Archive for the ‘Lens’ Category


HardCORR Optics

Choosing a correction collar objective upfront will save you a hassle in the future

“Clayton, I need you to get down to the lab. There’s a problem with the 40x objective.”

I get to the lab after looking over the original quote. My eyes race down the line-by-line configuration to “Objective HI PLAN 40x/0.65.” The numerical aperture, 0.65, says it all. Coverslips only.

I put a slide down on my customer’s Leica DMi1 microscope and visualize it easily for them through the digital camera.

“All good?” I blankly ask, suspecting there is more.

“Now put on a well plate and tell me what happens,” asks my customer.

I grab her flask with a confluent layer of kidney cells in it and place it on the stage. My heart beats faster knowing what’s going to happen next. I’m able to visualize the cells in phase contrast at 20x. Check.

Switch to the 40X objective lens and then, “SHING-CHUNK!” As the 40x objective slides into place, the surface of the lens housing grinds against the stage insert and crashes into the plastic flask.

“Ohhh that is bad,” the PI says to me, “is there a way to fix this?”

Sure enough, the objective bumped into the bottom of the 2 mm thick flask due to the short working distance of the lens. This 40X lens is designed for the 0.17 thickness of standard microscope coverslips and has a free working distance of 0.36 mm. Plastic dishes vary in thickness, but most are 1mm thick. Too thick for the plastic flask the PI is attempting to use with the 40X objective lens. I explain the optical limitations of the lens and we agree that the 20x will get the job done for this flask.


Coverslips or plastic dishes, an impossible choice!

Right about now, the PI is wishing he had chosen the long working distance version of the 40x objective lens. The long working distance version is designed to focus through plastic cell culture dishes and flasks. This objective has a lower numerical aperture than the objective he currently has (0.50 vs. 0.65), but enough working distance to focus through thicker flasks and well plates. These lenses, although ideal for cell culture vessels, are not ideal for slides. When this microscope was purchased the PI prioritized slides over plastic dishes at 40X. It’s clear now that he needs an objective lens for both plastic vessels and slides.


If only there were a lens that could adjust for coverslips and plastic…

The product managers at Leica recognized that users need a lens for both plastic and glass, which provides an adjustment for either application. Leica offers an option for mid-range magnifications on inverted microscopes called a “correction collar objective” (CORR). The CORR brings flexibility and savings to imaging. A twistable, ribbed ring encompasses the circumference of the objective, marked by the numbers “0, 1, and 2.” These numbers stand for the thickness of the vessel (mm) that light must go through in order to properly focus on the sample. For slides, the correction collar can be adjusted to the “0” position to compensate for the thin coverglass. For well plates and petri dishes, the objective can be adjusted to the “1” position for optimal focus. Lastly, for vessels that utilize thick 3D matrix media, the lens can be adjusted closer to the “2” position for optimal focus and contrast.


So much value in CORR lenses

The CORR method saves users headache, space, and the cost of purchasing two objective lenses. Alternatively, choosing one lens design will limit the types of compatible vessels ultimately causing frustration and limitations to the types of experiments that can be completed. The investigator could go out and buy a revolving microscope, but the user is still limited by the lens that is being switched!

The use of two 40x lenses, with clear tradeoffs of numerical aperture/working distance, takes up an extra objective lens position. Depending on the number of objective lens positions on the microscope, two lenses may force researchers to have to put another magnification on the shelf. Lastly, purchasing a CORR objective in the beginning means the lab won’t have to duplicate spending. Users won’t be forced to purchase a second 40x lens when it becomes apparent that current experiments require long plastic or glass.


Seeing is believing

Fast forward two weeks, my fingers clench a test slide and a CORR 40X lens rests safely in my jacket pocket. I take off the existing non-CORR 40x from the DMi1 and slip on the new one. The ribbed ring marking rests under “0”, and my slide sits on the stage.

Center.

20X

Focus.

40X

Focus.

Boom.

“Very good, Clayton. Now my flask.”

The plastic flask full of a neon red liquid comes out of the incubator and is placed on top of the DMi1. I twist that CORR to “1,” and go back to my 20x lens.

Focus.

40X

Fingers crossed now. My memory is vivid. I can hear the “SHING-CHUNK” of it grazing the harsh metal stage and crashing into the unforgiving plastic.

Flip.

Silence. No sound. Only a clear layer of phase cells after a tad of focusing. “Nailed it,” I quietly said to myself.


Trust W. Nuhsbaum

Be hardCORR and choose Leica’s specialized CORR lens when imaging at 20X and 40X for glass and plastic. Don’t be limited to the types of vessels that can be used on your microscope – be limitless! When it comes to CORR, always better to have it and not need it than to need it and not have it.

Trust the microscope specialsts and imaging specialists at W Nuhsbaum to learn about your application and recommend the CORRect lens for the application.

Color Infidelity: Why Choosing the Wrong Lens is Like Cheating on your Data

The Microscope Objective Lens is a Critical Decision

When choosing a microscope and the associated lenses, many microscope users understand that the objective lens is a critical component to producing an excellent image. However, many people, when faced with the ultimate decision of what objective lens to select, do not have all the information to make an informed decision.

Simple and easily digestible information provided by lens manufacturers include immersion and numerical aperture. This information is in large print on the lens and makes it into the lens description on every quote. Aspects that are less obvious are levels of planar correction, registration in Z, lens coatings, and correction collars. These items are less obvious on a quote but critical to an excellent image that produces equally excellent data.

The objective class you choose, such as an Achromat, Semi-Apochromat, or Apochromat, will affect the final image. The ability of a lens to efficiently pass light and have the respective wavelength reach an identical focal plane is what the user observes in every image. When light is passed inefficiently at different points in the light spectrum or when wavelengths of light reach different focal planes, the final image is degraded which influences resolution, contrast, and color reproduction.


Correcting Lenses for Color in Brightfield

Dominant sources of distortion change with magnification, however, as a general rule correction is more critical at higher magnification. The simple answer is that imperfections are, well, magnified. However, regardless of magnification there are critical distortions that need to be corrected for an ideal image. Although not a complete list, these include field curvature (flatness of field), spherical aberration, image distortion, and axial color.

Without proper correction images appear out of focus on the edges (field curvature), blurry (spherical aberration), oddly shaped (distortion), and color fringes (axial color). Many of these corrections are addressed in every objective lens, however, the degree to which they are corrected depends on the name of the lens. For instance, although imperfect, Plan Apochromat has a higher degree of correction than Plan Achromat. Both are corrected, just at different levels.

In addition, although there are industry standards for lens correction, there is variance between manufacturers. In other words, just because two companies are presenting Plan Apochromat lenses does not mean they are identical in the quality of their correction.

All of the sources of a degraded image come down to contrast. Assuming the microscope has been aligned for Kohler illumination, the contrast provided by the lens provides the clean edges, crisp and vibrant color in a final image.


Correcting Lenses for Color in Fluorescence

Fluorescence presents its own set of challenges because of the nature of single wavelength excitation and emission and low light. Since glass passes light at different speeds depending on the wavelength being used, correcting for spherical aberration is a critical component particularly in multi-channel fluorescence applications.

Without correcting for spherical aberration, features in different fluorescence channels will appear in different focal planes. This is most noticeable in achromat lenses, but it can be detected in Plan Apochromat lenses in applications such as confocal microscopy.

In addition to Z registration of each wavelength, the objective lens will also pass light more or less efficiently based on wavelength. For instance, assuming the light source is providing an equal number of photons at each wavelength of light, an objective lens will pass the 500nm wavelength more efficiently than 400nm. This mean that labels that emit at the 500nm wavelength will appear brighter simply because of the objective lens being used!

Brightness variability is present throughout the visible light range, however, it can be corrected – albeit differently by each manufacturer. A general rule of thumb is that an Apochromat will be superior, however, without the transmission data for each lens, or a spectrophotometer, it is impossible to know.


Immersion matters

Whether you are imaging histology slides, observing adherent cells on a live cell imaging system, or focusing deep into brain tissue, there is a lens that is specific for the application. One of the most important, and often overlooked features of the lens is immersion and numerical aperture (NA). Similar to camera megapixels, many assume the higher the NA the better the resolution. Not true! The better the contrast the better the resolution!

As explained earlier, eliminating aberrations and distortions improve contrast which in turn improve resolution. So although NA is an important feature to resolution, it is not the only feature.

When selecting an immersion lens it is important to consider the medium that will be used. Will there be an aqueous medium as with live cells, dehydrated and mounted tissue section, or fresh brain tissue in solution? Each medium will dictate whether oil, glycerol, or water immersion is required. Without the immersion medium matched to the mounting medium distortions will degrade the resolution. One is much better selecting a lens with a lower NA that matches the mounting medium!


Trust W. Nuhsbaum, Inc

When evaluating lenses and microscope systems having a person to guide you through the decision making process is critical. The ability to offer information and expertise will ensure that you, the user, will end up with a system that is optimized for your experiments.

The microscope and imaging specialists at W. Nuhsbaum, Inc. have the expertise and resources available to make proper recommendations that go beyond megapixels and numerical aperture. Trust W. Nuhsbaum, Inc. to guide you through the microscope configuration process.