Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (2023)

Introduction

Maximizing the on-sample field of view in fluorescence microscopy is of increasing relevance across a wide range of applications, including high content screening of large fields of cells, imaging of the developing embryo, neuron mapping and tissue imaging.

Selecting the right camera to match with your chosen objective for maximizing field of view, while maintaining good imaging clarity, can be a daunting endeavour to many. In this technical note, we cover the primary considerations and provide a guide to matching camera sensor properties to the microscope’s magnifying conditions, without going into exhaustive detail.

Key Parameters and Trade-offs

When matching camera to magnification conditions, there are some fundamental considerations to balance:

  • Resolving Capability (Nyquist Over-sampling) – This concerns matching magnification and objective Numerical Aperture (NA) to pixel size, such that we are sufficiently over-sampling the optical diffraction limit. However, the smaller the pixel, or the greater the magnification we use to achieve this condition, the fewer photons we will collect per pixel, thus adversely influencing signal to noise ratio. To compensate for reduced signal to noise, we are often tempted to use higher illumination intensity (not good for live cell phototoxicity or dye photobleaching) and/or longer exposure times (not good for following dynamic events).
  • Sensor size – Ideally we want to adopt an optical approach that fills the entire available sensor area, thus maximizing field of view. For sensors that are larger than the Field Number of the microscope, we can use an additional magnifying coupler, readily available from Andor for use with the large field of view Sona 4.2B-11 back-illuminated sCMOS (see Appendix). Again though, a trade-off to be borne in mind is that greater magnification, while helping attain Nyquist clarity, results in fewer photons per pixel.

Key camera parameters to consider in this exercise are:

  • Pixel size (given in µm)
  • Dimension of the sensor diagonal (given in mm)

Key microscope parameters to consider in this exercise are:

  • Objective magnification (e.g. 40x, 60x, 100x)
  • Objective NA
  • Additional coupler magnification
  • Field Number of microscope (given in mm)

Example Optical Configurations using Andor sCMOS Cameras

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Tables 1 and 2 show a series of example optical configurations that can be obtained using a selection of Andor sCMOS cameras. Table 1 focuses on the back-illuminated Sona 4.2B-11 (4.2 megapixel) and Sona 2.0B-11 (2.0 megapixel) cameras, each with 11 µm pixel size. Table 2 focuses on the microlens front-illuminated Zyla 4.2 PLUS and Zyla 5.5 cameras, each with 6.5 µm pixel size.

Key Input Parameters

  • Sensor format (horizontal pixels x vertical pixels)
  • Pixel size
  • Objective magnification and NA
  • Coupler magnification (if applicable)

Key Output Parameters

  • Field of View on sample (mm)
  • Oversampling (number of pixels oversampling the resolution limit)
  • Minimum port diagonal required

Assumptions

  • We have used the Rayleigh equation for calculating the resolution limit
  • We are assuming that ideal oversampling is 2.3 or greater, but that between 2 and 2.3 will also yield acceptable resolving capability.
  • While a wide range of objective lenses exist with various combinations of magnifications and NA, for the purpose of this exercise we have attempted to select some common objective lens parameters: 100x with 1.49 NA; 60x with 1.4 NA; 40x with 0.95 NA.

Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (1)

Figure 1: Schematic representation of how the sample area is captured and magnified onto the camera sensor. The challenge is to select an optical configuration and sensor selection that maximizes on-sample field of view while maintaining the ability to resolve fine structure.

Camera ModelRegion of InterestObjective Mag (x)NACoupler Mag (x)Over-SamplingFOV diagonal on sample (mm)Port Diagonal required (≥mm)
Sona 4.2B-112048 x 2048601.41.52.140.3521
2048 x 2048601.422.850.2716
2048 x 2048400.951.52.100.5321
2048 x 2048400.9522.800.4016
Sona 2.0B-111400 x 14001001.4912.230.2222
1400 x 1400601.41.52.140.2415
1400 x 1400601.422.850.1811
1400 x 1400400.951.52.100.3615

Table 1: Example optical configurations that can be obtained using Sona 4.2B-11 (4.2 megapixel) and Sona 2.0B- 11 (2.0 megapixel) back-illuminated sCMOS cameras.

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Key Conclusions from Table 1

  • Sona 4.2B-11, with the fully accessible 4.2 megapixel sensor, offers the largest on sample field of view while satisfying the NyQyist oversampling criteria for resolving clarity. For example, a 60x / 1.4NA objective combined with an additional 1.5x coupler magnification, means that a microscope port diameter of 21mm or greater can be used to image a large 0.35mm diagonal across the sample. Use of a 40x / 0.95 NA objective extends this to 0.53mm on-sample FOV diagonal, superb for developmental embryo imaging.
  • By carrying out the additional magnification onto the large 4.2 Megapixel sensor of Sona 4.2B-11, we can make use of relatively small microscope port sizes and Field Numbers, thus ensuring compatibility across a broad range of microscopes. See Appendix for details on the Andor Magnifying Coupler Unit, which can be ordered alongside the Sona 4.2B-11.
  • Sona 2.0B-11, with 2.0 megapixel sensor, offers a 22mm diagonal sensor and standard C-mount. This is an ideal match for modern microscopes that facilitate a 22mm c-mount port,1 maximizing the field of view available through this popular coupling standard.
  • The Sona 2.0B-11 model may be coupled with a 100x objective, without need for additional coupler magnification, yielding Nyquist oversampling. However, additional coupler magnification can of course be used to adapt the Sona 2.0B-11 for better use with 60x and 40x objectives.

Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (2)

Figure 2: Field of View comparison between Sona 2.0B-11 and Sona 4.2B-11. Captured using a Nikon Ti2 with 60x objective and integrated 1.5x tube lens. The Sona 4.2B-11 has 114% more pixels and is ideally suited to maximizing the on-sample field of view. The Sona 2.0B offers a 22mm field of view and is ideally suited to 22mm C-mount ports.

Camera ModelRegion of InterestObjective Mag (x)NACoupler Mag (x)Over-SamplingFOV diagonal on sample (mm)Port Diagonal required (≥mm)
Zyla 5.5 or Neo 5.52560 x 2160601.412.410.3622
2560 x 2160400.9512.370.5422
Zyla 4.2 PLUS2048 x 2048601.412.410.3119
2048 x 2048400.9512.370.4719

Table 2: Example optical configurations that can be obtained using Zyla 5.5 / Neo 5.5 (5.5 megapixel) and Zyla 4.2 (4.2 megapixel) microlens front illuminated sCMOS cameras.

Key Conclusions from Table 2

  • Zyla and Neo cameras each offer smaller 6.5 µm pixels, thus are ideally suited to 60x and 40x objectives, achieving Nyquist oversampling of the diffraction limit without need for additional coupler magnification.
  • Zyla 5.5 and Neo 5.5, each housing a 5.5 Megapixel sensor, yield the largest available field of view with 60x objectives (0.36 mm) and 40x objectives (0.54 mm). These models, particularly with 40x objectives are superb for high content screening, tissue section and embryo imaging with high clarity.
  • Zyla 4.2 PLUS requires only a 19mm (or greater) C-mount microscope port, thus ensuring compatibility across a broad range of microscopes.
  • Zyla 5.5 and Neo 5.5 each offer a 22mm diagonal sensor and standard C-mount. This is an ideal match for modern microscopes that facilitate a 22mm c-mount port,1 maximizing the field of view available through this popular coupling standard.

Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (3)

Figure 3: Field of View comparison between Zyla 4.2 PLUS and Zyla 5.5. The Zyla 5.5 has 32% more pixels and is ideally suited to maximizing the field of view from microscopes with a 22mm C-mount port.

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Back-illuminated sCMOS FOV: Competitive Comparison

(a) Sona 4.2B-11 – Largest Field of View

The Sona 4.2B-11 model offers the largest field of view solution, compared to competitive back-illuminated sensors that also use the same GPixel GS400 BSI sensor type.

The Sona 4.2B-11 is native F-mount and can be compared against “Competitor A” below, a camera using the same sensor but cropped down to 1608 x 1608 pixel format. By cropping the sensor down, this camera can avoid sensor glow issues that affect the edges of this sensor. However, Sona 4.2B-11 uses a unique Anti-Glow Technology approach that enables the full native 2048 x 2048 of the array to be harnessed. Figure 2 shows the 62% larger field of view advantage offered by Sona 4.2B-11.

The Sona 4.2B-11 is native C-mount and can be compared against “Competitor B”, a camera using the same sensor but cropped down to 1200 x 1200 pixel format. Figure 5 shows the 38% larger field of view advantage offered by Sona 2.0B-11, while still fitting within a C-mount aperture. This is ideal for microscopes that offer a 22mm C-mount port. For mounting on microscopes with smaller ports, the user can readily choose one of the pre-defined Region Of Interest (ROI) sizes, or alternatively, the port can be used alongside a magnifying C-mount coupler.

Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (4)

Figure 4: “F-mount competitive solutions” - Field of View comparison between Sona 4.2B-11 and a competitor Fmount camera, utilizing the same GS400B back-illuminated sCMOS sensor but restricted to 1608 x 1608 max resolution. Captured using a Nikon Ti2 with 60x objective and integrated 1.5x tube lens. The Sona 4.2B-11 has 62% more active pixels and offers a compelling field of view solution.

(b) Sona 2.0B-11 – One camera, Multiple ports

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The Sona 2.0B-11 is native C-mount and is adaptable to various microscope c-mount port diameters, up to 22mm. The 1400 x 1400 full array size of this model is suited to modern 22mm C-mount ports and maximizes the field of view available through this common mount type.

However, pre-configured, centrally positioned ROIs are available, directly relating to various smaller microscope port sizes:

ROI SizeC-Mount Port DiameterExample Microscopes
1400 x 140022 mmNikon Ti2, Olympus IX83/73
1220 x 122019 mmLeica DMi8
1157 x 115718 mmVarious research microscopes

Table 1: Pre-configured ROIs of the C-Mount Sona 2.0B-11 model, shown alongside the corresponding microscope Port Diameter / Field Number for which they are optimized.

Alternatively, smaller ports can be used with the full 1400 x 1400 array size by utilizing the Andor Magnifying Coupler Unit (see appendix). This is a coupler that can readily connect to the port, expanding the image available from the microscope onto the larger sensor area. A 2x coupler also has the benefit of achieving Nyquist resolution utilizing a 60x objective, which in turn further optimizes the on-sample field of view.

Appendix

Andor provide an optional Magnifying Coupler Unit (MCU) accessory which can be used alongside the Sona 4.2B-11 in order to utilize the full field of view of this large sensor with several common types of modern research fluorescence microscopes. It can be used to adapt both Sona 4.2B-11 or Sona 2.0B-11 for use with 60x and 40x objectives, thus increasing the on-sample field of view while also maintain Nyquist resolving clarity. Since the image is being 2x magnified onto a 32mm diameter sensor area, then the MCU can be attached to any port that offers an image output of 16mm or greater. This describes the vast majority of available ports.

For further details, please refer to the specification sheet for the Andor Magnifying Coupler Unit.

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Optimizing Field of View and Resolution in Microscopy- Oxford Instruments (5)

Figure 6: Andor’s Magnifying Coupler Unit

References

  1. Nikon Eclipse Ti2 dimensions: https://www.nikon.com/products/microscope-solutions/lineup/inverted/ti2/spec.htm

FAQs

How do you optimize the resolution of a microscope? ›

In order to increase the resolution (d=λ/2 NA), the specimen must be viewed using either shorter wavelength (λ) light or through an imaging medium with a relatively high refractive index or with optical components which have a high NA (or, indeed, a combination of all of these factors).

How do you increase the field of view on a microscope? ›

Higher power lenses will allow you to view tiny objects, so the angle of view will be small; low power lenses will do the opposite and let you view bigger (wider) objects.

What are three factors necessary to optimize to produce the highest quality image using a microscope? ›

Using the right equipment, color-balanced illumination, and good microscopy techniques will ensure quality images when practicing photography through the microscope.

How can you improve the quality of an image on a microscope? ›

In general, choosing a higher-magnification lens produces higher-resolution images that yield better-quality image analysis results. However, choosing a lower-magnification lens offers a larger field of view so that you can image more cells per image, which can improve the statistical robustness of your results.

What are three factors that affect microscope resolution? ›

The primary factor in determining resolution is the objective numerical aperture, but resolution is also dependent upon the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast-enhancing methodology either in the optical system of the microscope or in the ...

What are two ways we can increase the resolving power of a microscope? ›

Solution : (i) By using light of smaller wavelength, <br> (ii) By increasing refractive index of the medium between object and objective lens microscope.

Can we increase our field of view? ›

Changing the lens changes the FOV. To increase FOV and capture more of a scene, a wide-angle lens -- which displays a wider field of view than our human vision -- is used. Similarly, to decrease the FOV, a zoom lens can be used. In general, a smaller focal length lens increases the angle and the FOV.

When you increase magnification you increase the field of view? ›

In short, as magnification increases, the field of view decreases. When looking through a high power compound microscope it can be difficult to determine what you will see through the eyepieces at different magnifications.

What causes the field of view to reduce microscope? ›

The field of view is a rounded area measured by its diameter, typically in millimeters or micrometers. Importantly, as our microscope objectives gain higher magnification and look closer into a sample, their overall field of view shrinks because the precise area they are looking at is further defined.

How do you optimize images for the best performance and quality? ›

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What are the factors affecting the resolution power of a microscope? ›

The wavelength of light, refractive index, and angular aperture are the significant factors that affect the resolving power.

What parameter is most important for image resolution in a microscope? ›

The numerical aperture (NA) is an important value for microscope objectives, which defines their resolution and luminous intensity. It measures the ability of the objective to gather light and resolve fine specimen detail at a fixed object distance.

Which tool improve the clarity of an image? ›

Fotor's AI photo quality enhancer automatically enhances photo resolution instantly to make the blurry photo clear without quality loss.

What can be done to improve contrast and resolution when using a microscope? ›

Contrast may be improved by placing suitable apertures or filters within the optical path, either in the illuminating system alone (dark ground or Rheinberg illumination), or in conjugate planes in the imaging system (e.g. for phase contrast, differential interference contrast or polarised light microscopy).

Is there a way to improve image quality? ›

The best way to get high-resolution images is by using the right camera for the job. But when that's not an option — or you're looking to improve older digital photos — Adobe Photoshop and Adobe Photoshop Lightroom can help. Experiment with Super Resolution and resampling to see how far you can push your image quality.

What two factors determine resolution? ›

As discussed above, the primary factor in determining resolution is the objective numerical aperture, but resolution is also dependent upon the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast enhancing methodology either in the optical system of the ...

What limits the resolution of a microscope? ›

In the 1870s, Ernst Abbe explained why the resolution of a microscope is limited. Since the microscope uses visible light and visible light has a set range of wavelengths. The microscope can't produce the image of an object that is smaller than the length of the light wave.

What is the importance of resolution in microscopy? ›

Microscope resolution is the most important determinant of how well a microscope will perform and is determined by the numerical aperture and light wavelength. It is not impacted by magnification but does determine the useful magnification of a microscope.

How can we increase magnification and resolution? ›

A simple hand lens can increase the magnification and resolution by about 20 times their actual size by increasing the visual angle. A hand lens is often used to observe objects in the field. A compound microscope is capable of magnifying objects up to 1000 times their actual size.

How can resolving power of the instrument be increased? ›

Resolving power for the instrument is found to be 0. 61λμsinθ , UV light has short wavelength, hence higher resolving power. Oil is optically denser than air, that is, its μ is greater than that of air. Thus immersing in oil would increase the resolving power.

Which instrument does passes the resolving power? ›

Resolving Power of Microscope

The resolving power of a microscope is defined as the reciprocal of the distance between two objects which can be resolved when seen through the microscope.

What affects field of view? ›

Field of view (FOV) is the maximum area of a sample that a camera can image. It is related to two things, the focal length of the lens and the sensor size.

Why is field of view more important? ›

Well, in terms of photography it describes the amount of scene captured by the camera. This is important because to some extent it determines what we can see in a photo. A camera with a very small FOV will only capture a small part of the total scene, so we won't get to see much.

What is the maximum field of view? ›

The vertical range of the visual field in humans is around 150 degrees.

What is the relationship between resolution and magnification? ›

Magnification is the ability to make small objects seem larger, such as making a microscopic organism visible. Resolution is the ability to distinguish two objects from each other. Light microscopy has limits to both its resolution and its magnification.

Why field of view is smaller with magnification increases? ›

The light intensity decreases as magnification increases. There is a fixed amount of light per area, and when you increase the magnification of an area, you look at a smaller area.

Does increasing magnification decrease depth of field? ›

In digital and video microscopy, the shallow focal plane in the target of the camera tube or CCD, the high contrast achievable at high objective and condenser numerical apertures, and the high magnification of the image displayed on the monitor all contribute to reducing the depth of field.

What is the meaning of resolution and field of view in microscope? ›

There are two quantities that encapsulate a pretty good idea of a microscope's performance; they are the resolution and the field of view. Put simply, the resolution is the smallest feature you can see with the microscope, and the field of view is the size of the region of the sample you can see at once.

Does resolution affect field of view? ›

A larger horizontal resolution directly increases the horizontal field of view, and a larger vertical resolution increases the vertical field of view.

Which objective provides the greatest field of view in microscope? ›

The field of view is largest on the lowest power objective. When you switch to a higher power, the field of view closes in towards the center.

Why is image optimization important? ›

Optimizing your images helps to ensure a better user experience and that you meet users' expectations. SEO - Image optimization will help to ensure that your images rank in image searches on Google, and it will also be beneficial to the overall SEO (search engine optimization) of your website.

What elements of an image should be optimized? ›

10 Steps To Optimize Images for SEO
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19 Sept 2022

What does it mean to optimize image? ›

What is image optimization? Image optimization is about reducing the file size of your images as much as possible, without sacrificing quality, so your page load times remain low. It's also about image SEO—that is, getting your banner and product images to rank highly on Google and other image search engines.

What are the parameters affecting resolution? ›

Resolution is measured by dividing the difference in peak retention times by the average peak width. Resolution can also be expressed in the Resolution Equation as a combination of the factors (separation, efficiency, and retention) that affect this value.

Which Colour should be used to increase the resolving power of microscope? ›

According to the above formula, the resolving power will be more for the light having a minimum wavelength. As the wavelength of the violet light is minimum. So resolving power for violet will be maximum. Hence option 3 is correct.

Why is resolution more important than magnification? ›

While high magnification without high resolution may enable observers to view tiny microbes, it won't allow them to identify between microbes or their sub-cellular sections. Therefore, it's safe to say that microbiologists rely more on resolution because they want to distinguish between microbes and their subsections.

What are the 3 parameters in microscopy? ›

Regardless of the microscope technique employed to generate specimen contrast, the three fundamental parameters that govern image quality are resolution, magnification, and contrast.

Which microscopy technique has the highest resolution? ›

Spectral precision distance microscopy (SPDM)

This achieves an effective optical resolution several times better than the conventional optical resolution that is represented by the half-width of the main maximum of the effective point image function.

What unit is used to describe the resolution of a microscope? ›

Like the compound light, the resolution for a confocal microscope is about 1.2 nanometers. Scanning Electron Microscope Resolution: In a SEM, an electron beam scans rapidly over the surface of the sample specimen and yields an image of the topography of the surface. The resolution of a SEM is about 10 nanometers (nm).

How can I increase the resolution of an image without losing quality? ›

How to Enlarge an Image in Photoshop
  1. Open your image in Photoshop.
  2. Go to the Image Size dialog, check resample, and select "Preserve Details" in the corresponding dropdown menu.
  3. Make sure the Resolution is set to 300 Pixels/Inch.
  4. Set Width and Height to inches and adjust to enlarge your image.

Which tool determines the sharpness and clarity of an image? ›

Resolution is measured in megapixels. Generally, the more pixels, the more detail you'll see (considering the same sensor size, lens quality, and settings). Many times, especially when we talk about smartphone photography, we rely on the megapixels of the camera to determine what is best in terms of sharpness.

What are image sharpening techniques? ›

Unsharp masking (USM) is an image sharpening technique, first implemented in darkroom photography, but now commonly used in digital image processing software. Its name derives from the fact that the technique uses a blurred, or "unsharp", negative image to create a mask of the original image.

How do I increase the resolution and clarity of an image? ›

Change resolution by adjusting pixel dimensions.

If you have specific needs for the dimensions or size of your image — like a poster at a certain print size — check the box for Resample. This allows you to adjust the print size and resolution independently, which changes the number of pixels in the image.

How can you tell if an image is high resolution? ›

Right-click on the image and then select "Properties." A window will appear with the image's details. Go to the "Details" tab to see the image's dimensions and resolution.

What makes an image high quality? ›

Count your pixels

Hi-res images are at least 300 pixels per inch (ppi). This resolution makes for good print quality, and is pretty much a requirement for anything that you want hard copies of, especially to represent your brand or other important printed materials.

How does an electron microscope get better resolution? ›

Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light. Electrons have much a shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.

How can you increase the resolution on your microscope quizlet? ›

How can you increase the resolution of your microscope? Increasing the objective lens then adjusting the fine adjustment knob will increase the resolution.

How can you improve resolution at high magnification? ›

Further magnification of the image will not reveal more details. The only possibility to increase resolution is to switch to an objective with a higher resolving power, to use a shorter wavelength of light or to generally improve the optics.

Which type of microscope gives the best resolution? ›

Different types of microscope have different resolving powers. Light microscopes let us distinguish objects as small as a bacterium. Electron microscopes have much higher resolving power – the most powerful allow us to distinguish individual atoms.

Why is resolution so important in microscopy? ›

The resolving power of a microscope is the most important feature of the optical system and influences the ability to distinguish between fine details of a particular specimen.

Which objective provides the greatest field of view? ›

The 4x objective lens has the lowest power and, therefore the highest field of view. As a result, it is easier to locate the specimen on the slide than if you start with a higher power objective.

What is used to increase resolution? ›

Increasing the number of pixels is called upsampling, which adds data to the image. When you increase the number of pixels in an image without adjusting the dimensions, you add more pixels into the same amount of space and increase the resolution (or amount of detail) held within each inch.

Which objective provides the highest resolution and magnification? ›

Oil Immersion Objective Lens (100x)

The oil immersion objective lens provides the most powerful magnification, with a whopping magnification total of 1000x when combined with a 10x eyepiece.

What is resolving power of optical instruments? ›

It is defined as the inverse of the distance or angular separation between two objects which can be just resolved when viewed through the optical instrument.

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