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.)