Microscopy & digital imaging Flashcards
When observing a metaphase with a 10X objective, the image is clear. When a 100X
coverslip corrected oil lens is used, the image is not clear. What is the problem?
There is oil on the slide.
There is a coverslip on the slide.
The slide is upside down.(+)
The objective is damaged.
In the culture of CVS it is essential to remove all maternal decidua tissue. Which microscope
would you use?
Brightfield Microscope
Inverted Microscope
Fluorescent Microscope
Stereomicroscope(+)
Unstained slides need to be checked by:
Brightfield microscope
Phase microscope(+)
Fluorescent microscope
Darkfield microscope
The optimal objective for photography is:
Planapochromatic(+)
Apochromatic
Acromatic
Planacromatic
Why is an inverted microscope used to view flask cultures?
Because the condenser is under the stage
The light is coming from top
The objective is under what is being viewed(+)
It has greater resolution
Standard compound microscope
This microscope is also referred to as a brightfield microscope and is used to observe stained specimens. These microscopes are the most common microscopes used in a cytogenetics lab, which processes G-banded specimens. The light source for a brightfield microscope is a halogen tungsten bulb (usually 12V, 100W).
Phase microscope
This microscope is used to observe unstained specimens. Unstained specimens block the passage of light in a differential manner. The small differences in thickness and refractive index within a specimen are converted into differences in intensity and brightness, which can be observed under this type of microscope. The thicker an object is, the less light is allowed to pass through. Cells are composed of regions of various thickness and density, for example, the nucleus of the cell is very dense and allows little light to pass through and therefore appears darker than the surrounding cytoplasm.
Inverted microscope
This is a phase microscope with the light source and condenser above and the objectives below the stage. This feature allows tissue culturists to view cell cultures being grown in flasks and other culture vessels. These cultures can not be observed with the standard compound microscope due to the small working distance between the objectives and the stage
Stereomicroscope
This microscope is commonly called a dissecting microscope. Dissecting microscopes have a large distance between the optics and the specimen to allow dissection of tissues. These microscopes have the optics and the light source above the specimen.
Fluorescent microscope
The most common type of fluorescence used in cytogenetics is called epifluorescence. The term epifluorescence implies that the light source is above the specimen (If the light comes from a source below the specimen, it is called transmitted fluorescence.). The excitation light passes down through the objective, hits the specimen and then the reflected light from the stain is captured by the objective and is allowed to pass through a barrier filter (which in turn filters out the excitation light). This reflected light can then be observed by the user or the camera. The most common light source for fluorescent microscopy is a Mercury HBO 100W bulb.
Note: The exciter and barrier filters used is dependent on the stain being used. Different stains will be excited by different wavelengths of light. In addition, each stain will emit different wavelengths of light (always of a lower energy or longer wavelength due to energy lost as heat - Stoke’s Law).
Computerized equipment
Computerized imaging systems are becoming increasingly used in cytogenetic labs around the world. These systems allow visualization and manipulation of a microscopic image through the use of computers. Below are important components of these systems.
Video camera
This camera should have sufficient resolution and contrast for use in the photography of cytogenetic banding and staining techniques.
Digitization
Conversion of light information from an analog to a digital format. A digital image can be interpreted and manipulated because it is expressed in computer language. This digitization process occurs when variations in light intensity detected by the video camera are converted to number values.
Monitors
used for image manipulation must have a sufficient level of resolution
Resolution
is dependent on the matrix size or the number of pixels per inch of screen. A pixel is defined as a unit of screen. Recent developments in high definition TV and computer monitors will eventually revolutionize this field of image analysis, allowing an even better quality of image reconstruction.
Thresholding
Manipulation of the image is achieved by the use of a light histogram, which plots the different values representing the different light intensities the computer recognized during the digitization process. Through a process called thresholding it is possible to minimize background debris by setting a value below which all values will be considered white and a value above which all values will be considered black. This process can also be used to enhance the light and dark bands of G-banded chromosomes. In color imaging this process can be used to manipulate the various colors of DNA probes and counterstains.
Karyotyping programs
can assist in the karyotyping of G-banded and Q-banded cells. Many of today’s programs can attempt the identification of the banded chromosomes with various levels of success. These computer programs use area and arm ratios to determine the different chromosomes.
High quality printers
eliminate the need for the traditional darkroom thus saving a significant amount of time and expense incurred in cytogenetics laboratories.
Archiving
Archiving of images is another feature of imaging systems. Before the use of these systems, storage of negatives and photographic images for the period of time required by state regulations (5 - 7 years) required an enormous amount of storage space. Now images can be stored onto optical discs greatly reducing the amount of storage area required.
Maintain phase microscope, inverted microscope, stereomicroscope and/or computerized equipment
Microscopes and computer imaging equipment should be housed in a relatively dust-free environment. Dust on any surface in the path of the light will appear in the same focus as the image. In addition, oil from eyelashes on the ocular lenses can result in blurred images. Daily cleaning of the lenses on the microscope will result in clear images. In addition, the surrounding area should be maintained as free of dust as possible.
The equipment should be housed in an area free of vibrations. Nearby construction or the use of a centrifuge on the same countertop will result in blurry images and photographs.
As with all equipment, preventive maintenance is necessary for the long life of microscopes and computerized equipment.
Operate and maintain photographic equipment.
Many of the methods to operate and maintain photographic equipment have already been discussed. To reiterate, the camera used to take pictures on the microscope may be either a 35mm camera, a 4 x 5 Polaroid camera, or a video camera. To operate these camera systems, the user must have an understanding of the microscope, the film, the proper ISO and exposure settings, and the proper video camera settings.
In the darkroom, the enlarger should be maintained free of dust and should be the condenser type with two convex lenses. Convex lenses allow the maximum transmission of light to the surface of the paper and will give photographic images with sharp detail. (Concave lenses will diffuse the light given softer photographic images needed for portraits). The amount of light the paper is exposed to is controlled by the F-stop diaphragm, usually located in the lens of the enlarger and the timer system, which sets the exposure time.
Identify microscopic components
1.Field diaphragm (Field Stop)
This diaphragm is located at the base of the scope and is used to focus the light coming from the lamphouse. It also concentrates the light as it is closed. This ensures that the maximum amount of light is illuminating the desired field of view on the slide. (Typically, the field stop is closed 25% for taking photographs.)
2.Condenser
The condenser collects the light that passes through the field diaphragm and focuses it onto the slide. The condenser is located between the field diaphragm and the stage where the slide is placed.
3.Condenser or aperture diaphragm
This diaphragm is located within the condenser. This diaphragm changes the angle of light. When this diaphragm is open, the maximum amount of resolution can be obtained. When this diaphragm is closed, the maximum contrast can be obtained. (Typically, the aperture diaphragm is closed 25% for taking photographs.)
Filters
Green filters are used in the imaging of G-banded cytogenetic preparations in order to increase the contrast between dark and light bands.
Objectives
Most objectives are parfocal. Parfocal lenses allow for an object in focus on low power to be in focus on high power with the minimal adjustment of the fine focus knob.
Achromatic objectives
These objectives correct for two colors of visible light (blue and red). This implies that the objective brings these wavelengths to a common focus within the objective.
Apochromatics
These objectives correct for three colors of visible light (blue, green and red). Because a greater amount of light is brought to a common focus within the objective, apochromatic lenses have a greater inherent resolution than achromatic lenses.
Plano
These objectives correct for curvature of field (Photomicroscopy depends on a field of view entirely in sharp focus)
features of an objective are transcribed on surface of each and every objective
170/0.17 PlApo Oel 100/1.32
170 = Tube length in mm 0.17 = Coverslip width in mm Pl = Plano Apo = Apochromatic Oel = Oil lens 100 = Magnification 1.32 = Numerical aperture
Eyepieces
Eyepieces allow the image to be magnified for easier observation (Usually 10X or 12.5X). Eyepieces also allow the microscope to be adjusted to the eyesight of the user.
Other components include
stage, nosepieces, body tube, fine and coarse adjustments and centering screws.
Achieve optimum resolution
1. Resolution
The resolving power of the microscope is defined as its ability to distinguish two points as separate entities. This is represented mathematically by the equation:
r = 0.61lambda/N.A
Factors which affect resolution include:
1. The numerical aperture (N.A.) of an objective
2. The numerical aperture (N.A.) of a condenser
Note: The N.A. of both the objective and condenser should be equal for maximum potential.
- Numerical aperture
This is the index of the light gathering capacity of an objective or condenser. The larger the number, the more light gathered and the higher the resolution.
Koehler illumination - This is the proper alignment of the light through the microscope. Proper alignment of the light source and condenser ensures that the maximum amount of light possible will illuminate the field of view and that there will be an even distribution of light. Centering screws located in the condenser, in addition, are used to center the light.
Koehler illumination will ensure that the best image quality and the highest resolution possible are achieved.
The refractive index of the mounting medium (and immersion oil), the refractive index of the slide, mounting medium and coverslip should be within the range of 1.515 - 1.520. This allows the light refracted through the specimen to be passed directly up into the objective without being deflected. Light is the information seen by both the eye and the camera.
Appropriate magnification
1. Total magnification
Total Magnification = Ocular * Objective Magnification
Note: The greater the magnification of an objective, the less is the working distance between the objective and the slide. Therefore, for lenses of greater magnification, immersion oil is used to prevent damage to the specimen and the lens.
2.Empty magnification
Increasing the magnification does not always result in a more detailed image. Empty magnification results
When Total magnification is >1000 * N.A. of the objective
Select appropriate cover glass thickness.
The appropriate cover glass thickness is important when using high-powered oil immersion objectives. The proper thickness of coverslip to use is engraved on each objective (usually 0.17mm). Some older oil objectives can not be used with coverslips and will not have this information engraved on them.
Produce clear photographic or computer images
Images, which have good focus, resolution and contrast, are obtained only with attention to detail throughout the entire photographic process.
When pictures are taken on the microscope, care must be taken to use the proper ISO setting to allow the proper exposure of the film to light. In addition, to obtain the highest resolution, high quality lenses and Koehler illumination techniques must be used. Before exposing the film, the microscopist should check the proper setting of the condenser diaphragm. For photography of chromosomes, the camera system should be set for spot metering. The small circle in the middle of the frame reticule is the only area metered when the camera is set for spot metering. This allows the camera to focus only on the object placed in the circle (a piece of chromosome), not the background that usually comprises 70% of the field of view in a metaphase spread. In addition, the crosshairs located within the frame reticule should be in focus as well. The crosshairs allow the camera to adjust its focus to match the eyesight of the user.
Film should not be over- or under-developed
The image should be in proper focus before exposing the photographic paper. In addition, the proper use of filters or the proper selection of paper should be made to ensure that the image does not have too much contrast (no continuity of the dark bands with the light bands) or does not have too little contrast (a high gray level which prevents the distinguishing of the light and dark bands).
Computer images require not only the proper settings on the microscope as previously described, but also the proper settings of black and contrast levels of the video camera. Proper manipulation of image threshold is also important to obtain an image with the proper resolution and contrast.
Operate and maintain photographic/imaging equipment
1.Select appropriate film
The most common film used in cytogenetic laboratories is Kodak technical pan 2415 film (TP 2415). This is a panchromatic film (sensitive to all colors of light) with fine grain, which can be used at a wide range of ISO settings. This type of film is useful for the different types of photography that may be needed in a cytogenetic lab. For example, Giemsa stained chromosomes are photographed at an ISO setting of 50 - 100, whereas fluorescent stains such as quinacrine, acridine orange, DAPI/Distamycin A are photographed at an ISO setting of 160 - 400. (ISO may still be referred to as the older notations of ASA or DIN.)
The best film for black and white photography of chromosomes has a high resolution (fine grain observed after development) and high contrast (accutance is the term used to define sharpness of image).
Color photography - FISH and other fluorescent techniques require working with a lower light level than non-fluorescent techniques. This will result in longer exposure times. This longer exposure time is required because the fluorescent light emitted by the stain and reflected back up the objective in epifluorescence is often of low intensity. Films with a high ISO or a greater sensitivity to light are recommended in photographing color images from FISH to prevent these long exposure times. This also prevents reciprocity failure that can occur during long exposure time. Reciprocity failure is the inability of the film to become increasingly exposed over time in low light conditions.
2.Select and prepare chemicals for film processing
When the light strikes the film in the camera on the microscope it creates a latent image. A latent image is a chemical image, which can not be seen by the naked eye. If chromosomes are Giemsa stained, they will block the light and these regions on the film will be protected from the light. All other regions will be exposed to the light. Therefore, negatives obtained from Giemsa stained preparations after development will appear dark except for the chromosomes which will appear light.
Most black and white film used in the cytogenetic lab is panchromatic. Because this film is sensitive to all wavelengths of light, the development of the film must be performed completely in the dark without the aid of safelights.
Developer - Film has a layer of silver halides on its surface. This layer is called the emulsion. The developer is an alkaline solution that reduces exposed silver halides to metallic silver. The developing process is dependent on time, concentration of the developer, temperature of the developer and the amount of agitation during development. If all of the above is increased, the film will become more developed. These are the factors that often contribute to overdeveloped or conversely underdeveloped film. Overdeveloped film inherently has more contrast and underdeveloped film inherently has less contrast. Both will result in troubleshooting problems that will be discussed later. If film is left in the developer too long, eventually all of the silver halides will be reduced to metallic silver regardless of whether they were exposed to light or not.
Stop bath - The stop bath in the development of film may be only a water rinse, which stops the developer action by washing it away from the surface of the film. Some laboratories use a 1% acetic acid wash to neutralize the alkalinity of the developer. This increase in acidity prevents the developer from reducing the silver halides.
Fixer - Rapid fixers contain ammonium thiosulfate which removes the unexposed silver halides from the surface of the film. With the proper exposure to the fixer solution (usually five minutes), the film will no longer be light sensitive and only the metallic silver will remain. This metallic silver is visible to the naked eye and represents the dark regions of negatives.
Wash solution - The fixer must be washed away from the film, for eventually, the fixer will remove even the metallic silver from the surface of the film. Film may be washed in water for a minimum of 15 minutes or a solution, which neutralizes the fixer, must be used.
- Select and prepare chemicals and paper for manual and automated print processing
Manual print processing
The manual processing of prints requires the use of the same chemicals that are used in the development of film. However, the type of developer used will be different. Developer used for paper must be stronger that that used for film development. The stop bath will usually contain the 1% acetic acid. Finally, the fixer will be the same, but will be used at one-half the strength as is used for fixing film. The paper must also be washed for the appropriate amount of time. The process is similar because photographic paper also has a layer of emulsion, which is light sensitive.
Because photographic paper is orthochromatic, the processing of photographic paper can be done with the use of safelights that are red or yellow in color.
A darkroom enlarger together with a time is used to control the amount of light that will pass through the negative and strike the paper to form a positive latent image. (strikethrough: Safelights are used during the developing of photographic paper (red or yellow), and allow focusing of the image prior to exposure of the paper.) The negative is placed in a negative carrier, and the image should be focused prior to exposure of the paper. The paper is then placed on an easel, which hold the paper in place and prevents any movement that may cause a double image when the paper is being exposed to light.
Dodging is the term used to describe the manipulation of the image during exposure to light. Uneven illumination of the image on the microscope due to improper Koehler illumination will result in a negative image with one side lighter than the other. To get an even photographic image, it is necessary to dodge the lighter part of the image to prevent it from developing faster than the darker part of the image. Dodging can be accomplished by using the hand or a piece of paper to block the light from hitting the area of the paper that will be overdeveloped.
The photographic paper used in this method of print processing can have variable levels of contrast. These papers are ranked from the lowest level of contrast (F1) to the highest level of contrast (F5). In addition, different thickness of papers can be used (single weight or double weight).
Non-resin coated printing paper must be dried with a print dryer, however, resin coated paper may be used which can be air-dried (RC paper).
Automated print processing
Automated print processing requires the use of photographic paper which has developer impregnated within the emulsion. After exposure of the paper to light, the paper is placed into an automated print processor, which works by a conveyor belt system. Rollers carry the paper into the machine where it is first exposed to an activator that activates the developer. The paper is then exposed to a stabilizer that stops the action of the developer and makes the print insensitive to light.
Maintain permanent storage of photographic or computer images.
It is necessary to store images as a permanent record. Photos are stored with the patient records. Film should be stored in a dust free environment in a different location as a backup in the case of fire. Computer images can be stored temporarily on the hard drive of the imaging computer but should be copied to optical disk drives or digital tapes for permanent storage. Two copies should be stored in two different locations in the event of fire. This is a CAP regulation.
Troubleshooting
Microscopy
Brightfield
Image can’t be focused or doesn’t stay in focus
Make sure that there is sufficient immersion oil on the slide.
Make sure that two different types of immersion oils haven’t been used on the same slide.
Make sure that the slide has not been mounted with more than one coverslip by mistake.
Image is in focus on 10x objective but not on 100x objective
Make sure that the slide is not upside down. Also check that cells have not been dropped on the wrong side of the slide.
Image is not sharp and clear in appearance
Check Koehler illumination.
Check that the aperture diaphragm has not been closed more than 25%.
Fluorescence
Low fluorescence
Make sure that the bulb is centered.
Replace the bulb if it appears fogged.
Air bubbles or other factors that cause drying out of the mounting media.
Make sure that proper filter cube is being used.
Autofluorescence/Background
Lower the condenser to prevent light reflection.
Blot any excess mounting media from the slide.
Image can’t be focused on low power due to low fluorescence
Focus on the edge of the slide.
Focus first with a coversliped Giemsa stained slide.
Flickering light
Make sure that when a new bulb is mounted that it is left on for more than 2 hours. The arc of the light will not be burned into the bulb if less time is used.
Troubleshooting (cont.)
Photomicroscopy
1.Negatives
Out of focus
Check that the cross hairs are focused.
If there are vibrations, use a stability platform or place table legs in sand.
One side of negative is consistently lighter than the other.
Check Koehler illumination.
Fogged negative
Make sure that the proper safelights have been installed.
Make sure that a radio with a red indicator light is not being used during film development.
Debris shows up in the same spot on each frame of a negative or negatives.
Make sure that you clean all optical surfaces that fall within the light path.
Open the base of the microscope and check for spider webs.
Too dark or too light
Check that the temperature of the water is 20oC
Check the ISO setting on the camera.
If it is too low, the negative will be too dark.
If it is too high, the negative will be too light.
Make sure that the film is not overdeveloped or underdeveloped.
If overdeveloped, the negative will be too dark.
If underdeveloped, the negative will be too light
2.Prints
Out of focus
Improper focusing of the enlarger
Use the edge of the negative to help in focusing
Use a focusing tool to focus on the grains of the negative.
Focus under the brightest light
Too dark or too light
Adjust the exposure time or the F-stop of the enlarger
If print is too dark, raise the F-stop or decrease the exposure time.
If print is too light, lower the F-stop or increase the exposure time.
Too much or too little contrast
Change the filter that is being used in the enlarger
Use a photographic paper with either a lower or higher contrast
Double images
Make sure that the easel, which holds the paper, is not moved during exposure.
