Wednesday, August 31, 2011

LWD Objectives with Correction Collars

Long working distance (LWD) microscope objectives for inverted biological microscopes are either designed to be used with a standard thickness typical of common flasks and petri dishes, or they have correction collars that allow users to precisely focus on the specimen by turning an external ring on the barrel.

Long working distance objectives without correction collars are made to work through a thickness of approximately 1.0 - 1.2mm (the thickness of most flasks, petri dishes and multi-well plates). These are the routine lenses for inverted cell culture microscopes. These lenses are moderately priced compared to the LWD lenses with correction collars.

 60x Plan Fluor objective lens with correction collar.

At 40x and above, the aberrations resulting from a cover slip of less than or greater than 0.17mm noticeably degrade the image. The amount of media used in preparing the sample can also increase the distance required for optimal image quality. To compensate for these problems, more expensive objectives feature a correction collar that adjusts for this distance. The image below is a LWD objective with correction collar which allows a sharper image with more detail. The disadvantage to these lenses is that it takes experience and diligence to use the objective properly. If poorly adjusted the lens will result in an unusable image.

40x LWD microscope objective with correction collar.

How to Properly Use the Correction Collar:
The scale for the correction collar will be engraved on the barrel. Some start with 0 at one end which would correspond to no cover glass and go up to 2mm. Others start at 0.17, the thickness of a standard cover slip, while others start at 0.5mm.

With the microscope objective lens in place, note whether the smaller number is rotated all the way clockwise or counterclockwise. If you are viewing a slide turned upside down on an inverted microscope, you are looking through only a cover glass thickness of 0.17mm and the collar should be turned all the way in the direction of the lower number. Focus on a detail in the specimen as clearly as possible.

With one hand controlling the fine focus and the other adjusting the correction collar, turn the collar a small amount and refocus the specimen. If the image improves, turn the collar a little more in the same direction and refocus. If the image improves, repeat. However, if the image becomes worse, turn the collar in the opposite direction and refocus. Continue to do this until you get a sharp and clear image.

It is important to turn the collar, then refocus as two distinct steps. If you turn both the collar and the fine focus at the same time, it is very difficult to adjust the collar correctly.

If you are looking through a petri dish or flask, put the collar around the 1mm mark to start. If the lens is in place and the scale is 0-2mm, you can estimate the half way point by feel and start from there. It is good to get in the habit of turning either clockwise or counterclockwise first so that you systematically work as efficiently as possible.

A slide is approximately 1mm thick, so if you are looking through the thick part of the slide you should set the collar about half way between the petri dish setting.

Precisely setting the correction collar on your microscope objective can seem daunting, but if you take the time to practice the procedure, it quickly becomes routine. The difference is a poor image and disappointment in your equipment, or getting excellent images and using a relatively expensive objective in the most advantageous way.

Tuesday, August 30, 2011

Bichromatic Ferrography Analysis

Ferrography is one of the best predictive maintenance and analytical tools. This technique for wear particle analysis has become prominent in the paper industry, in the healthcare industry (particularly with artificial joints and limbs), and in industrial plants with automated operations. Bichromatic ferrography is the analysis of particles present in fluids to indicate mechanical wear. It enables plant managers, maintenance managers, and healthcare professionals to make critical decisions more effectively.

The ferrogram is a specially designed and prepared microscope slide that is examined under a polarized bichromatic microscope equipped with a camera. Red, green, and polarizing filters are used to distinguish the size, shape, composition and surface conditions of ferrous, nonferrous and non-metallic particles. With bichromatic illumination, metal particles (which reflect light) appear bright red, whereas nonmetallic particles appear green because light transmits through them. These particles are further studied and classified in order to determine the type of wear and the source of the particle.

Monday, August 29, 2011

Low Power Microscope Kids Activities

If you have a low power or stereo microscope, here are a few fun ideas of items to view with children.

  • Get a $5 bill and look at the back. Just above the pillars on the Lincoln Memorial you should see the names of some states. How many can you read? Take a $1 bill and see what other interesting items you can find on it with the microscope. Coins and stamps are other fun items to view.
  • Samples of fabric can be fun to compare. A plain weave looks like a simple cross pattern, while a twill weave (try blue jeans) or a pile weave (corduroy) will look different.
  • Sand samples from different beaches or a river bed. Try viewing them with bottom illumination, then put the sand on a black piece of paper and view it with only the top illumination.
 Sand under the microscope.

  • If you leave a small pan of water outside in the summer, you may end up with mosquitoes laying eggs in it. Once the eggs hatch, look closely for tiny wigglers - these are mosquito larvae. You can collect one and put it on a depression slide to view under the microscope.
  • Look for some other small insects - you might find a dead spider in a window frame, or an ant outside in the yard. Caterpillars are also great fun to watch under the microscope! When you are done - put your insects back outside so they can live in nature.
  • Take a color page from a magazine - can you see the dots of different colored ink that make up the photo? You can also try this with a newspaper.
  • Look at the center of a flower. Can you locate the stamen and the pollen?
  • Interesting rocks, especially those with multiple colors in them are fascinating to view under the microscope.
Use your imagination! Just about anything you find around your house might be interesting to view under you microscope. If you find some interesting items you may want to take them to school to share with your science class.

Thursday, August 25, 2011

IMA/USP Microscope Counting Reticle

The IMA/USP counting reticle is used for manual particle counting, most often in the pharmaceutical industry. Government guidelines (USP 788 Government Standards) require a manual microscope particle counting system must include a circular diameter reticle (installed in the microscope eyepiece) that is designed specifically for this purpose and calibrated using a certified stage micrometer.

The IMA/USP particle counting reticle is divided into four quadrants by crosshairs - each showing transparent and black reference circles 10um and 25um in diameter at 100x magnification. 

When manually counting particles, the user will place the filter under the microscope with the counting reticle in the eyepiece. By carefully comparing any particles seen under the microscope to the reference circles, the user can record the number of particles of particular sizes.

Wednesday, August 24, 2011

Microscope Objective Tube Length

Microscope objectives are typically either a fixed tube length, or Infinity Corrected. A fixed tube length objective might have the number 160 printed on the objective, while an infinity corrected objective lens would have this: ∞ printed on it.

A fixed tube length objective lens has a finite tube length. If 160 is printed on the microscope objective, this means that 160mm is the distance from the opening of the nosepiece (where the microscope objective is screwed into the microscope) to the top of the observation tube (where the eyepiece is inserted). If this distance were to be lengthened (for example by inserting accessories in the light path above the nosepiece), spherical aberration would result.

Infinity corrected optics are more common, and allow the distance between the nosepiece and the top of the observation tube to be altered. Infinity corrected optics project emerging rays of parallel bundles projected toward infinity.

Infinity corrected microscope objectives can not be used with fixed tube length microscope systems because the fixed tube length microscope system does not include a tube lens to bring the parallel rays of light into focus. Alternatively, using a fixed tube length objective lens on an Infinity optical system microscope may produce an image, but it most likely will be a deteriorated image.

You can learn more about mixing microscope objectives here.

Tuesday, August 23, 2011

Science Project: Mouth Cells

This is a simple science project you can do at home with just a few supplies. You will need:
Once you have gathered your supplies, take the Q-Tip and run it across the inside of your cheek to moisten it. Smear the swab on your blank slide, and put a cover slip on top of it. Place the slide under the microscope and focus at the lowest magnification first (40x), then move up to 100x and finally 400x. What do you see? There are several items you may be able to view including bacteria and your cheek cells.
This is an image that was captured at 400x magnification of cheek cells. Can you identify the nucleus of each cell? It is the small dot in the center of each oval shaped cell.

This is an image of bacteria. Usually bacteria cells are rod-shaped. You can identify some of the different types of bacteria shapes here. There is always bacteria in your mouth - and not all bacteria is bad. Even the yogurt you eat for a snack has bacteria in it!

Monday, August 22, 2011

Viewing Microchips

A customer of Microscope World recently needed an instrument to view quality control of their microchips. The system they ended up purchasing is a Meiji EMZ-5 stereo zoom microscope system with a C14+ microscope camera. The customer can use the software included with the camera to measure any scratches in the microchips, capture images of the scratches, and document these images.

Scratched microchip, captured with a stereo zoom microscope.

Friday, August 19, 2011

Steel Under a Metallurgical Microscope

Inverted metallurgical microscopes are great for viewing solid items (such as steel) that don't allow light to pass through them. In addition, the inverted microscopes are best for viewing large pieces of material, that would not typically fit on the stage of an upright metallurgical microscope.

This image of a steel plate was captured using the Meiji IM-7200 inverted metallurgical microscope and the Motic MC2000 microscope camera.

Thursday, August 18, 2011

Long Working Distance Objectives

Some microscope objectives have the inscription LWD or ULWD on them. This stands for Long Working Distance or Ultra Long Working Distance. Working distance is the vertical distance that must be between the tip of the microscope objective lens and the part of the specimen you wish to view through the microscope, in order for it to be in focus. Long Working distance microscope objectives require that this distance be greater.

So why would you want more distance? Having a longer working distance is invaluable when viewing specimens through a culture vessel or Petri dish while using an inverted biological microscope. When using a metallurgical microscope, a long working distance microscope objective is helpful if viewing IC wafers in order to avoid inadvertent contact with the wafer. Inspecting solder joints is another process that often requires use of a long working distance objective. Additionally, any time more working room is required under the microscope, long working distance objectives are usually preferred.

Wednesday, August 17, 2011

Diabetes Testing

This is a diabetes test strip. The image was captured using the SMZ-168 stereo zoom microscope and the 2 mega pixel MC2000 microscope camera at 40x magnification.

The circular part of the diabetes test strip is where blood sugar is measured. The manufacturer of this particular test strip uses a microscope during manufacturing to view the quality of the testing area, and to ensure that the entire circle is complete and able to accurately measure blood sugar.

Tuesday, August 16, 2011

Microscope Filters Explained

Microscope Filters are used for both observation and photo microscopy. Below are some common filters and their intended use.

  • Green Interference FilterAchromat and planachromat microscope objectives are best corrected spherically for green light, which means their performance improves with the use of a green filter. Phase contrast objectives are also created to give the best phase images in green light.
  • Daylight Blue Filter: This filter is for observation use only. It provides a pale gray-blue hue to the field of view and is often used to balance the light created by tungsten or halogen light sources. The daylight blue microscope filter was not created for use with photomicrography with daylight color film. Daylight color film requires a blue conversion filter that will boost color temperature of the light source and simulate light of daylight color temperature quality required for daylight balanced color film.
  • Ground Glass Filter: This filter is often placed over the illuminator to give a more even and diffused light. It is often used with Tungsten illuminators.
  • Neutral Density (ND) Filter: This microscope filter is used to reduce the light by a percentage. There are different numbers listed on the ND filters - such as ND8 or ND50. ND8 means that the light is reduced by 8%. Neutral density filters are often used in photomicrography.
  • Didymium Filter: This filter is also known as an “enhancing filter”. The didymium filter is made of didymium glass for increasing the intensity and saturation of red objects.  Since thin sections of biological tissue are often stained with one or more dyes to enhance visibility of various features in the specimen, if taking photographs of these specimens, it is desirable for the stained colors to appear in the picture. While most stains show up well on colored film, there are several that appear washed out. A didymium filter helps combat this problem.
  • Yellow Filter: The yellow microscope filter is commonly used to fine-tune the color balance of tungsten and halogen microscope light sources for photomicrography with color film. There are several metallurgical applications where the yellow filter is helpful in identifying failures in metal structures.

Sunday, August 14, 2011

Immersion Oil Microscope Objectives

You may have noticed the inscription “oil”, “oel” or “WI” on your microscope objective lens. This refers to an immersion objective, and is typically found on higher magnification objective lenses (50x – 100x). Most microscope objectives are created to allow you to view specimens by using air as the medium between the objective lens and the cover slip.

An immersion objective requires a drop of immersion oil, or sometimes water be placed between the tip of the microscope objective lens and the cover slip. This reduces the refractive index differences between the glass and the imaging medium, allowing the user to achieve a numerical aperature of 1.0 or above, while greatly improving the microscopy image.

When using an immersion microscope objective, the image appears very poor if not using the proper immersion liquid. The letters “WI”, “W”, “Water” and “Wasser” refer to an objective that requires water for the immersion medium. “Oil” and “Oel” objectives require immersion oil. “HI” refers to homogeneous immersion and “Gly” stands for glycerol immersion. Immersion objective lenses are especially useful when observing living biological specimens.

When finished using the immersion oil objective, be sure to clean it properly so it does not attract dust or form a thick foggy film on your lens. If the 100x oil immersion objective is used frequently, it is often a good idea to mount the dry 40x objective on the opposite side of the microscope nosepiece from the oil objective. This reduces the likelihood of accidentally dipping the 40x dry objective into immersion oil as you rotate the objectives.

Friday, August 12, 2011

Henrietta's Cancer Cells

In the 1940’s two researchers from Johns Hopkins School of Medicine, George Otto Gey and his wife Margaret, established one of the first Tissue Culture Research Labs in the world and spent the better part of three decades trying to find human cells that would grow in a cultured environment and could be used to study cancer.

In early 1951, Henrietta Lacks, a young African American woman with five children walked into Johns Hopkins complaining of unusual bleeding. She was diagnosed with cervical cancer and underwent radiation therapy, but the highly malignant tumor rapidly spread throughout her body and she died within a few months.
During surgery a piece of the tumor was collected and given to Gey’s Lab to see if this sample was able to grow and reproduce outside of the body. Gey concocted a feeding medium of blood from a placenta, beef embryo extract, and fresh chicken plasma which worked extremely well and her cells grew and reproduced at an unheard of rate. Henrietta’s cells, even now, 61 years later, are some of the strongest cells known to science. Most normal cells only divide 20-40 times before experiencing signals that lead to cell death. These HeLa cells show the presence of a version of telomerase which prevents the shortening of telomeres, normally leading to apoptosis, or cell death. Henrietta’s cells are immortal and will grow indefinitely. One researcher studying viruses says that over the 26 years he has used them, 600,000,000 HeLa cells are produced in his lab each week for a total of 800 billion cells.

With Gey’s success at creating a human cell line a medical revolution began. He shipped small tubes of her cells with instructions to colleagues around the world. Researchers now had the means to study many disease states and treat issues such as leukemia, viral growth, genetic expression, protein synthesis, radiation treatment, toxicology results, and polio vaccines. In a short period of time they became standard reference cells and are the most widely used cell line in cell and tissue culture labs today. There are more than 600,000 papers that cite HeLa cells in their research and over 300 papers a month continue to be submitted. Her cells were used to develop the polio vaccine by Jonas Salk and were on one of the first shuttles to outer space where they continued to grow vigorously.

Many researchers also began growing other cell lines, which over time became just as robust. In 1974 a researcher named Walter Nelson-Rees claimed that a closer examination of these other cell lines used to do unrelated research would show contamination by Henrietta’s cervical cancer cells. Denouncements were immediate and emphatic but upon investigation many cell lines thought to be breast cell lines or prostate cells, were in fact, HeLa cells. It turns out that these robust cells easily contaminated and overpowered any other cell culture they encountered and in labs where multiple cell lines were used, research became suspect. Forty different human cell culture lines used around the world were found to be contaminated. Many published papers addressing cancer research were discredited and the controversy is still on going.

Henrietta’s family was unaware for years that her donation to the cause of science had transformed research. The concept of informed consent was unknown in 1951 and the family has never benefitted from the multimillion-dollar industry that developed from the cells that killed her.

This image of HeLa cells shows the nucleus (blue) and the mitochondria (orange). The image was captured using an epi-fluorescence microscope with a Meiji 100x objective lens and a Jenoptik CCD microscope camera.

Images of mitochondria which produce power for the cell.

Thursday, August 11, 2011

Microscope Eyepiece Inscriptions

Microscope eyepieces often have several numbers and letters inscripted on them. For example, you may have a microscope eyepiece that has KWH10x/20 written on it.

Below are some of the text you may encounter on your microscope eyepieces and what it means:

K or C = compensating eyepiece. Some microscope objectives do not include correction for lateral chromatic aberration and in these cases, the compensating eyepiece completes this correction.

WF = widefield eyepiece. This means more of the specimen can be viewed at a given time.

H = high eyepoint eyepiece. The user's eyes do not have to be placed very close to the eyepiece lens during observation. This is especially helpful for those who wear eyeglasses.

20,22, etc. = This is the field number of the eyepiece. The higher the field number of the eyepiece that is used with a particular objective, the larger the field of view and the more of the specimen that can be seen through the microscope. The diameter of the field of view in millimeters is calculated by dividing the field number of the eyepiece by the magnification of the objective. So if you are using an eyepiece that says WF10x/20 and a 4x objective lens, your field of view would be 20/4 = 5mm. A larger field of view is often preferred by scientists.

Monday, August 8, 2011

Mixing Microscope Objectives

Microscope manufacturers advise against using other brands of objectives on their microscopes but in reality if the thread size of the objectives matches that of the microscope, it often works fine and provides good images. Manufacturers discourage this practice because designs and how optical corrections are made vary between companies and may result in a less than optimal image. This is often a minor issue, resulting in a colored ring around the outermost edge, with a good quality image in the center 90% of the field of view.

These are a few drawbacks to mixing microscope objectives:

  • Parfocality (the ability to change from one magnification to another without the image going out of focus) of the objectives is sacrificed when mixing objectives.
  • Special techniques such as phase contrast are also less likely to work.
  • Infinity corrected optics can not be mixed with fixed tube length microscope systems at all.
  • When mixing objectives from the older fixed tube length optical systems it is possible to install optics with different mechanical tube lengths. This results in a difference of magnification between what is printed on the objective and the actual image. For example, the lens might say 10x, but the magnification might actually be 12.5x. A calibration standard allows you to determine the correct magnification. These configurations of differing tube lengths can also result in an inability to focus using an objective at either the high or low end, so a 100x or a 2x objective might not be useable. 
Keeping these comprises in mind, try out a different objective and judge for yourself. It might be a great success. Below are some images from Microscope World's recent test.

This microscopy image of a privet leaf sample was captured using a Lumenera CCD camera and a 20x Motic objective on a Motic microscope.

This image was captured using the same leaf sample with a the same Lumenera CCD monochrome camera and a 20x Meiji objective on a the same Motic microscope.

Friday, August 5, 2011

Zebrafish Make Good Teachers

Lincoln K-8 Choice School in Rochester, MN joined researches from the Mayo Clinic to bring science into the classroom in a new way that excited students and piqued their interest in real research that can be applied to everyday life.

Dr. Stephen Ekker, a zebrafish researcher from the Mayo Clinic worked with the educators and students at Lincoln K-8 to develop class plans for the students to learn about zebrafish by working closely with them in the classroom. Students learned how to raise zebrafish and how scientists set up experiments to learn about everything from genetics, to how babies were affected when exposed to ETOH (alcohol) before birth. Even children as young as eight years old used microscopes to examine the problems of lower birth weights, lower heart rates and deformities associated with the exposure.

Zebrafish are a good research model because they share 75% of their genetic code with humans. Their reproduction cycle is extremely short, the equipment needed to study them is fairly basic, and students can study the fish from embryos to adulthood on a compressed time schedule. This low power Differential Interference Contrast (DIC) image was taken on Nikon equipment and shows blood flow in the tail of the zebrafish.

Older students visited Dr. Ekker's lab and got a better understanding of what research looks like in one of the most prestigious institutions in the world.

The students started identifying themselves as scientists and test scores went up! The number of eighth graders registering for Honors Biology went from 40% in 2006 to 86% in 2009 and the majority of the class achieved a rating of "exceeds expectations" in the Minnesota science standards. Overall science scores improved and resulted in the highest science scores in the state. Mr. Jim Sonju from Lincoln K-8 was named Principal of the year, and several teachers were recognized for outstanding work on the zebrafish project.

The goal of the collaboration between Mayo Clinic and Lincoln K-8 was to show that educators and scientists can work together to not only dramatically improve science proficiency in public education, but also to make science exciting and relevant.

If your school has an inspiring science collaboration story, send us an email and we may feature it!

Thursday, August 4, 2011

Microscope Objective Inscriptions

Microscope objectives usually have text printed on them, which can sometimes be confusing. The objective shown below is a Meiji metallurgical microscope objective.

This is what the microscope objective inscriptions stand for:
  • BD Plan EPI - this is a Brightfield / Darkfield Plan EPI objective lens. The BD means it can be used for both brightfield and darkfield microscopy. Plan EPI is a higher quality objective that has a flat field of view. You can read more about plan vs. semi-plan objectives here.
  • 50x / 0.75 - the objective magnification is 50x and 0.75 is the numerical aperture. This is a number that expresses the ability of a lens to resolve fine detail in an object being observed. It is derived by a complex mathematical formula and is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen. To get the best possible image, you should have a condenser system that matches or exceeds the N.A. of the highest power objective lens on your microscope.
  • ∞/0 - The objective lens is infinity corrected and the 0 is the thickness in mm of the cover slip that was used in computing the corrections for the objective. The image distance is set to infinity, and a tube lens is strategically placed within the body tube between the objective and the eyepieces to produce the intermediate image. Infinity optical systems allow introduction of auxiliary components, such as differential interference contrast (DIC) prisms, polarizers, and epi-fluorescence illuminators, into the parallel optical path between the objective and the tube lens with only a minimal effect on focus and aberration corrections. Older finite, or fixed tube length, microscopes have a specified distance from the nosepiece opening, where the objective barrel is secured, to the ocular seat in the eyepiece tubes. This distance is referred to as the mechanical tube length of the microscope. The design assumes that when the specimen is placed in focus, it is a few microns further away than the front focal plane of the objective. Finite tube lengths were standardized at 160 millimeters during the nineteenth century by the Royal Microscopical Society (RMS) and enjoyed widespread acceptance for over 100 years. Objectives designed to be used with a microscope having a tube length of 160 millimeters are inscribed with this value on the barrel.
  • F=200 - the objective focal length of the tube lens is 200mm.
  • WD 0.35 - the working distance of this objective lens is 0.35mm. This is the distance that must be between the tip of the objective lens and the specimen in order for the specimen to be in focus when looking through the microscope eyepieces.
If an objective is an oil immersion objective lens, it will typically have "oil" printed on the objective.

Wednesday, August 3, 2011

Coronary Atherosclerosis under the Microscope

Coronary artery atherosclerosis is the single largest killer of men and women in the United States and is the primary cause of coronary artery disease. Coronary artery disease is where vascular inflammation is present and the vessel walls have a buildup of lipids, cholesterol, calcium and other debris.

Image of coronary atherosclerosis captured with a biological microscope and a CCD microscope camera.

Tuesday, August 2, 2011

Printed Circuit Boards under the Microscope

Stereo microscopes are often used to view printed circuit boards. A typical setup uses a stereo zoom microscope and either an LED ring light or a 150w halogen dual pipe light. Transmitted light is not needed. A large base stand or a boom stand is used to provide working room under the microscope.

An electronic circuit board.

A connection point on a printed circuit board, captured at 70x magnification.

Printed circuit board captured with the Jenoptik PRC14+ microscope camera.

If you need to view printed circuit boards under the microscope, please give Microscope World a call, or email us and we would be happy to help you determine which stereo microscope works best for your needs.

Monday, August 1, 2011

Intestines under the Microscope

Some human intestine facts:

  • The small intestine is about 20 feet long.
  • The small intestine absorbs nutrients from food and water.
  • The large intestine is about 5 feet long.
  • The large intestine absorbs water from wastes, creating stool.

Image of the intestine captured with a digital biological microscope showing the intestinal villi. Villi increase the surface area of the intestinal wall, allowing for greater absorption of digested nutrients. The increased surface area of the intestine decreases the average distance traveled by nutrient molecules. Circulating blood then carries these nutrients away.