Publishers of technology books, eBooks, and videos for creative people

Home > Articles > Design > Adobe Photoshop

This chapter is from the book

Imaging on Stereo Microscopes

Stereo microscopes (also called dissection microscopes) provide an intermediate range of magnification somewhere between a 1:1 macro lens on a consumer camera and a microscope (with overlap). Depending on lenses that are used on the stereo microscope and the projection lens on the camera, specimen widths from approximately 8 cm (3 inches) to 1 mm (~1/16 inch) can be imaged.

The denotation of "stereo" refers to separate and angled optical paths used for the right and left eyepiece to provide depth perception. For that reason, stereo microscopes are also used as a magnification tool for working with small objects.

For specimens with dimensions that exceed the field of view for a stereo microscope but are still small objects at less than 30 cm (~12 inches) or so, a copystand is often used. With additional diopters or a bellows, a copystand can duplicate magnifications of stereo microscopes, but the working distance between the object and lens can present problems with handling objects and with lighting.

Digital imaging choices when using image acquisition software is identical to what was covered earlier. Differences lie in the creation of flatfield images for front-illuminated specimens, where a white card is used as a reflective surface. The white card is put in place and imaged either before the specimen is put into place, or the specimen is removed (if practical) once the lighting decisions have been finalized.

On three-dimensional surfaces, flatfield images may be useless, depending on how the object is lit. Test the efficacy by comparing a flatfield corrected image of a specimen and an uncorrected image. If the lighting is focused more like a spotlight and the position of the lighting changes depending on the position of the specimen, or when uneven illumination is the objective of lighting, flatfield correction is counterproductive.

Controlling Glare and Lighting

The major disadvantage of macroscopy (small object photography) is that glare can be produced by specimens no matter what kind of lighting is used. For that reason, no stereo scope or copystand should be sold without a means to control glare. Often the thought is to place a polarizing filter in front of the lens, but that only controls glare when lighting is at a specific angle.

For effective control of glare, polarizing filters must be placed in front of lights and the lens (Figure 4.15). The polarizer in front of the lens turns so that the level of polarization can be controlled. The polarizers on the lights need to be fixed at a single angle of polarization (sometimes marked on filters). If the angle of polarization is not marked, a reflective object, such as crumpled tin foil, can be placed under the lens and the polarizer in front of one light can be turned until the glare disappears (or diminishes as much as possible). The disappearance of glare indicates "cross polarization." Then the same can be done for another light, one at a time. Do not turn the polarizer in front of the lens when going through this process, because the polarizers on the lights are being cross-polarized with the (stationary) polarizer on the lens.

Figure 4.15

Figure 4.15 Illustration showing polarizing filters in front of lens and light source (fiber-optic light guide).

Once the polarizers on the lights are set at the same angle of polarization, the polarizer on the lens can be turned to vary the level of polarizaton.

The advantage of stereo microscopes and copystands is in the multitude of ways to light specimens (when the lights are not fixed). Typically, lighting is done by using a fiber-optic lamp. Several ways of viewing a quartz rock with flecks of crystalline mica and a mouse embryo follow (Figures 4.16 through 4.18).

  • One fiber-optic light at 45 degrees, no polarization. This lighting shows both topographical information on the surface of the rock and specks of glare from the mica (Figure 4.16A). The bright specks help identify the mica as a crystalline structure.
    Figure 4.16

    Figure 4.16 Three methods for lighting a specimen.

  • One fiber-optic light at 45 degrees, polarized. This lighting shows topographical information without glare from the mica by turning the polarizer in front of the lens to cross-polarize it with the polarizer at the end of the fiber optic (Figure 4.16B).
  • Two line (or comb) lights pointed across the surface. Comb lights enhance the surface topography of the rock (Figure 4.16C). These lights are also effectively used to create darkfield images from unstained and near-transparent samples.
  • Backlit by a single fiber-optic light. This method of lighting eliminates surface features to reveal only veins and mica (Figure 4.17A and B). A specimen of a mouse embryo is also shown (Figure 4.17C): This image is backlit off-axis by a single fiber optic.
    Figure 4.17

    Figure 4.17 Backlit rock and mouse embryo.

  • On-axis lighting. Light can penetrate into crevices when using a 45-degree mirror in front of the lens (Figure 4.18). This mirror can be half-silvered (one half of the light is reflected, one half transmits through the mirror) or coated so that particular wavelengths are reflected (on the way to the specimen) and others pass through (emitted or reflected by the specimen to the camera). Ring lights can also be used for this purpose, but they are yet slightly off-axis from the lens, and some shadowing can result.
    Figure 4.18

    Figure 4.18 Portion of 96-well plate showing on-axis lighting and penetration of light into wells.

Tips for Imaging Challenging Specimens

Depending on materials or composition of specimens, some can be very challenging to position and light in order to produce accurate images. Their appearance can be visually confusing because of reflections or because textures and roundness do not translate well in images. Here are some suggestions:

  • Specimens that reflect surrounding objects. A white tent can be created to surround highly reflective objects, such as highly polished metals. White paper can be made into a funnel. The camera is then focused through the opening in the funnel. Another alternative is to rub a thin layer of soft wax on reflective surfaces.
  • Removing glare via setting specimen underwater. Glare can also be removed by setting the specimen underwater. Some elongation of the specimen will result because of the difference in the refractive index of air and water.
  • Polymers and glass. Polymers and glass present difficult challenges for creating good representations of a specimen. Lighting is often pointed on the glass or the polymer itself. The better means for lighting may be to point a narrow beam of light on a part of the specimen that is not visible to the camera, and then to allow the light to "pipe" through the glass or polymer, or back light off-axis with a black background (as shown in Figure 4.18 with the embryo).

Peachpit Promotional Mailings & Special Offers

I would like to receive exclusive offers and hear about products from Peachpit and its family of brands. I can unsubscribe at any time.

Overview


Pearson Education, Inc., 221 River Street, Hoboken, New Jersey 07030, (Pearson) presents this site to provide information about Peachpit products and services that can be purchased through this site.

This privacy notice provides an overview of our commitment to privacy and describes how we collect, protect, use and share personal information collected through this site. Please note that other Pearson websites and online products and services have their own separate privacy policies.

Collection and Use of Information


To conduct business and deliver products and services, Pearson collects and uses personal information in several ways in connection with this site, including:

Questions and Inquiries

For inquiries and questions, we collect the inquiry or question, together with name, contact details (email address, phone number and mailing address) and any other additional information voluntarily submitted to us through a Contact Us form or an email. We use this information to address the inquiry and respond to the question.

Online Store

For orders and purchases placed through our online store on this site, we collect order details, name, institution name and address (if applicable), email address, phone number, shipping and billing addresses, credit/debit card information, shipping options and any instructions. We use this information to complete transactions, fulfill orders, communicate with individuals placing orders or visiting the online store, and for related purposes.

Surveys

Pearson may offer opportunities to provide feedback or participate in surveys, including surveys evaluating Pearson products, services or sites. Participation is voluntary. Pearson collects information requested in the survey questions and uses the information to evaluate, support, maintain and improve products, services or sites; develop new products and services; conduct educational research; and for other purposes specified in the survey.

Contests and Drawings

Occasionally, we may sponsor a contest or drawing. Participation is optional. Pearson collects name, contact information and other information specified on the entry form for the contest or drawing to conduct the contest or drawing. Pearson may collect additional personal information from the winners of a contest or drawing in order to award the prize and for tax reporting purposes, as required by law.

Newsletters

If you have elected to receive email newsletters or promotional mailings and special offers but want to unsubscribe, simply email ask@peachpit.com.

Service Announcements

On rare occasions it is necessary to send out a strictly service related announcement. For instance, if our service is temporarily suspended for maintenance we might send users an email. Generally, users may not opt-out of these communications, though they can deactivate their account information. However, these communications are not promotional in nature.

Customer Service

We communicate with users on a regular basis to provide requested services and in regard to issues relating to their account we reply via email or phone in accordance with the users' wishes when a user submits their information through our Contact Us form.

Other Collection and Use of Information


Application and System Logs

Pearson automatically collects log data to help ensure the delivery, availability and security of this site. Log data may include technical information about how a user or visitor connected to this site, such as browser type, type of computer/device, operating system, internet service provider and IP address. We use this information for support purposes and to monitor the health of the site, identify problems, improve service, detect unauthorized access and fraudulent activity, prevent and respond to security incidents and appropriately scale computing resources.

Web Analytics

Pearson may use third party web trend analytical services, including Google Analytics, to collect visitor information, such as IP addresses, browser types, referring pages, pages visited and time spent on a particular site. While these analytical services collect and report information on an anonymous basis, they may use cookies to gather web trend information. The information gathered may enable Pearson (but not the third party web trend services) to link information with application and system log data. Pearson uses this information for system administration and to identify problems, improve service, detect unauthorized access and fraudulent activity, prevent and respond to security incidents, appropriately scale computing resources and otherwise support and deliver this site and its services.

Cookies and Related Technologies

This site uses cookies and similar technologies to personalize content, measure traffic patterns, control security, track use and access of information on this site, and provide interest-based messages and advertising. Users can manage and block the use of cookies through their browser. Disabling or blocking certain cookies may limit the functionality of this site.

Do Not Track

This site currently does not respond to Do Not Track signals.

Security


Pearson uses appropriate physical, administrative and technical security measures to protect personal information from unauthorized access, use and disclosure.

Children


This site is not directed to children under the age of 13.

Marketing


Pearson may send or direct marketing communications to users, provided that

  • Pearson will not use personal information collected or processed as a K-12 school service provider for the purpose of directed or targeted advertising.
  • Such marketing is consistent with applicable law and Pearson's legal obligations.
  • Pearson will not knowingly direct or send marketing communications to an individual who has expressed a preference not to receive marketing.
  • Where required by applicable law, express or implied consent to marketing exists and has not been withdrawn.

Pearson may provide personal information to a third party service provider on a restricted basis to provide marketing solely on behalf of Pearson or an affiliate or customer for whom Pearson is a service provider. Marketing preferences may be changed at any time.

Correcting/Updating Personal Information


If a user's personally identifiable information changes (such as your postal address or email address), we provide a way to correct or update that user's personal data provided to us. This can be done on the Account page. If a user no longer desires our service and desires to delete his or her account, please contact us at customer-service@informit.com and we will process the deletion of a user's account.

Choice/Opt-out


Users can always make an informed choice as to whether they should proceed with certain services offered by Adobe Press. If you choose to remove yourself from our mailing list(s) simply visit the following page and uncheck any communication you no longer want to receive: www.peachpit.com/u.aspx.

Sale of Personal Information


Pearson does not rent or sell personal information in exchange for any payment of money.

While Pearson does not sell personal information, as defined in Nevada law, Nevada residents may email a request for no sale of their personal information to NevadaDesignatedRequest@pearson.com.

Supplemental Privacy Statement for California Residents


California residents should read our Supplemental privacy statement for California residents in conjunction with this Privacy Notice. The Supplemental privacy statement for California residents explains Pearson's commitment to comply with California law and applies to personal information of California residents collected in connection with this site and the Services.

Sharing and Disclosure


Pearson may disclose personal information, as follows:

  • As required by law.
  • With the consent of the individual (or their parent, if the individual is a minor)
  • In response to a subpoena, court order or legal process, to the extent permitted or required by law
  • To protect the security and safety of individuals, data, assets and systems, consistent with applicable law
  • In connection the sale, joint venture or other transfer of some or all of its company or assets, subject to the provisions of this Privacy Notice
  • To investigate or address actual or suspected fraud or other illegal activities
  • To exercise its legal rights, including enforcement of the Terms of Use for this site or another contract
  • To affiliated Pearson companies and other companies and organizations who perform work for Pearson and are obligated to protect the privacy of personal information consistent with this Privacy Notice
  • To a school, organization, company or government agency, where Pearson collects or processes the personal information in a school setting or on behalf of such organization, company or government agency.

Links


This web site contains links to other sites. Please be aware that we are not responsible for the privacy practices of such other sites. We encourage our users to be aware when they leave our site and to read the privacy statements of each and every web site that collects Personal Information. This privacy statement applies solely to information collected by this web site.

Requests and Contact


Please contact us about this Privacy Notice or if you have any requests or questions relating to the privacy of your personal information.

Changes to this Privacy Notice


We may revise this Privacy Notice through an updated posting. We will identify the effective date of the revision in the posting. Often, updates are made to provide greater clarity or to comply with changes in regulatory requirements. If the updates involve material changes to the collection, protection, use or disclosure of Personal Information, Pearson will provide notice of the change through a conspicuous notice on this site or other appropriate way. Continued use of the site after the effective date of a posted revision evidences acceptance. Please contact us if you have questions or concerns about the Privacy Notice or any objection to any revisions.

Last Update: November 17, 2020