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III Components, Microscope Setup December 2008

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1 III Components, Microscope Setup December 2008
Rudi Rottenfusser – Carl Zeiss MicroImaging

2 Categories 1 Stands, Base Plates 2 Stereo stands & Accessories
3 Fiber Optics Light Sources 4 Power Supplies 5 Lamp Housings, Coll., Sockets, Adapters 6 Bulbs, Arc Lamps, Burners 7 Inserts for Stands, Sliders 8 Filters, 42mm & 32mm diameter 9 Filters, 25mm & 18mm diameter 10 Filters, others 12 Condenser- and Illuminator Carriers 13 Condensers, Lenses, BF, DF, Ph, Pol, DIC 14 Stage Carriers, Stages, Specimen holders 15 Scanning stages, Spec.hldrs, Controllers 16 Cooling / Heating Stages & Accessories 17 Objectives 160mm 18 Objectives ICS (covered specimens) 19 Objectives ICS (for non-covered samples) 23 Objectives for SteMi's, Luminars 24 Objectives (Spc. e.g. Hoffmann; McCrone) 25 Nosepieces, Rings, Adapters 28 Compensators, 6x20mm-type Analyzers 30 Fluorescence Reflectors and Filter Sets 31 Other Sliders & Reflectors f/Infinity Space 32 Components f/ Analyzer slider receptacle 33 Intermediate tubes & modules 34 Randomizers, Tube Carriers, Tube Mounts 35 Tubes, Tube panels, Tube heads, Access. 38 Eyepieces & Projectives - ICS & Stereo 40 Eyepiece reticles, Micrometers 42 Adapters for Still and Video Cameras 43 Attachment Camera Systems 44 Digital High Resolution Camera Systems 45 Zeiss Video Camera Systems 47 Cases, Dust Covers, Cover Plates, Cables 49 Miscellaneous Micro Items In an attempt to find a structure into which all of the items of the microscopy products would fit, the above categories were established. You can find them in SCOPES, our quote writing program. The categories are but one way to search and find items. The sequence follows essentially the beam path on a microscope, from the illuminator towards eyepieces. In this tutorial we will touch on the categories which are showing in bold characters. Categories above 50 are used for various systems such as Confocals, Imaging etc. In this tutorial we will follow the beam path of a transmitted light microscope, and we discuss the hardware options in a very basic fashion. We will purposely NOT cover the principals of the Optical system. This will follow in the next tutorial, #5.

3 Topics The major Optical Components of the Microscope Light Sources
“Natural” Tungsten, Halogen Arc Lamp LED Condenser Resolution, Numerical Aperture Objective (more details in part 4) Eyepiece Useful Magnification Markings Parfocal Setting

4 Topics Setting up the Microscope for optimal Performance “Contrast”
Basic Setup for Brightfield Koehler Illumination Conjugate Image Planes on Microscopes

5 Cross-section through an ∞ corrected Microscope

6 Light Source for a typical Laboratory Microscope (late 1800’s to mid 1900’s)
To start out with, we will need a source of illumination. Long before electricity was invented, people already used microscopes, and the source was the sun, of course. Actually, a cloudy sky is better because it provides for perfectly even illumination of the condenser aperture. Daylight has another advantage besides its uniformity – its spectrum is shifted more towards blue than artificial light. This helps to resolve details as we will learn later. If you ever come across one of these old microscopes, don’t look down on them. If they were well cared for, the optical performance may rival the performance of a modern microscope! This applies to observations in Brightfield, Oblique, Darkfield, Pol, the only illumination variations available before the introduction of phase contrast microscope in With the invention of the light bulb, external illuminators which contained a tungsten bulb, were adapted to the mirror which had to be carefully aligned to reflect the source towards the center of the optical axis. Later, built-in illuminators made life easier. 1,000’s of the famous Zeiss Standard microscope which was sold between 1955 and 1980, are still in use. Most of them were equipped with a pre-aligned 6V, 15W tungsten filament lamp. In the 80’s, tungsten halogen bulbs began to replace the older tungsten lamps. A brief summary about the various sources follows in the next slide > Perfect even illumination Perfect Color Temperature (“Daylight”) Evenings? Nights? Intensity?

7 Artificial Light Sources (incoherent)
Tungsten Tungsten Halogen Common Light Sources for microscopes are: Tungsten > During the lifetime of a tungsten bulb, it changes its spectral output as it gets older, due to deposits of tungsten inside the bulb. Tungsten lamps have been universally replaced by Tungsten Halogen Lamps > The major advantage of a halogen lamp over regular tungsten is that its spectral output never changes. It is made of fused quartz and filled with inert gases. The halogen reacts with the tungsten deposit to produce tungsten halides, which break down when they reach the hot filament. The breakdown releases tungsten back to the filament—known as the tungsten-halogen cycle—and maintains a constant light output over the life of the lamp. Halogen lamps are either simple bulbs or bulbs mounted in reflectors. These are used mostly for fiber optics light sources. LED’s > Due to the advances in LED technology, these bluish-white sources with a lifetime of 100,000+ hours, are the wave of the future. They produce very little heat and don’t change their spectral output even when they are dimmed down. They will make their debut into fluorescence microscopy, with a patented new Zeiss Source named “Colibri”, to be introduced in 2007. Mercury arc > These “bulbs” are often called “burners”, because they are very hot and very bright at the center of the arc. The emitted light is not a continuous spectrum, but it has distinctive peaks, the best known probably being at 546nm in the green. Because of its intensity, it has become the standard for excitation of Fluorescence. The X-cite is also a Mercury arc lamp but it is combined with other gases for a more even light output. It is merged with a reflector to form one unit and delivers its intense light via a liquid fiber cable. Xenon arc > Xenon burners work very similar to mercury burners, but they are not quite as bright. Their spectrum, however, is different, and more even than that of mercury. Lasers > are the sources for our LSM Confocal Microscopes LSM, and also for TIRF Total Internal Reflectance Fluorescence. They are extremely bright and by design only emit at one specific wavelength at a time.

8 Tungsten – Halogen Principle

9 Tungsten-Halogen Lamp
Visible Light Inexpensive Easy to replace Temporally Stable Spacially Stable No change of Spectral Output during Life Low UV output High IR output

10 Arc Lamps Tungsten Tungsten Halogen Mercury Arc Xenon Arc
Common Light Sources for microscopes are: Tungsten > During the lifetime of a tungsten bulb, it changes its spectral output as it gets older, due to deposits of tungsten inside the bulb. Tungsten lamps have been universally replaced by Tungsten Halogen Lamps > The major advantage of a halogen lamp over regular tungsten is that its spectral output never changes. It is made of fused quartz and filled with inert gases. The halogen reacts with the tungsten deposit to produce tungsten halides, which break down when they reach the hot filament. The breakdown releases tungsten back to the filament—known as the tungsten-halogen cycle—and maintains a constant light output over the life of the lamp. Halogen lamps are either simple bulbs or bulbs mounted in reflectors. These are used mostly for fiber optics light sources. LED’s > Due to the advances in LED technology, these bluish-white sources with a lifetime of 100,000+ hours, are the wave of the future. They produce very little heat and don’t change their spectral output even when they are dimmed down. They will make their debut into fluorescence microscopy, with a patented new Zeiss Source named “Colibri”, to be introduced in 2007. Mercury arc > These “bulbs” are often called “burners”, because they are very hot and very bright at the center of the arc. The emitted light is not a continuous spectrum, but it has distinctive peaks, the best known probably being at 546nm in the green. Because of its intensity, it has become the standard for excitation of Fluorescence. The X-cite is also a Mercury arc lamp but it is combined with other gases for a more even light output. It is merged with a reflector to form one unit and delivers its intense light via a liquid fiber cable. Xenon arc > Xenon burners work very similar to mercury burners, but they are not quite as bright. Their spectrum, however, is different, and more even than that of mercury. Lasers > are the sources for our LSM Confocal Microscopes LSM, and also for TIRF Total Internal Reflectance Fluorescence. They are extremely bright and by design only emit at one specific wavelength at a time.

11 Arc Lamps

12 Arc Lamps HBO 100 XBO 75W Metal Halide Courtesy – Michael Davidson

13 Light Sources Tungsten Tungsten Halogen Mercury Arc Xenon Arc LED’s
Common Light Sources for microscopes are: Tungsten > During the lifetime of a tungsten bulb, it changes its spectral output as it gets older, due to deposits of tungsten inside the bulb. Tungsten lamps have been universally replaced by Tungsten Halogen Lamps > The major advantage of a halogen lamp over regular tungsten is that its spectral output never changes. It is made of fused quartz and filled with inert gases. The halogen reacts with the tungsten deposit to produce tungsten halides, which break down when they reach the hot filament. The breakdown releases tungsten back to the filament—known as the tungsten-halogen cycle—and maintains a constant light output over the life of the lamp. Halogen lamps are either simple bulbs or bulbs mounted in reflectors. These are used mostly for fiber optics light sources. LED’s > Due to the advances in LED technology, these bluish-white sources with a lifetime of 100,000+ hours, are the wave of the future. They produce very little heat and don’t change their spectral output even when they are dimmed down. They will make their debut into fluorescence microscopy, with a patented new Zeiss Source named “Colibri”, to be introduced in 2007. Mercury arc > These “bulbs” are often called “burners”, because they are very hot and very bright at the center of the arc. The emitted light is not a continuous spectrum, but it has distinctive peaks, the best known probably being at 546nm in the green. Because of its intensity, it has become the standard for excitation of Fluorescence. The X-cite is also a Mercury arc lamp but it is combined with other gases for a more even light output. It is merged with a reflector to form one unit and delivers its intense light via a liquid fiber cable. Xenon arc > Xenon burners work very similar to mercury burners, but they are not quite as bright. Their spectrum, however, is different, and more even than that of mercury. Lasers > are the sources for our LSM Confocal Microscopes LSM, and also for TIRF Total Internal Reflectance Fluorescence. They are extremely bright and by design only emit at one specific wavelength at a time.

14 LED Light Sources

15 LED Light Sources Long life (10,000+h?) Stable Output over time
Clean spectrum Cool No lamp alignment No need for shutter – no vibration Quick switching

16 Colibri Fast Switching FL Source

17 Light Sources Tungsten Tungsten Halogen Mercury Arc Xenon Arc LED’s
Laser (coherent) Common Light Sources for microscopes are: Tungsten > During the lifetime of a tungsten bulb, it changes its spectral output as it gets older, due to deposits of tungsten inside the bulb. Tungsten lamps have been universally replaced by Tungsten Halogen Lamps > The major advantage of a halogen lamp over regular tungsten is that its spectral output never changes. It is made of fused quartz and filled with inert gases. The halogen reacts with the tungsten deposit to produce tungsten halides, which break down when they reach the hot filament. The breakdown releases tungsten back to the filament—known as the tungsten-halogen cycle—and maintains a constant light output over the life of the lamp. Halogen lamps are either simple bulbs or bulbs mounted in reflectors. These are used mostly for fiber optics light sources. LED’s > Due to the advances in LED technology, these bluish-white sources with a lifetime of 100,000+ hours, are the wave of the future. They produce very little heat and don’t change their spectral output even when they are dimmed down. They will make their debut into fluorescence microscopy, with a patented new Zeiss Source named “Colibri”, to be introduced in 2007. Mercury arc > These “bulbs” are often called “burners”, because they are very hot and very bright at the center of the arc. The emitted light is not a continuous spectrum, but it has distinctive peaks, the best known probably being at 546nm in the green. Because of its intensity, it has become the standard for excitation of Fluorescence. The X-cite is also a Mercury arc lamp but it is combined with other gases for a more even light output. It is merged with a reflector to form one unit and delivers its intense light via a liquid fiber cable. Xenon arc > Xenon burners work very similar to mercury burners, but they are not quite as bright. Their spectrum, however, is different, and more even than that of mercury. Lasers > are the sources for our LSM Confocal Microscopes LSM, and also for TIRF Total Internal Reflectance Fluorescence. They are extremely bright and by design only emit at one specific wavelength at a time.

18 Lamp Housings and its optical components
100W, 35W Halogen 100W HBO 75W Xenon Colibri (4 LED + HBO) Collector Fixed Focusable, 3-lens or aspheric Heat Filter Heat absorbing or reflecting Light sources emit not only the light which is desired, but also heat, which is generally undesirable for “life” specimens and polarizers in the microscope. That’s why every lamp housing now is equipped with either a heat absorbing or heat reflecting filter. For special applications such as IR the heat filter can be removed, but then it’s important to keep a watchful eye on all of the components of the microscope’s illumination path to prevent excessive heat build-up. Light sources, other than lasers or diodes, emit light in all directions. To harness their light for the microscope, a collector is used. Its name is very appropriate because it “collects” the available light and sends it towards the sample. Two types of collectors are used in Zeiss microscopes, spherical and aspherical types. Traditional spherical three-lens collectors are incorporated in the large Mercury or Xenon lamp housings, and aspherical single-lens collectors are used in the 100W Halogen lamp housing and the 50W Mercury housing. In addition to collectors, concave mirrors behind the lamps send the light rays back towards the collector and almost double the light output of the system.

19 Cross-section through an ∞ corrected Microscope

20 Components between Light Source and Specimen
Internal Light Path incorporates: Transmitted Light: Light Shutter Filter Turret or Filter Slider with Neutral Density or Color Filters Luminous Field Diaphragm Reflected (Incident) Light: Light Shutter Filter Turret or Filter Slider with Neutral Density, Color Filters, Attenuator Aperture Diaphragm Luminous Field Diaphragm Light sources emit not only the light which is desired, but also heat, which is generally undesirable for “life” specimens and polarizers in the microscope. That’s why every lamp housing now is equipped with either a heat absorbing or heat reflecting filter. For special applications such as IR the heat filter can be removed, but then it’s important to keep a watchful eye on all of the components of the microscope’s illumination path to prevent excessive heat build-up. Light sources, other than lasers or diodes, emit light in all directions. To harness their light for the microscope, a collector is used. Its name is very appropriate because it “collects” the available light and sends it towards the sample. Two types of collectors are used in Zeiss microscopes, spherical and aspherical types. Traditional spherical three-lens collectors are incorporated in the large Mercury or Xenon lamp housings, and aspherical single-lens collectors are used in the 100W Halogen lamp housing and the 50W Mercury housing. In addition to collectors, concave mirrors behind the lamps send the light rays back towards the collector and almost double the light output of the system.

21 Cross-section through an ∞ corrected Microscope

22 The Condenser Why do we need a condenser and what does it essentially do? >

23 (minimum resolved distance between two points):
Resolution (minimum resolved distance between two points): Without Condenser: Objective Specimen Specimen b With Condenser: It provides illumination, ideally up to the same angle (NA) as the objective, which means that it will help us to see details which are only ½ the size as before, without condenser. Of course, it also provides for many contrasting techniques besides brightfield, but these issues will be covered in later modules. Condenser When Condenser NA matches Objective NA Highest Resolution !

24 The Objective More details later… Why do we need a condenser?
The objective is the microscope’s most important optical element. The final image quality is always limited by the quality of the objective. No other elements which follow will be able to improve a mediocre image from the objective! To provide for an image which comes as close as possible to the way the object looks like, the objective doesn’t look even close to the single bi-convex lenses we have been looking at for the purpose of understanding optics. A fair amount of individual lenses may be needed to produce the best image possible. The example on the next slide is a cross-section through a Plan-Apochromat > Why do we need a condenser?

25 NA = sin a · n Numerical Aperture (NA) b a c  Refractive Index n
90° a c  Refractive Index nair = 1 nwater = 1.33 nglycerin = 1.47 noil = 1.518 The Numerical Aperture, that number behind the magnification of the objective, is the most important number associated with the objective. It indicates how small of a detail the objective can resolve. The NA is directly related to the angle of the cone which is formed between the detail on the specimen and the periphery of the front lens. Please follow the animation on the drawing to see how it is defined. The sine is nothing else but a numerical expression of an angle. Some of you may remember the term “sohcahtoa” from Geometry class refering to sine, cosine and tangent functions. “soh” stands for “opposite over hypotenuse”. At angle “alpha=0”, the sine is “0” because line “b, opposing the angle alpha, is “0”. As the angle approaches 90 degrees, “b” will be approaching the size of the hypotenuse “c”, this means that the sine of 90 degrees is “1”. It can never get any larger than 1, because when you follow the circle, after 90 degrees the ratio between b and c will get smaller again… As Ernst Abbe determined, the resolution of the microscope is directly related to the Numerical Aperture – the larger the angle, the smaller the details which can be resolved. To go past NA 1, one can accomplish this by applying a medium between specimen and objective, which has a larger refractive index than air (1), as we see in the next slide > n How is a sine function defined ?

26 Why immersion medium affects NA
Plan-Apochromat 40x/0.95 corr.   Plan-Apochromat 100x/1.46 Oil   No Oil “Dry” a1 a2 Oil Immersion 2 1 3 Objective Cover Slip + slide Again, please follow the animation. Let’s compare two Zeiss objectives with the highest possible acceptance angles into the front lens (72-74 degrees =~146 degrees for the total cone angle). Both objectives happen to be Plan-Apochromats. No immersion: On the left side, the ray path of a “dry” objective is coming into play. The rays from the specimen, a red dot, go through the cover slip and at the interface between the cover slip and air. they are deflected away from the normal, according to the laws of refraction. At ~72o, which corresponds to 0.95 NA, the ray will still be able to enter the objective. Steeper rays don’t make it into the objective any more, and as the rays exceed the critical angle, they are totally reflected back down. Unfortunately, the actual angle alpha1 which contributes to the light as it is “gathered by the objective”, is smaller by the factor of the refractive index of air (1) over the refractive index of the cover slip (1.52). 1/1.52 = 0.62 > So the actual angle alpha1 which is captured from the specimen is only 39o. With oil immersion (n=1.518): The scenario with immersion oil is seen on the right side. Since oil has essentially the same refractive index as the cover slip, there is no “bending” of the rays taking place on top of the cover slip. Consequently, the objective “sees” the total angle . The given NA of the objective is based on its use with oil (1.46). So we can divide it by the refractive index of glass (1.52), arriving at 0.96 > This means that the actual angle alpha2, collected from the specimen, is 74o. Immersion Oil No immersion (dry) Max. Objective aperture ( = 72°) Captured Aperture of specimen below cover slip: 0.95/1.52 = 0.62 (1 = 39°) With immersion oil (3) n=1.518 No stray light, no total reflection ! Max. Objective aperture 1.46 (oil) Captured Aperture of specimen below cover slip: 1.46/1.52 = 0.96 (2 = 74°)

27 Cross-section through an ∞ corrected Microscope

28 Infinity Space Components in Infinity Space: DIC sliders
Compensator Sliders Fluorescence Filters Analyzer . The objective itself which follows the specimen in the light path, will be covered in detail during Module 6. With the exception of the special “fixed stage” microscope, the objective threads into a revolving nosepiece. A standard for the thread size was established by the Royal Microscoy Society in 1866(!), and it has been the same ever since, hence the “RMS thread” or “society thread”. It has a diameter of 0.8” and a pitch of 1/36”. Nosepieces on Zeiss microscopes used to be equipped with these threads. The need to use larger diameters was brought out primarily in industrial applications when “Darkfield” through the objective required a larger diameter. All Zeiss research microscopes have now consistent 27mm thread nosepieces to accommodate any objective of our program. Simple intermediate rings adapt the RMS objectives to M27. At the point where the objective threads into the nosepiece, the “infinity space” begins. Many components can be added to the infinity space. Depending on how versatile the microscope system is, up to 4 different levels incorporate receptacles which can be utilized for components. Level 1 – in the nosepiece itself –Neutral Density Filter Sliders, making it possible to equalize intensities between objectives, DIC Sliders, for DIC techniques Level 2 – 6x20mm receptacle for compensator sliders which are needed in Pol microscopy, or for a simple polarizer or 18mm filter holder Level 3 – Filter turret – it can be outfitted with combinations of Fluorescence Filter Sets, Analyzers for Transmitted and/or reflected light, a Bertrand lens to observe the back focal plane of a 50x Pol objective, magnification changers (Optovar) 1.25, 1.6, 2.5x Level 4 – a receptacle for a large filter slider or various analyzer sliders. Since all of these receptacles are in the infinity space, none of the inserted components will deteriorate the image or cause any image shift, as long as the inserts are plane-parallel. Requirement for co-localized Images: Components need to be plane-parallel ! Infinity System Specimen off-center

29 Cross-section through an ∞ corrected Microscope

30 Intermediate Tubes and Tubes
We have now arrived at the top of the microscope stand. In most cases, the viewing tube will be mounted at this point. The first element of the viewing tube is the “tube lens” which will now bring the image back from infinity into the front focal plane of the eyepiece, the intermediate image plane. Zeiss tube lenses have a focal length of 164.5mm. For additional flexibility, “intermediate tubes” are available which go between the top of the microscope stand and the viewing tube. It is important to remember that all intermediate tubes come equipped with tube lenses. So it will be necessary to remove the tube lens of the viewing tube, otherwise two tube lenses would be in play! Intermediate tubes are available to have Tube Lens Turrets 1.6x, 2.5x, 4x magnification + Bertrand lens, and for multi-discussion, where as many as a dozen additional binoculars may be “daisy-chained” for teaching purposes. Tube Lens Turret with up to 3 tube lenses in addition to standard 1x, such as 1.25x 1.6x 2.5x 4.0x

31 Tube Mounts – Upright Microscopes
Primostar Axiostar / Standard Line Axiostar tubes fit old (160) Zeiss microscopes, converting them to Infinity Optics ! Standard, GFL, RA, WL, ACM can get “upgraded”! Old (finite) condensers work with new objectives! No upgrade to infinity possible for Universal, Photomicroscope, Ultraphot or UEM Axioskop 1, Axioskop 40, Axioskop 2FS, Axioplan, Axiophot Forward and Backward Compatibility between c) and d) via tube adapters! Axioplan 2, 2i, 2ie AxioImager A1, D1, M1, Z1 Stereo Microscopes SV6, SV11, SR, SV8 SteReo Discovery, Lumar There are 7 different tube mounts for Zeiss Upright microscopes. The Axiostar has the same size mount as the old style 160mm Standard microscopes, therefore one can upgrade a microscope to infinity optics from as far back as 1955 (the GFL) ! No other company offers so much flexibility ! Tubes for the larger Axio-type research microscopes prior to the Axio Imager can be interchanged. Because of the current Axioskop 40 and Axioskop 2 FS microscopes, any tube for them will fit directly on the Axioskop 1, Axioskop 2, Axioplan 1, Axiophot, and via adapter plate on the Axioplan 2, 2i, 2ie. This concludes Module 4

32 Binocular Tubes (example - Axio Imager)
Note: Tube Lens always included All Zeiss tubes can be folded up or down Optimum angle for most comfortable viewing: 15-20º Finally, we have arrived at the place where we can look into the microscope. All tubes nowadays are “binocular” which makes it more comfortable for the eyes. Ergonomy is another issue. It is known today that a viewing angle of about 20 degrees results in minimum stress for the neck muscles. As shown in the diagram, many interchangeable tubes are available, so one can choose the most useful one for the applications. Noteworthy are tubes which allow for vertical and horizontal adjustments, to provide the most comfortable position for the microscope user. Some tubes deliver a side-correct and upright image which is very useful if the operator needs to manipulate samples by hand under the microscope. All tubes are designed according to the “Siedentopf” principle which means that there is no change in the optical path when the Interpupillary Distance (IPD) is changed by the operator. And, remember to remove the tube lens if an intermediate tube is added.

33 Dual Video Adapter 2 camera ports with 60mm interface – one is adjustable (x, y, z) interchangeable beam splitting cube for neutral or spectral image or signal splitting We have now arrived at the top of the microscope stand. In most cases, the viewing tube will be mounted at this point. The first element of the viewing tube is the “tube lens” which will now bring the image back from infinity into the front focal plane of the eyepiece, the intermediate image plane. Zeiss tube lenses have a focal length of 164.5mm. For additional flexibility, “intermediate tubes” are available which go between the top of the microscope stand and the viewing tube. It is important to remember that all intermediate tubes come equipped with tube lenses. So it will be necessary to remove the tube lens of the viewing tube, otherwise two tube lenses would be in play! Intermediate tubes are available to have Tube Lens Turrets 1.6x, 2.5x, 4x magnification + Bertrand lens, and for multi-discussion, where as many as a dozen additional binoculars may be “daisy-chained” for teaching purposes. attaches to all camera ports with 60mm interface

34 Camera Adapters for 60mm Interface
C-mount Adapters 1x, 0.63x, 0.5x, 0.4x, Zoom T2-mount Adapters 1x, 1.6x, 2.5x, 4x ENG-mount Adapters 1x, 0.8x Eyepiece tube (for digital cameras) Adapter for Digital Cameras with built-in objective (37 and 52mm thread diameters) Adapters to fit the standard 60mm interfaces of the Axiovert 200 are available for a variety of different mounts: C-mounts (most video and many digital cameras) T2-mounts (Single lens reflex cameras will fit to microscope, if a camera-specific T2-mount is supplied) ENG-mounts (Broadcast type cameras, two different mount sizes) Cameras with built-in objective – requires interface to microscope which projects specimen image to infinity

35 Cross-section through an ∞ corrected Microscope

36 Who needs eye-pieces any more?
Eyepieces (Oculars) Who needs eye-pieces any more? Field of View “Presence” Detect fine nuances in color shades Stereo Dynamic Range of the Eye After discussing the role of objective and condenser, we finally arrive at the eyepiece. Who needs an eyepiece any more, with all of the modern imaging possibilities? As it turns out, even with the best imaging systems and monitors, microscopists generally agree that the image as it is seen in the eyepiece, has a “presence” which cannot be duplicated by other means. For this reason, pathologists like to set up multiple sets of co-observation tubes for teaching. Every students can see the same image through “his” eyepieces, for discussions about fine nuances in detail and color. So, what is there to know about eyepieces?

37 Magnification In order to see small objects with the eye the small objects must be magnified to an appropriate size In order to clearly see small objects with the eye they must be magnified to an appropriate size. These 3 images with different enlargements of just one region clearly illustrate the effect of increasing magnification on how cellular details in this cross-section of the embryo of a young mouse can be recognized.

38 Useful Magnification Rule of Thumb:
Limitation #1 – The eye You will miss fine nuances in the image if the objective projects details onto the intermediate image, which are smaller than the resolving power of your eye (typical at low magnification / high NA) Limitation #2 – The microscope You will reach “empty magnification” if you enlarge an image beyond the physical resolving power of the optics. Since the eyepiece produces the second stage of magnification in our microscope, it should magnify the image sufficiently to allow our eyes to see all of the details which the objective provides in the intermediate image plane. The ideal lower and upper ideal eyepiece magnifications are expressed in the term “Useful Magnification Range”. This range applies not only to eyepieces, but in general to any magnified image. Here are the limitations: Our eyes are limited by the size of our rods and cones in the retina. We will miss available information if the objective projects these details at a size which is smaller than the resolving power of our eye (typical at low magnification / high NA) The microscope optics has a given resolution (Abbe’s equation). “Empty magnification” will be reached if we enlarge an image beyond the physical resolving power of the optics. To stay within these limits, consider the simple rule of thumb: The Total Magnification should be between 500 – 1000x NA of the objective ! While eyepieces are available in different magnifications, only few applications nowadays call for eyepieces other than “10x”, generally considered a good compromise. Besides, there is an optical device available for all current microscopes, called an “Optovar”. The Optovar which could be part of an intermediate tube, or a “push and click type” system which fits in the fluorescence filter turret or slider. It is available in magnifications of 1.25x, 1.6x, 2.5x on any Zeiss microscope, up to 4x on the Axio Imager. In the days before infinity optics and Optovars, quite a number of eyepieces with different magnifications were available. For convenience, they could even be arranged on a turret, just like the objectives. A typical arrangement might be 6.3x, 8x, 10x and 12.5x eyepieces mounted in the turret. > Rule of Thumb: Total Magnification of an image to the eyes should be between 500 and 1000 times the objective’s Numerical Aperture

39 1939

40 Eyepiece Characteristics
Example: W PL 10x/23 Foc. W Wide Angle PL Flat Field (“Plan” > old style: “KPL” or “CPL”) 10x Magnification 23 Field of View diameter in mm Foc Focusable Another characteristic of an eyepiece is its Field of View Number (fov#). It refers to a diaphragm which restricts the diameter of the intermediate image inside the eyepiece. It is expressed in mm. So, an eyepiece labeled 10x/23 has a field of view diameter of 23mm. If you wish to know how much of the specimen you can see in the eyepiece, you divide the objective (+Optovar) magnification into the fov#. Example 1: Eyepiece fov# = 23mm, objective 20x > Visible object diameter = 23mm / 20 = 1.15mm = 1150 µm. Example 2: Eyepiece fov# = 18mm, objective 50x, Optovar 1.6x > Visible object diameter = 18mm / (50x 1.6x) = 18,000µm / 80 = 225 µm Plan-Apochromats are corrected for a field of view up to 25mm. Less expensive objectives are generally corrected for a smaller field of view, hence it is recommended to use the less expensive objectives with eyepieces which have fov#’s 23mm, 20mm or 18mm. On the Zeiss Optics website, under “Description of Classes of Objectives”, these limits are publicized. In general, 23mm is considered an overall comfortable field size for viewing.

41 Eyepiece Reticles Useful for: Centering Stage (Pol) Counting
Measuring distances, circles Discussions (movable pointers) Setting of Parfocality Eyepieces may be equipped with reticles for measurement, marking, counting etc. These glass discs may have fine micrometers, cross lines, squares, pointers and many more special designs printed on them. The most popular reticles are available through Zeiss and, when ordered together with the whole system, are usually factory installed. Custom-built reticles are commercially available and are quite easy to retrofit by unscrewing the bottom ring of the eyepiece, dropping the reticule into a receptacle and screwing the ring back on. The reticle diameter for all current Zeiss eyepieces, no matter what their fov# is, is 26mm . One of more popular US supplier of reticles is “Klarmann Rulings, Inc.” (www.reticles.com). They have a big stock, ship fast and support their products well.

42 Go to highest magnification possible with your system 2
Setting your microscope to be “parfocal” Required: Two focusing eyepieces and/or focusing camera adapter 1 Go to highest magnification possible with your system 2 Focus carefully via focusing knobs 3 Go to the lowest magnification possible; leave focus alone 4 Refocus system with your two focusing eyepieces (or camera focusing adjustment) One of the first things everyone who sits down on the microscope should do: Set up the eyepieces for his or her eyes! This means the focusing sleeves have to be set properly. This setting is different for every individual! There are three different scenarios on how to do this If there is a properly attached digital camera on the system, this is easy: Go to a low-power objective, focus the image “live” on the monitor, and set each eyepiece until you see the image sharp through the eyepieces as well. If there is no camera on the microscope, but one of the focusing eyepieces has a properly installed reticle, make sure that the reticle is in sharp focus and set the other eyepiece accordingly. If there is neither a camera nor a reticle with the microscope, follow the described procedure on this slide. Afterwards, note the number on the focusing sleeve, so next time when you sit down on a microscope, you only have to dial in that specific number, and you won’t have to repeat the whole procedure. The same procedure will apply when you add a camera to the microscope which has a focusable adapter (i.e. Optronics). Always see to it that a microscope has two focusing eyepieces. Sometimes inexpensive scopes are equipped with one focusing eyepiece and one non-focusable one. This would only be ok if all of the operators of this microscope had 20/20 vision… So, what are we actually doing by focusing the eyepiece? We are setting it up that we are actually looking at the intermediate image which is located 10mm from the edge of the tube, within the eyepiece. Since the objectives + tube lens will produce a well corrected image at that intermediate plane, it means that in a plane other than the intermediate image the image is not well corrected any more. The first sign that “something is wrong” is lack of “parfocality”.

43 Questions? Short break?

44 0 Units 50 Units 100 Units C ONTRAST 50 Units 50 50 50 – 100 / = -0.33 50 – 0 / = 1 To understand “contrast”, you will find the word “contrast” being displayed over a background which changes gradually from “black” on the left side to “white” on the right side. The intensity of the letters which make up the word “contrast” is constant. Since it is somewhere between 0 (black) and a arbitrary number of 100 (white), let’s assume it is “50”. It is easy to see when we apply the formula for “contrast” that the letters on the left side, against a black background, appear bright, and at the right side, against a brighter background, they appear darker. In the middle where the background intensity is the same as the intensity of the letter, there is no contrast. Unfortunately in Biology, many specimens, particularly live cells, are hardly distinguishable from the background. Hence, special illumination techniques have been developed over many years to utilize the special properties of light and its interaction with different types of material. 50 – 50 / = 0

45 Contrasting Techniques
Examples Brightfield Phase DIC Fluorescence These 4 images show the same details on a specimen, illuminated with different techniques. Brightfield will render almost no contrast. Phase Contrast typically produces good contrast, but also a “halo” around structures. DIC, when displayed at its optimum, produces a pseudo 3-D image, and fluorescence allows for the separation of different components within one structure, if these components have been tagged with different fluorochromes.

46 Brightfield For stained or naturally absorbing samples
specimen condenser objective Brightfield For stained or naturally absorbing samples True Color Representation Proper Technique for Measurements Spectral Dimensional Koehler Illumination !

47 “Koehler” Illumination (since 1893)
Prof. August Köhler: “Koehler” Illumination (since 1893) Provides for most homogenous Illumination Highest obtainable Resolution Minimizes Straylight and unnecessary Iradiation Allows adjustment of optimal Contrast Defines desired Depth of Field Helps in focusing difficult-to-find structures Establishes proper position for condenser elements, for all contrasting techniques August Koehler, who developed his famous illumination technique while he was a professor of Zoology at the University in Giessen, joined the Zeiss works and contributed his “invention”. To this day, no other illumination method beats the “Koehler” method for optimizing the illumination through a microscope. It provides for most homogenous Illumination, Highest obtainable Resolution, minimum effects from straylight. I also Allows for adjusting Contrast and required Depth of Field and, after it is all set and done, it can help to see difficult-to-find structures. Particularly important for modern microscopy, by following the Koehler method, one establishes the proper position for condenser elements, not only for Brightfield, but essentially for all contrasting techniques.

48 Necessary components to perform “Koehler” Illumination:
Adjustable Field Diaphragm Focusable and Centerable Condenser Adjustable Condenser Aperture Diaphragm To be able to perform “Koehler” illumination, one needs to have available on the microscope: An adjustable “field diaphragm” A Focusable and Centerable Condenser An Adjustable Condenser Aperture Diaphragm

49 Conjugate Planes (Koehler)
Retina Eye Eyepoint Eyepiece Intermediate Image TubeLens Imaging Path Objective Back Focal Plane Objective Specimen These diagrams show the beam path which is associated with the specimen – on the left, and the “illumination beam path” which starts with one spot of the actual light source – to the right. Elements which are visible simultaneously in the imaging path are: Field diaphragm, Specimen, Intermediate Image (where a reticle would be), Retina of the Eye – we can see it. Elements which are visible simultaneously in the illuminating path are: Light source, Condenser Aperture Diaphragm, Back Focal Plane of Objective, Eyepoint behind eyepieces. We can only see these planes by removing an eyepiece or using a focusable telescope in place of it. Condenser Condenser Aperture Diaphragm Field Diaphragm Illumination Path Collector Light Source

50

51 Conjugate Planes in the Upright Microscope
Image Planes Aperture Planes This is a cross-section through an Axioskop microscope. The blue arrows show the planes which are conjugated to the specimen; the red arrows show the planes, conjugated to the light source.

52 Conjugate Planes in the Inverted Microscope
1 Intermediate image plane (photo tube) 2 Eyepiece (Intermediate Image inside) 3 Intermediate image plane (front port) 4 Intermediate image plane (base port) 5+6 Imaging Beam Path switchers 7 Tube lens 8 Analyzer 9 Reflector 10 Field stop (Reflected light = RL) 11 Aperture diaphragm (RL) 12 Filter slider (RL) 13 HBO Illumination (Arc) 14 HAL Illumination (Filament) 15 Field stop (Transmitted light = TL) 16 Polarizer 17 Aperture diaphragm (TL) 18 Condenser 19 Objective (Back Focal Plane inside) This is a cross-section through an Axiovert 200 microscope. The blue arrows show the planes which are conjugated to the specimen; the red arrows show the planes, conjugated to the light source.

53 Koehler Illumination Steps:
Open Field and Condenser Diaphragms Focus specimen Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) Here is the sequence: Open Field and Condenser Diaphragms Focus specimen

54 Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) 3) At this time, you may have to adjust the intensity of the light source for comfortable viewing. If you turn down the transformer or rheostat setting too much, the background of the image turns more and more yellow. So, at this point you may wish to engage the necessary amount of neutral density filters to preserve a more neutral background. Only LED illuminators will not change their spectral output when you decrease power so, if you use one of those, you may skip this step.

55 Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) 4) You close the field diaphragm until you see the illuminated field appear like a “hot spot”.

56 Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm by moving condenser up or down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) 5) You focus on the leaves of the field diaphragm by moving the condenser up or down. At this point you should see the specimen and the field diaphragm in focus at the same time.

57 Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Stop by moving condenser up or down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) 6) You center the field diaphragm by moving the condenser in its mount via the centering screws which are part of the condenser carrier. These screws are typically located at 4 and 8 ‘o clock of the condenser mount. You may have to go back to step 5 and re-focus the condenser slightly to get a truly sharp image of the diaphragm leaves.

58 Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view of observer Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) 7) Now you can open the field diaphragm again, but only as large as you can see it, until it just disappears beyond the edge of the viewed field in the eyepieces. If you are using a camera, it should be just slightly larger than the field seen by the camera. At this point, you are done with the more mechanical part of Koehler illumination. The next steps will address the individual control of resolution, contrast and depth of field.

59 BFP Open Field and Condenser Diaphragms Focus specimen
Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture BFP 8) You need to set the aperture diaphragm of the condenser for optimal results. To see this diaphragm, you will have use a Phase telescope in place of an eyepiece, or a Bertrand lens, or to simply remove one of the eyepieces. When the aperture of the condenser matches the whole aperture of the objective, you will see a bright circle. This would be the setting for highest resolution. 9) To get a better contrast, with minimum loss of resolution, you should close the condenser aperture diaphragm to fill about 75% of the fully visible field. This is a good starting point. With added expertise, you may wish to play with the settings of this diaphragm until you get the best compromise between resolution on one hand and contrast / depth of field on the other hand. The details to be seen on the specimen should dictate how to set the aperture! Better: Depending on specimen’s inherent contrast, close condenser aperture to: ~ x NAobjective

60 Done ! Koehler Steps: Open Field and Condenser Diaphragms
Focus specimen Correct for proper Color Temperature Close Field Diaphragm Focus Field Diaphragm – move condenser up and down Center Field Diaphragm Open to fill view Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture Observe Image ! Now you can return the eyepiece again to its position, and you have set the microscope to perfect “Koehler”. Repeat these procedures every time when you change objectives for optimum results. Done !

61 This is how to optimize contrast by Koehler Illumination
Let’s Review the steps to achieve Koehler Illumination… This is how to optimize contrast by Koehler Illumination

62 Koehler Illumination Steps:
1 2 3 4 5 6 7 8 9 10 Turn light on; open field and condenser diaphragms Focus specimen Consider neutral background (set rheostat to 3200K, use neutral density filters for comfort) Close field diaphragm Focus field diaphragm – move condenser up or down Center field diaphragm Open to fill view Observe objective’s back focal plane via Ph telescope or by removing ocular Close condenser diaphragm to fill approx. 3/4 of objective’s aperture Done! The most important part!!!!!


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