 objective and a UW10 eyepiece lens are used. Actual field of view = 25 / 100 A circular area of diameter 0.25mm can be seen through the eyepiece lens. 3-3 Numerical aperture (NA) Numerical aperture is an important value which determines the performance of the objective and the condenser, and also relates to the resolution and the depth of focus, as well as to the brightness. Numerical aperturewhich is normally referred to as NAis defined as follows. Numerical aperture (NA) = n sin Here, n is the refractive index of the medium between the specimen and the front lens of the objective: this medium is air (n = 1.0) for a dry objective, and immersion oil (n = 1.5) for an oil-immersion objective. There are also water-immersion objectives (n=1.333), and hence the refractive index of the medium varies according to the type of objective used. is the angle between the optical axis and the light rays that pass through the outermost edge of the lens. From the equation given, it can be seen that numerical aperture is proportional solely to the refractive index of the medium and the conversing angle. Since the maximum value of sin is 1, the maximum NA value is the refractive index of the medium.

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Consider for example the maximum NA of a dry objective. Assuming that the objective is set on the surface of the specimen, the maximum value for n (when = 90) yields a theoretical result of NA = 1.0. For actual operation, however, a slight space is required between the surface of the specimen and the front lens of objective. As a result, NA is around 0.95 in practice. Nikon CFI Plan Apo 40 and 60 objectives perform as this level.

3-4 Image brightness The brightness of image varies with total magnification and numerical aperture, as well as with the intensity of illumination. In an optical microscope, the higher the numerical aperture or the lower the total magnification, the brighter the observed image. For example, given two objectives with the same magnification, if one has a numerical aperture that is twice the numerical aperture of the other, it will produce an observed image that is four times as bright. The relationship between numerical aperture (NA), the total magnification (M), and the image brightness of the brightfield observation (I) is expressed by the following equation: I (NA/M)2 In the case of a fluorescence observation, both excitation light and fluorescence pass through the objective. Hence the brightness is given by the following equation: I NA4/M2

As a result, when observing a dark specimen, such as in a fluorescence observation, selecting an objective with a high NA allows a bright image to be observed.

Angular aperture

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Example 7 Compare brightness of the Plan Apo 20 objective (NA 0.75) and the Plan Fluor 20 objective lens (NA 0.50). Call the brightness of the Plan Apo 20 image Ia and the brightness of the Plan Fluor 20 image Ip. k is proportionality factor Ia = k (0.75 / 20)2 Ip = k (0.50 / 20)2 Comparing the two, it can be seen that the object will be 2.25 times brighter through the Plan Apo objectives. 3-5 Resolution Resolution is indicative of an optical microscopes performance. Resolution refers to the ability to distinguish two points that are very slightly apart as being separate, and is defined as minimum distance between two such points. Hence, the smaller the value of a microscopes resolution, the more minute the objects that can be observed. In specific terms, when two points emerging from pinholes are very close to one another, an image of two points overlapping and connected with each other is visible, as shown below. According to Rayleighs definition, the limit of resolution is reached when the overlap between these two points reaches 74%.

Brightness

Coordinates

R

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In Rayleighs equation for resolution limits, the resolution (R) is expressed in terms of the wavelength of the light (). Resolution (R) = 0.61/ NA The equation for resolution capability varies slightly with the adopted criterion value for resolution limit and the shape of the light source and specimen. In general, the greater the magnification of objective, the larger the numerical aperture. Hence, in order to observe in more detail, it is better to switch to a higher magnification objective with a larger numerical aperture. [Additional information 11] Variations in resolution due to the shape of the light source: The type of light source used is a key consideration when defining resolution. Rayleighs definition is premised on a (circular) point source of light. If the light is imagined as passing through a narrow slit, the spreading of zero-order and 1-order rays will be even smaller. Resolution =/ 2NA However, Rayleighs definition of resolution is widely used. Example 8 What kind of resolution does an objective have in practice? The resolution capability for an oil-immersion objective with NA 1.49 is calculated as follows, using green light with a standard wavelength of = 550nm as an example. Resolution = 0.61 550 / 1.49 = 225 (nm) As can be seen from the equation for resolution, the shorter the light wavelength used, the smaller the resolution. However, if green light is used as a standard-wavelength light, even an oil-immersion objective with a numerical aperture of 1.49 gives a maximum resolution of 225nm. Alternatively, for a dry objective with a numerical aperture of 0.95, the maximum resolution would be approximately 350nm. Since the resolution of an optical microscope is essentially determined by the numerical aperture of the objective, it is not possible to discern minute structures that are beyond its resolution simply by increasing the magnification. There is also a limit on the capability of the human eye to discern size. Although the resolution of the human eye varies from individual to individual, it is quoted as being approximately 0.15 mm (or 2 of angle) at the reference viewing distance (a position 250mm from the eye).

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The magnification of a microscope can be said to be appropriate if the image size (its resolution its total magnification) slightly exceeds the resolution of the eye. This is termed the effective magnification and is generally of the order of 5001000 the numerical aperture of the objective. Any greater enlargement than this simply involves the enlargement of a blurred image and is termed ineffective magnification. Since the maximum numerical aperture of objective is approximately 1.4 and maximum resolution is approximately 0.2m, the maximum total magnification of a microscope is approximately 1500. However, it is preferable to use a microscope with magnification of up to approximately 1000 in order to minimize eyestrain. [Additional information 12] Resolution and resolving power The performance of the objective on a stereoscopic microscope is expressed by its resolving powernot its resolution. For the HR Plan Apo 1.6, which has a numerical aperture of 0.21, this is the number of visible lines that can be resolved in 1mm. Since resolving power is given by dividing 1mm by the resolution, in this example the resolving power = 625 lines / mm. 3-6 Working distance In practice, smooth operation requires a space between the front lens of the objective and the surface of the specimen. This space is known as the working distance, which is abbreviated to WD. The WD is generally larger with lower NA and lower magnification objectives. A cover slip is normally placed on top of a biological specimen, in which case the working distance is from the front lens of the objective to the top surface of the cover slip. 3-7 Depth of focus When a specimen is observed with an optical microscope, there is a certain distance range within which focus will be maintained when the objective is moved. This range is known as the depth of focus. The precise calculation for depth of focus is quite complex; however, for an optical microscope with a total magnification of M, the depth of focus (in m) is given by Martins equation as follows: = (n / (2 NA2)) + (n 1000) / (7 M NA) The first term, which comprises the first half of this equation, is known as the physical depth of focus. For photography or TV viewing, this term alone represents the depth of

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focus. The second term, which comprises the second half of this equation, represents the depth of focus for the human eye, and is thus required when the eyepiece lens is used for observation. Example 9 Let us calculate the depth of focus for observation by eye using a Plan Apo 4 objective and a Plan Apo 40 objective. Here, the NAs for the objectives are taken to be 0.20 and 0.95, and the magnification of the eyepiece lens is taken to be 10. In order to keep the units consistent, the wavelength used in the calculation is 0.55m. 4 = 1.0 0.55 / (2 0.22) + 1.0 1000 / (7 (4 10 0.2) = 6.87 + 17.85 = 24.72m 40 = 1.0 0.55 / (2 0.952) + 1.0 1000 / (7 (40 10 0.95) = 0.30 + 0.37 = 0.67m It can be seen that the depth of focus for the 40 objective is tiny in comparison with that for the 4 objective. [Additional information 13] Image-side depth: The term depth of focus can be used to refer to both object-side (specimen-side) depth and image-side depth. The explanation above relates to object-side depth. Image-side depth is expressed by the following equation. Image-side depth = physical depth of focus (magnification at image surface)2 By way of reference, if the image-side depth is calculated for the 4 and 40 objectives mentioned above, respective values of approximately 110m and approximately 480m are obtained. Hence, it can be seen that if the magnification is high, the object-side depth will be shallow, while conversely the image-side will be extremely deep. 3-8 Aberration Light rays close to the optical axis and in a lens of zero thickness will behave in accordance with theory. In practice, however, a lens has a certain thickness and light rays may pass through the periphery of the lens. Phenomena known as aberrations occur, in which light rays do not converge at a single point. These aberrations can be divided into two types according to their cause: monochromatic aberrations, which are caused by the nature of the lens, and chromatic aberrations, which are caused by wavelengths of light.

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Objectives and eyepieces lenses adequately correct these aberrations to allow for observation. The types of aberration that occur in optical microscopes are as follows: Categories of aberration Spherical aberration Coma Monochromatic aberrations Astigmatism (The five Seidel aberrations) Curvature of field Aberration Distortion Axial chromatic aberration Chromatic aberrations Lateral chromatic aberration Monochromatic aberration Spherical aberration: Unrelated to the angle of view. The only type of aberration that is evident in the center of the field of view. Circular blurring. The higher the NA, the more difficult to correct. NA3 Coma: More pronounced at larger angles of view. Comet-shaped blurring NA2 y1 Astigmatism: More pronounced at larger angles of view NA y2

Shifts the focus and produces longitudinal, circular, and lateral blurring. Curvature of field: More pronounced at larger angles of view NA y2

Point of focus shifts both in the center of the field of view and at the periphery. Distortion: More pronounced at larger angles of view. y3 The only type of aberration that is evident even when NA is reduced. Rectangular shapes are distorted into barrel-like and spool-like shapes.

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Chromatic aberration Axial chromatic aberration: Unrelated to the angle of view NA Circular blurring due to the focal point shifting according to color Lateral chromatic aberration: Unrelated to the NA y Color shifts at the periphery of the field of view, due to the image-forming magnification varying according to color.

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4. Functions and Usage of the Illumination System

4-1 illumination optics The illumination system that is commonly used in microscopes is known as the Khler illumination system. This system forms an image of the light source (an image of the filament of a halogen lamp) at the rear focal point of the objective, and forms an image of the field diaphragm on the specimen plane. The specimen is uniformly exposed and the observation area is limited by the field diaphragm, enabling observation that is free from flare or ghosting. Khler illumination Advantages

Illumination is uniform. Field diaphragm and aperture diaphragm can be set independently. A wide area can be exposed, even at low magnification.

Disadvantages Structure is complex and expensive.

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Critical illumination This method forms an image of the light source on the specimen plane. Advantages

Construction is simple. Bright illumination is achieved.

Disadvantages Illumination is not uniformly concentrated. There is no field diaphragm. It is not possible to expose a wide area at low magnification.

As shown in the diagram below, in a microscope the specimen is illuminated by the Khler illumination system and the field of view has maintained uniform brightness.

Optical path diagram for the Khler illumination method

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4-2 Light source Nikon microscopes are now equipped with halogen lamps for illumination of brightfield observation. In the past, some microscope models have used tungsten lamps (which are also a thermal light source). However, halogen lamps have proved superior in terms of brightness, size and life span, and have consequently become standard. The use of LED illumination, which generates little heat and provides long life and constant color temperature, has also increased. However, halogen lamps are superior in terms of brightness. There are also especially durable long-life halogen lamps. Once installed, these can be used for up to a year, as they have a life span of over 2,000 hours (although this depends on frequency of use). Because a halogen lamp bulb is made of quartz, dirt or grease from fingerprints can burn onto it, with the result that the amount of light from it will be reduced. When changing the bulb, you should be careful not to touch it directly, and wipe off any attached dirt using alcohol or petroleum benzine.

Mercury lamps Mercury lamps have characteristic line spectrum in the wavelength range from ultra-violet to visible light. With prominent emission peaksso-called spectral lines (near 254nm, 365nm and 405nm) this lamp is far superior to other lamps in terms of output at these spectral lines.

Spectrum for mercury arc lamp

365nm 405nm

546nm

577nm 435nm

Reference 6

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Xenon lamp Characterized by a continuous spectrum from the ultra-violet region to the infra-red region. White light is obtained that is close to sunlight. A further characteristic is the stable output in the ultra-violet region. Halogen lamp Has high color temperature and is used for microscope illumination or as the light source for an overhead projector or movie projector. Metal halide lamp Greater luminous efficiency than a halogen lamp and well balanced in the visible-light wavelength range.

Spectrum for xenon lamp

Spectrum for halogen lamp 400 500 600 700

Spectrum for metal halide lamp

Reference 7

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LED lamp The advantage of LEDs is that it is possible to change the spectrum by changing the color combination. Another major characteristic is that, with a life span of 100,000 hours, an LED lamp will last approximately 10 times as long as any other light source. 4-3 Color and brightness of illumination One of the roles of illumination equipment is to illuminate using light of a color that is close to natural white light, thereby faithfully reproducing the colors of the specimen for observation. With the halogen lamps used in microscopes, varying the voltage applied to the light source also varies brightness and color of output light. This is referred to as color temperature. Most modern microscopes use either a 12V/100W or 6V/30W halogen lamp. However, when microscopic images are shot using traditional film, it is necessary to set the voltage at 9V with the 12V type and at 5.5V with the 6V type because the NCB filter is designed to yield most natural transparent illumination at that voltage. In these cases, it is also necessary to adjust the brightness using an ND filter to prevent varying color temperature. A feature of white-light LED illumination is that varying the voltage does not change the color temperature, as it does with halogen lamps. However, light intensity control for comfortable observation is made by inserting an LA60 filter. Nowadays, digital cameras are mostly used. It is necessary to use the cameras white balance function after adjusting the color temperature, just as it is with film cameras.

Reference 8

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[Additional information 14] What is color temperature? Given an ideal black body, the distribution of light wavelengths radiated by the body at specific temperatures can be deduced. At low temperature, the color of this light is a dark orange, which becomes a yellow-tinged white as the temperature increases, and then almost a bluish white as the temperature rises even higher. Thus, the color of light can be expressed by the temperature of the black body, and this temperature is known as the color temperature. There is an analogy to color temperature in the color of celestial objectsstars that shine bluish-white have higher surface temperatures than stars that shine red. The effects of NCB (neutral color balance) filters NCB filters compensate for the characteristic color temperatures of a light source. Halogen lamps are widely used in microscopes nowadays due to their brightness and long life. The pictures below illustrate the effect of using an NCB filter on a 12V, 100W halogen lamp.

Reference 9

Illumination using the halogen lamp alone

Illumination using the NCB filter

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4-4 Condensers A condenser does not merely condense light. It also affects factors such as image resolution, contrast, depth of focus and image brightness and is indispensable for demonstration of the full capability of the objective. The many types of purpose-specific condenser are distinguished by their numerical aperture, object distance (OD) and the magnification of compatible objectives.

Type Numerical

aperture Object distance (mm)

Magnification of compatible objective (for field number of 22)

Abbe condenser 0.9 1.9 4100 Achromat condenser 0.85 4.2 4100 Achromat swing-out condenser 0.8/0.12 3.2 1100 Achromat/Aplanat condenser 1.40 1.6 10100 LWD achromatcondenser 0.65 10.2 440 Low power condenser 0.15 10.2 14 Darkfield condenser (dry) 0.80.95 4 1040 Darkfield condenser (oil) 1.21.43 1.5 20100 Phase contrast condenser 0.9 1.9 10100 Universal condenser (dry) 0.9/0.13 2.3 2100 DIC condenser (oil) 1.4 1.6 10100 Motorized universal condenser (dry) 0.9/0.13 2.3 2100

Examples of condensers for upright microscopes

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4-5 Functions and usage of diaphragms Illumination systems are equipped with field diaphragms and condenser lens aperture diaphragms. The principal function of these diaphragms is to eliminate unnecessary light so that high-quality images can be obtained. The field diaphragm is in the microscope body and acts to limit the illuminated area. When photographs are being shot or when an image is being output to a TV monitor, a clear image with no extraneous light can be obtained by using the diaphragm to exclude illumination areas that are unnecessary for observation. Flare or ghosting will occur if even the faintest light generated on the lens surface or inside the lens is reflected and reaches the image. Closing the field diaphragm until it circumscribes the field of view prevents this from occurring and gives favorable results.

The condenser diaphragm is an aperture diaphragm, it is equivalent to a camera lens diaphragm in that closing it reduces the resolution and brightness and increases the contrast and depth of focus. In general, closing the aperture diaphragm to around 7080% of the NA of the objective enables a well balanced image to be obtained.

Condenser is closed to around 7080% of the NA of the objective.

Making adjustments while looking into the pupil of the objective Alternatively, the condenser is closed to around 7080% of fully open while the viewer looks into the pupil of the objective.

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4-6 Flare and ghosting Flare and ghosting are two phenomena that are hard to perceive during observation, but which are often apparent when photographs are shot with a digital camera or when image enhancement is carried out. Flare: A phenomenon in which light reflected in an uneven fashion at the surface or the inside of a lens affects a photograph or image. The contrast is reduced and the entire image appears washed out. Ghosting: Light traveling through the microscope is reflected internally and appears as spurious image informationfor example, in terms of the image shape or image spectrum.

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5 Objectives and Eyepieces

5-1 Objectives Since the objective is the most important element in determining the performance of an optical microscope, there are many different types, which vary in terms of their degree of aberration correction and their intended use. Hence, it is vital to use an objective that is appropriate for the task at hand. There are indications regarding the characteristics, the uses, and the standard on the lens body. The following categories are used to classify objective types in terms of their uses and aberration correction. Achromat Plan Achromat Fluor S Fluor Plan Fluor Plan Apochromat Plan Apo S Plan Apo IR Achromat objective This is a reasonably priced objective with a simple structure intended for general observation. It emphasizes optical performance in the center of the field of view and corrects for various aberrations. The basic meaning of the name is that the lens performs chromatic aberration correction on the C-line (red) and F-line (blue). Since the lens does not normally correct for curvature of field, the focus is accurate in the center of the field of view, but sometimes with blurring at the periphery. However, flatness has been improved significantly in CFI objectives. Plan Achromat objective The lens performs chromatic aberration correction in the center of the field of view, just like the Achromat. Since the lens also corrects for flatness within the field of view, a blur-free image can be observed with a sharpness that extends from the central area to the periphery. Fluor, S Fluor, and Plan Fluor objectives These objectives were principally developed for fluorescence microscopy. They thus realize high transmittance even in the shortwave ultra-violet region, and are made out of optical materials that exhibit little autofluorescence and have less deteriorative against powerful ultra-violet light rays. The numerical aperture is greater than that of Achromat objectives. This is advantageous for fluorescence microscopy, with the result that bright,

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high-contrast images are obtained with any excitation light wavelength. In particular, the S Fluor has high transmittance in the ultra-violet region up to 340nm and a large numerical aperture that enables bright fluorescent images to be obtained, even with UV excitation. Plan Apochromat objective This lens performs sufficient correction for chromatic aberration throughout the entire visible wavelength region, even including the g-line (bluish violet). It has a large numerical aperture and performs ideal correction for various aberrations right up to the edge of the field of view, making it a top-of-the-range objective. The basic meaning of Apochromat is that complete chromatic aberration correction is performed for three wavelengths: the C-line (red), the F-line (blue), and the g-line (bluish violet). Since these objectives have a large numerical aperture and exhibit outstanding resolution capability, image flatness and color reproduction, they are ideal for observing and photographing samples with highly detailed structures. Type General No cover slip (NCG) Correction ring (C) Long working distance (LWD) Dry types Phase contrast (DLL, DL, DM, BM (PHL, PH1, PH2)) Apodized phase contrast (ADL (PH1, PH2), ADM (PH1, PH2)) Differential interference contrast (DIC (N1, N2)) Polarized light (P)

General (H)

No cover slip (NCG) Phase contrast (DLL, DL, DM (PH3)) Oil Apodized phase contrast (ADH (PH3)) Differential interference contrast (DIC (N2)) Polarized light (P) Evanescent wave (TIRF) Immersion S

types General (W, WI) Phase contrast (DLL (PH2)) Water Differential interference contrast (DIC (N2))

S IR

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Phase contrast objective This type of objective enables observation of detailed structures and shapes of transparent samples (such as living cells and tissue, and unstained and faintly stained specimens), by converting their phase difference into light and darkness of image. There are two types of phase contrast method: the bright-contrast method (B), in which the phase of the direct light is delayed by 1/4 wavelength so that the phase contrast segment appears as a bright image against a dark background; and the dark-contrast method (D), in which the phase of the direct light is advanced by 1/4 wavelength so that the phase contrast segment appears as a dark image against a bright background. There are four types of phase contrast objectives that employ the dark contrast method, and these are categorized according to the degree of the contrast. Starting with the type which produces the low contrast, these are: DLL, DL, DM and DH. There are five types of condenser phase ring corresponding to each objective. Starting with the ring corresponding to the lowest magnification objective, these are: PHL, PH1, PH2, PH3 and PH4. Apodized phase contrast objective This is an objective that successfully reduces halo artifactsa task that is theoretically difficult in phase contrast microscopy. This type of objective has enabled observation of specimens that would otherwise be covered by halo artifacts and thus difficult to observe, such as the intracellular structure during cell division or thick-phase objects. There are three types of apodized phase contrast objective: ADL, ADM and ADH. As with phase contrast objectives, a condenser phase ring is required. Differential interference contrast objective This lens enables three-dimensional observation of the very highly detailed structures of colorless and transparent specimens (the same types of sample that phase contrast is used for) by optically applying shadow to them. Since there are no halos as there are when phase contrast methods are used, the detailed sections of the specimen can be observed. The interference color can be displayed using a lambda plate. Since this type of lens makes use of polarization, it is unsuitable for observing specimens in containers made of materials that disturb polarization, such as plastic. The type of Nomarski prism (N1 or N2) corresponding to the objective is indicated on the objective's body. A differential interference contrast objective makes use of polarization and needs to have little strain. However, the degree of strain is designed to be of an intermediate level between objective for general use and objective for polarization use. Polarizing objective A polarized-light microscope is used to examine the polarization characteristics of a specimen. Since a slight strain in the optical system will affect the specimens polarization characteristics, objectives used for polarized-light microscopy are with as little strain as possible.

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No-cover-glass objective This type of objective is used for observing specimens without a cover slip placed over them, such as blood specimens. Other general objectives used for biological microscopy are corrected aberrations to yield the best possible imaging result when a 0.17mm-thick cover slip is used. Therefore, if observation is carried out without a cover slip using this kind of lens and NA = 0.65 or above (for dry objectives) or NA = 1.3 or above (for oil-immersion objectives), aberrations will be conspicuous and there will be a deterioration in the image quality. Long working distance objective This type of objective is often used for the observation of tissue cultures through the bottom of a culture dish with an inverted microscope (principally using phase contrast microscopy). This type of objective is compatible with various types of observation vessel, since dish bottom thickness in the range 0mm2mm can be compensated for using a correction ring. Evanescent wave objective (Total internal reflection fluorescence objective) When total internal reflection illumination is performed using a laser light from underneath a dish that has a bottom made of a 0.17mm-thick cover slip and containing cells, only the ultra-thin layer nearest the cover slip will be excited by the evanescent waves. Since the layer is so thin, a fluorescent image with extremely good contrast can be obtained. An objective with a numerical aperture that is greater than the refractive index of water is required in order to effect total internal reflection. Nikon provides two objectives (100 and 60) with a numerical aperture of 1.49. S objective The name denotes wavelength () and spectrum (S). This type of objective has the capability to perform chromatic aberration correction over a wide wavelength range (405nm) as well as or wider than VC objectives and to realize high transmittance from the UV region up to the near-infra-red region. It is suitable for confocal spectral imaging. Infra-red objective This type of lens corrects for chromatic aberration in the 435nm1064nm range. Like the S objective, this type of lens has the capability to realize high transmittance from the UV region up to the near-infra-red region and is suitable for confocal spectral imaging.

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Markings on objectives CFI objectives are slightly larger than conventional CF objectives. The meaning of the information engraved on an objective is explained below:

Markings on an objective Type: Type as determined by aberration corrections performed by

objective: e.g. Plan Apo Working distance (WD):

Distance from front lens of objective to surface of cover slip. The longer this distance, the easier it is to change specimens, and the more user-friendly the system is.

Correction ring: Objectives are designed for use with a cover slip of a standard thickness (0.17mm). This is a mechanism for compensating the cover slip thickness deviation.

Numerical aperture (NA):

This value represents the brightness of objectives. Affects resolution and depth of focus of the objective.

Parfocal distance: Distance from the objective mount plane to the specimen surface. Each manufacturer has its own fixed proprietary values; objectives provided from the same manufacturer have the same parfocal distance.

Application markings:

Phase contrast objectives have markings such as DL, DLL, DM, while differential interference contrast objectives have markings DIC.

Immersion objective identifier:

Black line indicates that space between objective and specimen must be filled with immersion oil. All manufacturers use the same marking.

Color codes: Color codes line on the lens barrel indicates the magnification of the objective: 1: black, 2: gray, 4: red, 10: yellow, 20: green, 40: light blue, 60 cobalt blue, 100: white

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5-2 Eyepiece lenses An eyepiece lens fulfills the function of a magnifying glass to enlarge the real image produced by the objective to create a virtual image at the reference viewing distance (a position 250mm from the eye). The eyepiece lens also has the important function of diopter adjustment. Before observation, the observer turns the diopter adjustment ring and compensates for differences of eyesight between both eyes so that the two eyes can focus at the same time. There are several different types of eyepiece lens, with varying magnifications and field numbers. A type of eyepiece lens with a reticle in it is also available. If the eyepiece lens is removed, the pupil of the objective can be seen. By observing the objective pupil, it is easy to check for air bubbles in the immersion oil on the front lens of objective. The top surface of the eyepiece lens is the area where dirt and stains from eyelashes most readily adhere. Stains should be wiped off using absolute alcohol, and dirt should be removed with a blower.

Name (magnification) Field number Summary CFI 10 22 CFI 10M 22 With mask CFI 15 14.5 CFI UW 10 25 CFI UW 10M 25 With mask

Eyepiece lens types

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Eyepiece lens Reticle diameter CFI 10 27mm CFI UW10 27mm CFI 15 27mm E2-CFI 10 27mm E2-CFI 15 27mm C-W 10 25mm

Reticle diameter of eyepiece lenses [Additional information 15] Magnification and focal length: Since the reference viewing distance is 250mm from the eye, the relationship between magnification and focal length (f) is described by the following equation. f = 250 / magnification Taking a 10 eyepiece lens as an example, f = 25mm. The real image in this position is enlarged and can be seen at 250mm from the eye (the reference viewing distance). Eyepiece reticle Some eyepiece lenses can have reticles installed in them. Many different types of reticle (like the one shown in the illustration below) are available for different applications. The reticle is installed at the position in which the image is formed. 5-3 Eyepiece tube There are binocular eyepiece tubes for eye observation, while trinocular eyepiece tubes enable photography or TV monitoring. There are two forms of eyepiece tube structure, and these are distinguished by their respective eye point (exit pupil).

Reference 10

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Siedentopf type (for high-grade microscopes) The prisms P1 and P2 (with P3) are rotated about the 00 optical axis to vary the interpupillary distance EE. The advantage of this type of eyepiece tube is that, even when the interpupillary distance is adjusted, the imaging point does not change. As a result, it fully demonstrates the capabilities of the objective. The disadvantage, however, is that when an eyepiece lens containing cross hairs is used, the cross hairs will also rotate, thus necessitating an adjustment mechanism.

Siedentopf-type eyepiece tube

Jensch type The central prism P1 (which divides the optical path) is fixed, while the prisms P2 and P3 can simultaneously be moved left and right horizontally to vary the interpupillary distance EE. The advantage of this type of eyepiece tube is that adjusting the interpupillary distance does not rotate the eyepiece lenses. And these eyepiece tubes can be designed to be relatively small. Their disadvantage is that since the imaging point changes, either the viewer must change the position of his/her eyes or an adjustment mechanism is required.

Jensch-type eyepiece tube

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Name Division of optical

path Field number ISO tube

Binocular eyepiece tube B

100% in binocular section

22

Trinocular eyepiece tube FUW

100% in binocular section/ 100% in photo tube section

22/25 Can be mounted

Trinocular eyepiece tube TUW

100% in binocular section/ 100% in photo tube section 20% in binocular section/80% in photo tube section

22/25 Can be mounted

Ergonomic eyepiece tube

100% in binocular section 50% in binocular section/50% in photo tube section (if DSC port is used)

22

DSC port (for use with Ergonomic eyepiece tube) C-mount 0.7 lens installed

Eyepiece tube types [Additional information 16] Interpupillary distance: Interpupillary distance varies from person to person, however the average is recognized as 55mm70mm.

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6 Types of microscope

Optical microscopes can be divided into biological microscopes and industrial microscopes according to use. Research in biology and medicine, and clinical examination (chiefly in hospitals) correspond to the former, while semiconductor research and component inspection by manufacturing companies correspond to the latter. In the biological microscope, transmitted illumination is mainly used, as many of the specimens are translucent objects, such as cells. In the industrial microscope, reflected illumination is mainly used, since the samples are often objects through which light cannot pass, such as machine components and wafers. 6-1 Upright microscopes and inverted microscopes Biological microscopes can be divided into upright microscopes and inverted microscopes, according to application.

The specimen is observed from below. Used for observing culture specimens, etc. in Petri dishes.

Inverted microscope Upright microscope

The specimen is observed from above. The specimen is normally fixed on a slide mount.

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6-2 Transmitted light illumination and reflected light illumination Since virtually all biological specimens are thin enough to allow light to pass through them, they are observed using transmitted light. When the surfaces of metal and mineral samples are observed, reflected light is used.

Reflected illumination model Transmitted illumination model Light that is reflected off a specimen is observed. Principally used for observing the surfaces of metals and minerals.

Light that passes through a specimen is observed. Principally used for observing the cells of fungi, animals and plants.

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7 Observation Methods

A major characteristic of optical microscopy is that there is a wide range of observational methods intended for specific purposes.

Brightfield microscopy Phase contrast microscopy Differential interference

contrast microscopy

Stomach wall (HE dye) 10 Abdominal wall muscle 10 Abdominal wall muscle 10

Fluorescence microscopy Polarized-light microscopy Darkfield microscopy

Nerve cells from the neck of a cow 40

Polypropylene 10 Abdominal wall muscle 10

Modulation contrast

microscopy Asbestos microscopy Stereoscopic microscopy

Egg cells 10 Amosite 10 Water flea

Culture microscopy

HeLa cells 10

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7-1 Brightfield microscopy This is the most common observation method using an optical microscope. This kind of observation is carried out with transmitted illumination by a halogen or tungsten lamp when it comes with a biological microscope. Since virtually all biological specimens are colorless and transparent when unstained, the area to be observed is generally stained specifically before observation. At this time, the lamp is set to the recommended voltage, transparent illumination is applied using an NCB filter, and the brightness is adjusted using an ND filter. The most common staining methods are H&E staining and Giemsa staining, which has been used for a long time. Giemsa staining is used on blood samples and results in red blood cells and blood platelets staining between bluish red and blue. H&E hematoxylin stains basophilic cell nuclei and bone tissue bluish purple, while eosin stains acidophilic cytoplasm and red blood cells red or pink. Please refer to the specimen images in Section 12. 7-2 Phase contrast microscopy Unstained specimens that are colorless and transparent are observed by converting phase differences due to the specimens thickness or refractive index into intensity differences. This method is suitable for the observation of detailed structures with phase differences, and features a very high level of detection capability. The visibility is not affected by the orientation of the specimen. With the exception of methods that use external phase contrast systems, phase contrast microscopy generally involves the use of a dedicated objective with a phase plate inserted, which delays the phase by 1/4 wavelength, and the insertion of a phase ring into the condenser. The disadvantage of this method is that spurious bright areas known as halo effect can occur at the boundary sections of specimens that induce large phase shifts, making it difficult to view detailed structures. However, Nikon's Apodized phase contrast objective involves a new phase contrast method that reduces halo effecteven with thick specimens. The phase contrast method requires the use of a dedicated phase contrast objective and a corresponding phase ring (condenser annulus). Objective types DLL: low contrast (general-purpose) DLL: medium contrast (general-purpose) DM: high contrast ADL: for use in observing cell interiors and thick specimens

Reference 11

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ADM: for use in observing specimens of medium thickness ADH: for detailed observation with high magnification 7-3 Differential interference contrast microscopy Like the phase contrast method, the differential interference contrast method is used for observing colorless and transparent specimens. The basic principle involves splitting a polarized light into two light rays and visualizing the existence of phase objects by allowing the two light rays to interfere with one another after they pass through a specimen. The light is polarized with a polarizer and is split into two rays by a Nomarski prism. The two rays travel through slightly shifted points (depending on the amount of the shear) of a specimen. The rays are recombined with the second prism and interfere with one another when they pass through an analyzer, converting a specimen's optical path gradients and small steps on the surface of a specimen into intensity differences. This method achieves highly sensitive visualization. It does not suffer from the halos that appear when the phase contrast method is used and creates images for observation with a shaded three-dimensional effect. The differential interference contrast method requires a dedicated objective and Nomarski prisms that correspond to each objective. 7-4 Fluorescence microscopy This method involves labeling a specimen using a fluorochrome and observing the fluorescence of characteristic color for fluorochrome. A mercury lamp or xenon lamp is used for obtaining excitation light that induces fluorescence. It also requires a fluorescence filter cube that incorporates a dichroic mirror for separating the excitation light and the fluorescence. The fluorescence method is an excellent observation method, as even if an object is smaller than the resolution of the objective, its presence will still be revealed by the fluorescence. The fluorescence antibody method was once standard; however, the method using fluorescent proteins (such as GFP) is becoming common now.

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The Stokes shift phenomenon is quite characteristic of fluorescence, whereby the wavelength of the fluorescence is longer than the wavelength of the excitation light. Filters are selected in view of this phenomenon. Excitation filter: Filter that selects the excitation light Dichroic mirror: Beamsplitter that separates the excitation light and the fluorescence Emission filter: Filter that selects the fluorescence 7-5 Polarized-light microscopy Polarized-light microscopy involves illuminating the specimen with polarized light and observing the changes in the polarized light after it has passed through the specimen. The polarized light is produced by the polarizer, which only allows light vibrating parallel to the polarizing direction of the polarizer to pass through it. The light that has passed through the specimen then passes through an analyzer that is oriented orthogonally to the polarizer (in a configuration known as crossed Nicols). Polarized-light microscopes are most often used for research into rock composition; in the biological market they have long since been used for the inspection of the crystals that cause gout. Recently, they have also been used on biological tissue such as muscle, which has polarization properties. With specimens that exhibit biorefringence, as seen in crystals such as rock, light is split into two rays that travel orthogonal planes of vibration. The phase difference that arises between this split light rays is referred to as retardation. Measuring this retardation enables physical properties of the sample to be identified. There are two polarized-light microscopy methods: orthoscopic observation, in which the image plane is viewed, and conoscopic observation, in which a specimen-specific optical property is observed.

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7-6 Darkfield microscopy The specimen is illuminated obliquely using a dedicated condenser and the light scattered by the specimen is observed. In contrast to brightfield microscopy, the background of the image is dark, since the illumination light does not enter the objective directly. The illumination is in the form of a hollow cone of light, and the NA of the condenser must be higher than the NA of the objective. A: Light path with maximum numerical aperture B: Light path with minimum numerical aperture C: Light path with numerical aperture of objective Selecting an objective The numerical apertures of a darkfield condenser for oil-immersion use range from 1.201.43. For darkfield microscopy, the numerical aperture of the objective must be less than the condensers minimum numerical aperture of 1.20, so that illumination light does not enter the objective directly. Hence, either an objective with a numerical aperture of less than 1.20 or an objective that is equipped with a diaphragm will be used. Objectives that are equipped with a diaphragm have a numerical aperture that can be adjusted within the range 0.501.30. Similarly, since dry condensers have numerical apertures in the range 0.800.95, only objectives with a numerical aperture of less than 0.80 can be used with them. 7-7 Modulation contrast microscopy In addition to differential interference microscopy and phase contrast microscopy, modulation contrast microscopy can also be used for observing colorless and transparent living cells and bacteria. The basic optical system resembles the system for phase contrast microscopy. However, it produces a pseudo-relief image that yields an asymmetrical transmission distribution at the pupil. This method is specifically used for the sperm and egg cell specimens used in in-vitro-fertilization, and which are usually conducted in plastic containers. Plastic containers cannot be used for DIC microscopy, and since fertilization operations require three-dimensional images, phase contrast microscopy cannot be used either.

Objective

A BC Specimen

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Like differential interference contrast microscopy, modulation contrast microscopy produces a three-dimensional view and allows the use of plastic containers. When this method is used, attention should be paid to the direction in which the contrast is applied.

7-8 Asbestos dispersion staining microscopy An asbestos microscope (or a dispersion microscope, also known as the Schlieren microscope after the principle on which it is based) is used for observation of asbestos. Virtually transparent, the asbestos is optically stained by utilizing its dispersion characteristic. Illumination is carried out using the ring diaphragm on a phase contrast microscope, and the objective incorporates a shielding plate instead of a phase plate. This arrangement yields a darkfield effect, with the phase object brightly visible. Select the immersion liquid (which contains a specimen) that has a different dispersion curve (which shows how the refractive index varies with wavelength) to the dispersion curve of the specimen (the phase object). Then exclude the light on the wavelength where the dispersion curve for the specimen and that for the liquid intersect from the light passing through the specimen, and capture the remaining dispersion light. Observation of the specific dispersion colors, which can be observed by varying the immersion liquid, enables the asbestos type (or other substance) to be identified.

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Samples from an asbestos microscopy are shown below. White asbestos (chrysotile) Brown asbestos (amosite) Blue asbestos (crocidolite) Immersion liquid: nD = 1.550 Immersion liquid: nD = 1.680 Immersion liquid: nD = 1.680

7-9 Stereoscopic microscopy With an ordinary microscope it is not possible to observe a sample in three dimensions easily. Observing in three dimensions requires changing the angle of observation and viewing the specimen from at least two different directions. The stereoscopic microscope enables binocular stereo vision by incorporating two angled light paths. The greater the convergence angle, the more pronounced the three-dimensional effect. There are two types of stereoscopic microscope: the Greenough type (features a convergent optical system) and the parallel-optics type. The former is usually a lower-grade model, while the latter is usually a higher-grade model.

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7-10 Cell culture observation systems There have been various problems with the traditional method of removing a specimen container from an incubator and placing it on a microscope for observation, such as variation in temperature and other changes in environment, the stress on cells due to vibration when the specimen is moved, contamination, and the difficulty of repeatability of the observation position. With a cell culture observation system, it is possible to observe samples and to record images of them according to a preset schedule in a culture environment. In general, the culture environment must meet the following conditions: Cell culture environment Temperature: 37C Humidity: 90% CO2: 5%

BioStation CT

Observation section

Operations screen

BioStation IM

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8. Adjustment Method and Use of Accessories

8-1 Alignment of mercury lamp A mercury lamp can be aligned as follows. When fluorescence microscopy is carried out, there can be insufficient brightness or uneven intensity of the fluorescence principally caused by the lamps alignment. High-intensity lamphouse part names

Arc image adjustment knobs Horizontal lamp movement

Vertical lamp movement Lamp focus adjustment Fixing screw

Mirror image adjustment knobs Mirror diagonal movement Mirror focus adjustment Mirror diagonal movement

Notes: Arc image adjustment knobs: When the arc image adjustment knobs are used to move the lamp or adjust the focus, the mirror image will also move. Mirror image adjustment knobs: These knobs only move the mirror image.

Centering tool (for inverted microscope)

Mercury lamphouse

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Adjustment sequence (1) Fit the centering tool onto the revolver. (2) Turn on the power to the mercury light source. (3) Insert FITC or TRITC fluorescence filters into the light path for better image

visibility. (4) Locate the arc image (a real image) at the center of the field of view using the

arc image adjustment knob. (5) Locate the mirror image and real arc image side-by-side as shown in Figure 2

below using the mirror image adjustment knob and the arc image adjustment knob, and then focus the two images.

(6) Turn the arc image horizontal adjustment knob so that two images overlap exactly, as shown in Figure 4.

(7) Fix the knob used to adjust the arc image focus in place using the screw.

Arc image (bright and clear in shape) Mirror image

Figure 1 Figure 2

Figure 3 Figure 4

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8-2 Adjusting the Khler illumination (by adjusting the field diaphragm and aperture diaphragm) The Khler illumination method is a means of obtaining uniform illumination without any unevenness on the specimen plane by adjusting both the field diaphragm and the aperture diaphragm. Adjustment sequence

(1) Put the specimen on the stage and focus on it using a 10 objective. (2) Close the aperture diaphragm and field diaphragm. (3) Raise or lower the condenser to focus the field diaphragm image on the

specimen plane. The edge of the diaphragm will appear blue or red depending on the focus position. Set the focus position so that the color is neutral.

(4) Center the field diaphragm image within the field of view. Adjust the position of the image using the two condenser-centering screws (alignment screws) on the condenser holder.

(5) Open the field diaphragm so that the diaphragm circumscribes the field of view. If the field diaphragm is opened wider than is necessary, excess light will hit the inside edges of the objective, causing flare.

(6) Open the aperture diaphragm to approximately 70%80% of the numerical aperture of the objective.

Appearance of field diaphragm

Initial appearance Centered image

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8-3 Diopter adjustment for the eyepiece lens Since virtually everyone has a different diopter in each eye, it is necessary to adjust the diopter before using the microscope so that both eyes can focus at the same time. Ideally, the system has a diopter adjustment that can be operated separately for each eye. Some microscopes allow diopter adjustment on each tube of the eyepiece, while others allow adjustment on one tube only. Adjustment sequence when there are adjustment rings on both eyepiece tubes

(1) Turn the diopter adjustment ring on the eyepiece lenses until the ruling line aligns with the end surface of the diopter adjustment ring. (Diopter correction is 0)

(2) Turn the nosepiece and focus on the specimen using a 40 objective. (3) Next, switch to the 4 or 10 objective and turn the diopter adjustment ring on

each eyepiece lens so that the sample is properly focused. (4) For even better results, perform the entire operation a second time.

Adjustment sequence when there is only an adjustment ring on one eyepiece tube

(1) Turn the nosepiece and focus on the specimen using a 40 objective through the eyepiece lens without an adjustment ring.

(2) Next, set the objective to 4 or 10 magnification and turn the diopter adjustment ring so that the sample is properly focused.

8-4 Adjusting the correction ring on the objective Objectives are designed to achieve their optimum optical performance with cover slips that are 0.17mm thick. However, since there is variation in the thickness of cover slips, high-magnification objectives are equipped with a mechanism to correct for this. Depending on the numerical aperture of the objective, the cover slip thickness variation can cause major deterioration in image quality. Please refer to 2-5 Cover slip standards.

Ruling line

Adjustment ring

Eyepiece lens with diopter adjustment ring

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Adjustment sequence (1) Set the correction ring to the

0.17 position. (2) With the aperture diaphragm fairly

open, focus the lens on a minute object in the specimen.

(3) Turn the correction ring very slightly, refocus the lens, and check to see whether the image has improved or deteriorated.

(4) Repeat the above steps and assess whether you are currently turning the ring in the right direction to improve the image, or whether it is deteriorating.

(5) If the image quality is deteriorating, repeat the above steps, turning the correction ring in the opposite direction, and find the position which yields the best possible resolving power and contrast.

8-5 Checking and adjusting the cross-Nichols for differential interference contrast microscopy The differential interference contrast method is an alternative to the phase contrast method for observing unstained living cells. To obtain clear differential interference contrast images, it is essential to adjust the polarizer and the analyzer together with the Khler illumination, and select the differential interference contrast prism that best suits each objective.

Adjustment sequence

(1) Set up the microscope for normal brightfield observation. (2) Turn the nosepiece, set a 10 objective in place, and focus on the specimen. (3) Switch to a 40 objective. (4) If a DIC prism is installed at the bottom of the objective, remove it. (5) Insert the analyzer and the polarizer into the light path. (6) Remove the specimen. (7) Loosen the rotation screw on the polarizer and rotate 1/4 wave plate so as to

align the marking for center position with the arrow. Analyzer (for upright microscope) Polarizer (for upright microscope)

Rotation knob and rotation fixing screw

Align arrow

Polarizer fixing screw

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For an inverted microscope (8) Turn the dial below the binocular eyepiece tube to change the setting from O

(observation) to B (Bertrand lens) and adjust the focus. (9) Fully open the aperture diaphragm, rotate the polarizer-equipped condenser,

and keep making adjustments until a dark cross appears, as shown in the figure below.

For an upright microscope

(8) Remove the eyepiece lens and mount a centering telescope. Close the aperture diaphragm to a minimum and rotate the helicoid on the centering telescope to focus on the image of aperture diaphragm.

(9) Open the aperture diaphragm to a maximum and rotate the polarizer. Keep making adjustments until a dark cross appears, as shown in the figure below.

(10) Fix the polarizer position using the rotation fixing screw. (11) Install the DIC prism at the bottom of the objective.

Dark cross

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8-6 Centering of the objective Polarization color and retardation are measured with a polarized-light microscope using a rotating stage. However, the center of rotation of the stage and the center of the objective is not always properly aligned. If they are used while misaligned, the center of image will move and be difficult to observe when rotating the stage. Thus, it is necessary to align the center of each objective using the nosepiece's eccentricity correction mechanism. Adjustment sequence

(1) Set up the microscope for normal brightfield observation. (2) Set a 10 objective in place. (3) Focus on the specimen. (4) Position the intersection of the crossed hairs in the eyepiece lens over a

landmark object in the specimen. (5) Insert the dedicated centering tools provided with the nosepiece into the

nosepieces centering screw holes.

(6) Rotate the stage through half a turn (180) and use the nosepiece centering tool

to move the objective so that the center of the cross moves midpoint of the target object's travel distance.

(7) After this, move the specimen and position the target object under the center of the crossed lines.

(8) Repeat the above steps several times. Do the same for each objective.

Turn the tools from both sides of the objective and adjust the position of the objective.

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8-7 Adjusting the position of the phase ring The phase contrast method is the most common method for observing unstained living cells. To obtain clear phase contrast images, it is essential to adjust the phase ring and to accurately adjust the Khler illumination. If the condenser sections ring diaphragm and the objectives phase plate are misaligned, light will escape (as shown in the figure below), making it impossible to observe phase contrast images properly.

Adjustment sequence

(1) Set up the microscope for normal brightfield observation. (2) Set a 10 objective in place. (3) Rotate the condenser turret to line up with the PH1 mark. (4) Turn the coarse-motion handle and raise the stage as high as it will go. (5) Remove either of the eyepiece lenses. (6) Insert a centering telescope in place of the removed eyepiece lens. (7) While looking through the centering telescope, turn the focusing helicoid and

images of the phase plate and the condenser annulus can be seen clearly. (8) Turn the two condenser annulus centering dials and check to see whether the

condenser annulus image moves. (9) Move the condenser annulus image until the center of phase plate and the

center of the condenser annulus image are perfectly aligned.

After adjustment Without adjustment

Position the condenser annulus image on the phase plate.

Condenser annulus

Phase plate

Centering dials

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8-8 Adjusting the darkfield condenser For darkfield microscopy, condenser adjustment is all-important. Since the field diaphragm cannot be seen (just as with brightfield adjustment) in the field of view, it is necessary to adjust the darkfield condenser by viewing the image of the specimen. Adjustment sequence

(1) Set a 10 objective in place. (2) Focus on the specimen. (3) Turn the condensers up-down handle so that a round black spot appears in the

field of view.

(4) Adjust the condenser centering dials so that the spot is centered in the field of view.

(5) Turn the condensers up-down handle and keep making adjustments so as to minimize the unevenness in the field of view.

Darkfield condenser Underside of darkfield condenser

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8-