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What Type of Microscope Produces 3 Dimensional Images?


We have all seen the amazing 3D images of biological specimens displayed, but how are they produced and what microscopes are capable of creating such 3D imagery?

Electron microscope

 

In this article I will discuss the techniques and technology used to produce microscopic 3D images and which types of microscopes can perform this task.

In general, microscopes that produce 3D images include dissecting microscopes, confocal microscopes, and scanning electron microscopes.  Other types including transmission electron microscopes and light microscopes may output to 3D utilizing extra steps.  3D images such as volume or surface renderings may be produced in realtime or through the process of 3D reconstruction.

Microscopy has opened the doors to a whole new world of discovery. Prior to the invention of the first microscopes, most information about the unseen, microscopic world was merely theoretical at best. With microscopy, we were now able to get a closer look at these highly impactful organisms and gain more information. However, these microscopic techniques have some limitations.

While the more common light microscopy was able to illuminate the microscopic world, it came at a cost: 2-dimensional (2-D) imaging. Most of the samples had to be flattened in order to allow the light to pass through them in order to create a clear image. Specifically in cytological (cells) or histological (tissue samples), the sample size has to be super thin before being placed between two glass slides in order to be seen due to light penetrance and other issues.

Then on top of that, for even better resolution the investigator would use oil immersion. A drop of specialized oil is placed on the slide to reduce the air between the actual slide and the lens of the microscope. All of that is used to generate a 2-D image.

For 3-dimensional (3-D) images, there has to be a bit more work involved. At one point it was up to the investigator’s imagination to generate these images and simply draw them to the best of their ability. With the use of electron microscopy, we have also been able to view the 3-D surface of cells. As technology has evolved, more has been achieved to go from 2-D to 3-D including developing microscopy that generates real time 3-D imaging.

What do we mean by 3D?

There may be some confusion as to what is actually meant by 3D.  3D refers to viewing something in three dimensions- X, Y and Z and usually being able to rotate the scene, such as with a model of a 3D cell.

This can be achieved by various methods, including viewing in 3D in microscope, realtime 3D rendering, non realtime volumetric or surface rendering, or viewing and manipulating a virtual slide.

Stereo microscopes- Real Time 3D Viewing

The stereo microscope has been around since the late 1600’s. While not as refined as the ones today, it was still a valuable piece of equipment in generating 3D images in real time without as much of the preparation as the light microscopes and the later electron microscopes. Most people today know of these types of microscopes as dissecting microscopes.

The magnification for these microscopes is considerably low. Where compound light microscopes can reach magnifications into the thousands and electron microscopes into the millions, stereo microscopes can only achieve a magnification of about 10x-50x. This is still greater than an actual magnifying glass (2x-3x). This is because the light is directly reflected from the specimen. The lights on the microscope are used to either illuminate the surface of the specimen (top light) and in cases of more translucent specimens illuminate structures within the specimen (bottom light).

The stereo microscope allows for the investigator to look at a specimen that is normally seen with the naked eye.   The image that shows due to the fact that two separate optical paths are utilized including two objectives appears three dimensional, as if you are looking at an object in all its three dimensional reality under an extremely powerful magnifying glass.  The reason things appear 3D is light does not go through a flattened specimen on a slide but rather an intact thick specimen lit from all sides. Stereoscopy therefore allows for the illusion of a 3D image.

dissecting microscope
Stereo microscopy can be very helpful when examining specimens before sampling and preservation. In many ways it can be used to identify certain structures like those seen on these insects.

Scanning Electron Microscopy (SEM): 3-D Images

Scanning electron microscopy (SEM) is one of two types of electron microscopy. What separates electron microscopy from light microscopy is the source used to visualize the image. In light microscopy the source is actually light. The light can have different wavelengths that will produce different variations to the image, but the concept is the same.

The light is passed through the sample. The refracted, or bent, beams pass through a one (simple light microscopy) or more commonly a series of lenses (compound light microscopy). The lenses are what allow for resolution (clarity) and magnification (making the image appear larger) of the image.

Electron microscopy uses a different source: electrons. A beam releases a stream of electrons at the sample. The electrons penetrate the sample. The sample then reflects secondary electrons and X-rays that are then detected to generate an image. This is all done in a controlled environment with very expensive machines.

Scanning electron microscopy is used to take images of the surface of the cells. The scanning electrons are meant to penetrate the surface of the cells slightly in order to produce an image. Many of the images seen include structures such as the cell membrane, cell surface proteins, motility structures like cilia or flagella, and other external parts of the cytoskeleton.

The result is a 3D image of extremely small objects which cannot be seen with any other type of microscope.  Even though the image appears to be 100% 3D, it cannot be measured as there is no depth information.  SEM images can be converted into true 3D objects through the process of 3d surface reconstruction using the data from the SEM image if you want to rotate them.

Because SEM is used to take images of the surface of the cells. The cells have to remain intact. That means these images represent true 3-D images of the cells under examination. Unlike other forms of microscopy, these super thin samples are actually counterproductive since we want to see the intact structures of the cells. The sample sizes could be virtually any size and still have an image generated. The magnification is not as large as transmission electron microscopy, but it is still very impressive and gives a more accurate representation of what happens with the cell membrane and/or cell wall.

SEM microscope image
SEM can be used to see the surface of cells and how those components like the phospholipids and surface proteins interact with other substances including infectious agents.

Transmission electron TEM- Through 3D Surface Rendering

On the other hand, transmission electron microscopy (TEM) requires a bit more work in order to achieve a 3D rendering. TEM requires a more involved sample preparation before the microscopy can even be performed. The sample must be thin so an ultramicrotome is called for to produce a sample that is less than 150 nm in thickness.

Transmission electron microscopy is also more specialized and requires trained professionals in order to use them. Once the sample is prepared and placed, transmitted electrons are passed through the sample. The electrons that get through the sample are then collected in a lens below the sample and an image is generated. Picture a stencil where you place different colors of sand on top. Once you lift the stencil, you are given an image.

In order to generate a 3D image with TEM, serial sections need to be photographed and from there a 3d  model can be created based on the images in the stack . The basic concept is that multiple images are taken of multiple sections of the samples. From there computer software is used to generate a 3D image of the cells and corresponding structures. This process does take time and additional labor, but has been shown to be fairly successful.

TEM microscope image
TEM can be used to locate viruses within certain cells just like in the image above.

Confocal Microscopy – 3D Surface or Volume Rendering

Confocal microscopy is part of the spectrum of fluorescence microscopy. It is used to give more detailed and defined images of cellular structures at different depths within the cells. Unlike light microscopy and electron microscopy, live specimens can be used and are typically still viable after the process is completed with minimal light damage.

confocal microscope image
Volume rendering from a confocal microscope

Confocal microscopy uses fluorescence optics but the process is more refined. Instead of releasing a widespread light over the specimen and using the refracted light to generate the best image possible, a very small focused stream of light is directed through the sample at a very specific depth. The light or point of illumination is then reflected back to the lens without the remaining unfocused light yielding a very small yet highly focused image. This process is repeated throughout the area of the sample to at the same depth to reveal a 2D image with higher resolution.

In order to generate a 3D image, this process must be done multiple times at different depths to generate many 2D images in a “3d stack”. Those images are then combined to generate a 3D rendering of the specimen.  Such a rendering can be a volume rendering or a surface (polygon) rendering.

With newer confocal microscopes, however, it is now possible to render the image on the fly in 3D in the included microscope software.  The future seems very bright for 3D in microscopy.

Virtual Slides

There is another form of 3D microscopic imaging that requires extra work- virtual slides (whole slides).  These are scans of an actual specimen slide taken at different depths and magnifications to give you a digital version of a physical slide which will not degrade over time and can be easily transmitted from computer to computer.  Virtual slides can be viewed using online or software based viewers.

 

Here is an interesting video on how a scanning electron microscope works:

 

Conclusion

As microscopy has grown the technical aspects of generating high resolution images with detailed impressions of structures has become an even more accessible reality. With the application of software technology, a lot of those super detailed while flat images can be utilized to generate 3D renderings of microscopic specimen samples. In some cases, those images can even be taken of live specimens while still leaving them viable for use in other experiments.

As the technology around microscopy grows, perhaps it will come to meet the ingenuity of those that utilize it.

Click the following link to learn what hardware you need for 3D rendering.

How Long Does a 3D Printer Last?


Nothing lasts forever, or so they say. 3D printers aren’t an exception; some last longer than others. 3D printers are not inexpensive, whether its the Ultimaker, Ender 3, or Prusa 3D printer. That is why you have to carefully consider the one that will serve you the longest before you have to replace it.

3d printer printing

Careful use, proper cleaning, and replacements of the 3D printer impact how long the machine will last. So, how long should a 3D printer last and what determines it?

Various 3D printer brands have different lifespans. A good quality brand printer should last you 5 to 10 years.  Stay away from unknown brands and prices too good to be true.  Maintenance is key; otherwise, your printer may break down even before completing the one-year warranty the brands usually offer customers. Longevity of your printer depends on how often you use it.

The longest lasting quality 3D printer brands include Makerbot, Ultimaker, Monoprice, Formlabs, XYZ, Creality, Flashforge, and 3D Systems.  If you have never heard of a 3D printer brand, its best to stay away from it.  There are cheap imitation printers out there that look like  better known ones but will not last long at all, not even a year.

Maintaining your 3D printer shouldn’t be a hassle, especially if you have the right information to guide you. This article will help you learn how to maximize the longevity of your precious 3D printer fully. Read on to find out more!

How Many Printing Hours Does a 3D Printer Have?

A good printer should last approximately 10 years, printing at least four hours each day. The total number of hours the machine could hit in its lifespan could be about 7,500-15,000. But, you have to put in the work to ensure the printer lasts long. The work entails replacements, repairs, and fine-tuning the machine’s parts to secure a long lifespan.

You gain greater benefits beyond extending its lifespan when you maintain the machine. For instance, the machine could have increased printing capabilities, improved speed, and quality. These improvements also mean you cut down on costs spent on seemingly endless repairs when the machine keeps breaking down due to lack of proper maintenance.

Individuals regard older 3D printing machine models as those prone to frequent breakdowns. Technology has improved the brands being churned into the market today. Hence there are 3D printing machines that are more reliable and break down less. However, older brands are still in use even today, the key reason being proper care and maintenance. Without maintenance, even the newer models will break down as often as the older ones are prone to.

What Causes 3D Printer Breaking?

Most 3D printers can last several years if you do your part. “How?” you ask.

A 3D printer has several parts. Some are long-lasting, meaning you don’t have to replace them frequently, while others are the parts that break down often if you don’t maintain them properly. Some wires on the 3D printing machine can break, especially due to their nature as parts that experience constant bending and movements. Breakages happen if your printer doesn’t have the longer-lasting flex cables.

Also, some individuals, especially those in the printing business, love to experiment with various designs and upgrades to maintain a competitive edge in the printing business. While this could be a good idea, you push the machine to its limits, leaving it vulnerable to breakdowns that reduce its lifespan.

Other reasons that contribute to a 3D printer breaking down include:

  • Lack of proper storage for the machine

  • Ignoring minor underlying issues, making them worse as time goes by

  • General carelessness

  • Bumping the machine on hard surfaces

  • Not following the printer’s usage guidelines

What Can You Do to Ensure Your 3D Printer Lasts Longer?

This may seem obvious, but buying a reputable 3D printer is the first step toward a long-lasting machine. A good brand is reliable and durable and doesn’t break down even when a minor issue occurs. Reading other customers’ reviews on the internet about the brand is a good way to know the best brand you can trust for your printing needs.

Next, have the right knowledge to know what to do and when to do it when servicing the 3D printing machine. Knowing the right action to take is key to securing an extended life span for your machine. For instance, you may need to replace certain parts over time.

moving parts of 3d printer

The parts that need frequent replacements include:

  • Fans

  • Linear bearings

  • Nozzles

  • PTFE tubing

  • RAMPS boards

  • Extruders

  • Heatbreak tubes

  • Wires

  • Print bed surface/sheets

  • Stepper motors

  • Belts

Now that you have learned how to ensure a longer-lasting 3D printer, it is important to learn the proper maintenance tips for the machine.

Proper Maintenance Practices for Your 3D Printer

The following are important maintenance tips to ensure the longevity of your 3D printer:

1. Always Keep the Filament Nozzle Clean

A clean filament nozzle is important to maintain a high print quality on the printer. Always clean it to prevent clogging, which may deteriorate a print’s design, affecting its structure. Also, nozzles are one of the 3D printer’s consumables, meaning they need replacing if the print’s quality has severely deteriorated. Replacing the nozzle is the best you can do if cleaning the filament doesn’t improve its function.

   2. Replace Nozzles

There isn’t a specific rule of thumb guiding individuals on the specific time frame necessary to replace or change a 3D printer’s nozzle. However, it is good to change this consumable every three to six months, depending on several factors. The factors include the quality of your printer, what kind of filaments the machine has, and how often you use the printer.

 3. Regularly Lubricate the Machine

When did you lubricate your 3D printer? If you haven’t done so in a while, you could lower the machine’s lifespan. A 3D printing machine comprises various moving metal parts that need lubrication. Lubricating them prevents friction, which is the major culprit for wear and tear on your machine. Don’t overly lubricate the moving parts, as the lubricant could gum up, attracting dust and grime.

 4. Update the Software for Reduced Printer Model Aging

Keeping your 3D printer’s firmware updated is one of the easiest ways to maintain the machine in to-notch shape without breaking a sweat. A 3D printer is similar to your smartphone or laptop, requiring frequent updates to function well. You can single out the machine requiring a software update if the hardware is in good condition, but you feel like something is amiss with the printer. Updated software ensures the machine runs smoothly, preventing certain mishaps that lead to the printers aging prematurely.

5. Regular Cleaning of the Extruder and Build Plate

Operating the 3D printer entails applying glue to the build plate to avoid the prints moving. Glue is a fantastic agent for attracting and sticking unwanted substances like molten plastic to the build plate, resulting in improper prints if not cleaned. The 3D printer’s build plate needs to be perfect if you intend to use the machine longer without experiencing frequent breakdowns.

Some materials may also stick to the extruder when the filament passes through the rollers. These materials wear off from the rollers, altering the rollers’ geometry and ruining the extrusions. Proper cleaning of the rollers ensures the extruder remains spotless, functions properly, and lasts longer.

Also, keep all the other parts clean to prevent dirt and material build-up, causing them to shift and get stuck in other areas. A shift in the machine’s components alters how the printer works, reducing its longevity.

 6. Replace the 3D Printer’s Worn Out Parts

Replacing the machine’s worn-out parts may seem like a no-brainer, but some individuals overlook this aspect until the damage is too extensive to correct. Your machine is made up of several parts with different life expectancies.

Therefore, you need to replace those as soon as they wear out. Failure to do so causes a strain on the machine as it continues to function, the long-term effect being a massive breakdown that will be costly to repair, much like with your car.  The worst-case scenario is that the damage may be irreversible. Utilize frequent replacements to secure the longevity of your 3D printing machine.

 7. Tighten up the Printer’s Screws

Your 3D printer has screws installed to keep the pulleys intact. The screws are placed on the machine’s X and Y axes to facilitate a continuous motion of the pulleys. As the motions continue, so is the likelihood of the pulley screws loosening up over time, resulting in a misaligned 3D printer system. Misalignment hampers the quality and longevity of the printer, a situation you can easily avoid by tightening the pulley screws if they loosen up.

Here are some more tips on 3D printer maintenance:

Bottom Line

It is frustrating to part with your money and buy a printer, only to break down when you need it most. You can take several steps to ensure your 3D printer lasts longer, starting with buying a good quality one in the first place. Then, perform proper maintenance practices based on your machine’s needs, preventing incidents that tamper with how the printer works and lasts in the long run.

orthopedic 3d print

Do you need more information on all things 3D printers? You are in the right place! Our blog has more 3D printing articles for you to sample. See you there!

Why is Weight Painting Difficult?


Weight painting is a clever way to apply weight information to your model smoothly in a pretty intuitive way in order to rig a 3d mesh.

weight painted model

But why is weight painting difficult? Read more to find out why and the solutions to this.

Weight painting can be difficult for a number of reasons including not understanding the basic concept, having many weighted influences affecting a model, using automated weighting unsuccessfully, not having enough practice, and experiencing software errors. Let’s discuss and see how to make it less difficult.

Some of the difficulties you may encounter during the process of weight painting include:

Difficulties in Maya

Despite being one of the most used 3D animation software globally, Maya has a complex toolset.

If it’s your first time working with this software, you might be overwhelmed with the learning curve and spend more time looking for information than actually doing your project.

Another challenging part of using Maya for weight painting is that it requires a user to assign weights to every vertex manually. This process takes time and is prone to errors.

The best way to go around this challenge is;

  • Copy the weights
  • Remove the geometry
  • Copy back the weights to the original geometry

Difficulties in Blender

Although Blender is a powerful tool that you can use to create 3D animations and objects, it is also known for being complicated to use.

The learning curve is so steep that many people avoid using it altogether.

Depending on the video card you use on your computer, Blender may present several challenges such as

  • A black stripe on models – This typically happens when normals for one of the faces point in the wrong direction. You can solve this by having the blender recalculate the normals.
  • Hidden objects – Some images may not show up in your 3D view. If you accidentally press H while searching for another key, you will hide the selected object. To solve this, press Alt+H

Default weights not working

Default weights are a set of weights that can be used to quickly and easily create a new character. All the vertices and faces of the character can be created by painting their colors and adjusting their weights. The default weight is applied when there are vertices in the model with no weights or insufficient weights to correctly define all vertices in the object.

A default weight failure in weight painting occurs when there is no weight defined on any faces or vertices in an object, causing an incorrect surface to be generated when it gets exported.

In the weight painting process, default weights should be applied to the skin, hair, and eyes. But if you have a specific goal in mind for your character’s skin weighting, you have to select all the other bones in the body and clear the default weights before painting them.

There is a way to fix this by applying a new set of default weights which depending on the software can be done with a script.

This script will scan through your mesh automatically and change any bone whose weight is set to “default,” which means it has been cleared from painting by someone else.

Glitches in software

Glitches happen when a program malfunctions and fails to work correctly.

These glitches can be caused by;

  • Trying to save a file in a different format, like PNG instead of JPG, and save it with an extension that is not compatible with your device.
  • Using different software on an incompatible operating system
  • Using the brush resize tool to change the size of the brush in the wrong way

In all honesty, in programs like Maya weight painting may crash the program for any number of unknown reasons out of nowhere

Many possible solutions are available if you need to fix your weight painting issue, such as saving in another format or using another software compatible with your operating system.

To make your weight painting experience pleasant, try these solutions:

1. Proper 3D mesh for animation

Make sure that the mesh is clean, not too heavy, no intersections, no holes.

2. Correct joint placement

It is essential to know how to use joint placement properly for your object to be weighted correctly. If joints are not placed perfectly, they will not be taken into account by the paint system, resulting in incorrect weights.

To properly place the joints in Maya;

Select joint name from paint “skin weights tool.”

Right-click the joint you want to paint, then select influence in the marking menu

Drag across the skin. The joint influences painted vertices in relation to other joints.

3. Correct density in areas of deformation

The correct density in weight painting can be found by how closely the painted strokes are together. If they are too far apart, then it looks airy or feathery and will take up too much surface area on your object. It will be difficult to see what you have painted and look sloppy and unprofessional if they are too close.

Correct density ensures that there are no holes in the mesh due to inconsistent thicknesses.

To be used as a reference for the density of a given area in an image, the user will first need to paint a weight map. The color should represent how dense the pixels within this area should be.

Different areas of an image may have different densities, for example:

  • A black and white image or a line drawing that has no shading will have a uniform density
  • Areas with conspicuously higher contrast may need higher densities to prevent the lines from appearing too thin due to insufficient contrast.

If you have a brush node with node path A to B, Node Path A is set to 100% opacity, and Node Path B is set to 50% opacity. When this brush node moves from point A to point B on your mesh, a stroke begins at 100% opacity and gradually fades out at 50% transparency.

If you want this stroke to be more even or gradual, you can adjust the Density value by going into the properties window and changing the value from 0.0 to 1.0, making it so that your brush will have no deformation weight painting.

4. Even spans

Even spans in your model will make sure that the weight painting process goes as smoothly as possible and doesnt run into unnecessary issues performing the calculations which are complex behind the scenes of the intuitive process.

5. Start with rigid

If you are having problems you can make your weight equal 1 on each joint to begin with and then knowing what you started with add other weights.

6. Practice

To get proficient at weight painting and get over the fear, complete a dozen skinnings of characters, including higher resolution ones. Remember that practice makes perfect.

 

Here is a lesson on how to paint weights effectively in Maya:

 

Conclusion

Despite its benefits, the process of weight painting is not always straightforward and may seem like a daunting task with lots of trial and error.

You can use existing software to create weight-painting, but it requires human intervention and manual labor.

 

Click the following link to learn what 3d rigging is and why its important

Is Light Sheet Microscopy Confocal? Differences and Similarities


Are you confused by the terms light sheet and confocal? Are they completely unrelated or does one fall under the definition of the other?

confocal microscope objectives

In this article I will define the terms and explain exactly how they are related, along with additional information such as comparisons between the two.

In general terms, in light sheet microscopy samples are illuminated by a thin sheet of light emitted from the side.  In confocal microscopy, point illumination as well as a pinhole to block out-of-focus light is used. Both methods use fluorescence and are capable of producing stacks and 3D renderings, with light sheet being more capable of scanning larger tissue volumes at higher speed while confocal being better at depicting subcellular detail at higher magnification.

To understand how this works, you first need to know how a standard SPIM works. As its name implies, in SPIM, the specimen is illuminated with a thin, wide sheet of light to observe the specimen’s fluorescence over time.

In confocal SPIM, there is an additional feature: a pinhole positioned between the detector and the objective lens that allows only fluorescence from one focal plane to be detected at a time.

The pinhole blocks out-of-focus light from above and below the focal plane, which improves image resolution compared to non-confocal SPIMs.

Light Sheet Microscopy Basics

Light-sheet microscopy is a method for non-invasive, high-resolution imaging of biological samples. In this technique, cells are illuminated by a thin sheet of light propagated orthogonally to the light path for image acquisition.

This results in high penetration with minimal photobleaching and phototoxicity and thus allows long-term imaging of live samples.

Light-sheet microscopy benefits from the use of several specialized optical components. These include a condenser to generate the light sheet, an objective to collect emitted fluorescence or reflected light, and a tube lens in between to relay the image from one to the other.

The system can be arranged in two configurations: single plane illumination (SPIM) microscopy, where only one side of the sample is illuminated at a time, and dual-plane illumination (DPIM) microscopy, where both sides of the sample are illuminated simultaneously.

In addition, SPIM can be divided into two types: off-axis SPIM and reflective SPIM. Off-axis SPIM illuminates the sample from 90° with respect to the detection axis.

Reflective SPIM illuminates the sample from below through a specially designed glass-bottom dish or a coverslip coated with a scattering material.

Uses of Light sheet

Cell biology

If you want to visualize organelles in 3D, you can do that with a light-sheet microscope.

This is the most common application that people use it for.

To observe cellular dynamics

The microscope is designed to observe cellular dynamics over a long period in living specimens.

It employs a laser light sheet, a technique that uses a sheet of laser light illuminating the sample from an angle, as opposed to conventional microscopy, where the specimen is bombarded with illumination from all directions.

This means that the cells are exposed to less light than conventional microscopy to be observed for more extended periods without damage.

The critical advance lies in adaptive optics, a technique first developed to improve astronomical images taken by ground-based telescopes.

By measuring distortions caused by turbulence in the atmosphere, adaptive optics can alter the wavefront of incoming light using a deformable mirror.

The result is an image that is much sharper and more detailed than possible.

To visualize gene expression

Light-sheet fluorescence microscopy (LSFM) is a fluorescence imaging technique. It uses a thin sheet of light to illuminate a single plane of the specimen, while fluorescence from other aircraft is suppressed.

LSFM can provide optical sectioning similar to confocal microscopy or multiphoton microscopy by scanning the light sheet with less photodamage and photobleaching.

Moreover, LSFM enables fast 3D imaging of entire cells and organisms without damaging them through photobleaching and phototoxicity. It also allows for visualizing gene expression in whole embryos, with minimal disturbing light penetration into the specimen and surrounding area.

To perform super-resolution imaging

Light-sheet microscopy has been used to perform super-resolution imaging. This is achieved by using a pulsed laser to illuminate a thin section of the sample and taking images at the axial focal plane.

The sample is moved slightly in the z-axis, and the process is repeated. The resulting images are then combined using computational methods, such as iterative deconvolution or super-resolution optical fluctuation imaging (SOFI) analysis, to obtain an image with resolution better than that achievable with conventional microscopes.

Light sheet vs Confocal- Differences and Similarities

Light-sheet and confocal microscopes have several differences and similarities. Here are some of the differences.

Differences

Image Quality

With confocal microscopy the specimen must be illuminated through a pinhole and viewed under a highly narrow angle of view.  This results in a high-quality image because it blocks scattered light from outside the focal plane, which can degrade image resolution.

In contrast, a light sheet is a fragile sheet of light that illuminates the entire sample from the side, creating a more uniform image.  However, because the light is much brighter than in a confocal microscope, it is harder to control the scattered light outside the focal plane (especially for large specimens).

Light Efficiency

The efficiency of a laser can be increased by spreading it over a large surface area. A light sheet microscope takes advantage of this by using lasers to create fragile sheets of light that can cover large areas.

These sheets are created using cylindrical lenses and cylindrical mirrors and go through the specimen one by one. In contrast, only a tiny fraction of the laser energy makes it through the pinhole aperture to illuminate the sample in confocal microscopy.

With confocal microscopes, there are issues of light penetration through thick tissues.  Even if the tissue is fully permeated by a fluorescent antibody marker, it may not be visible for this reason.

Speed

In traditional confocal microscopy, the laser beam is scanned across one sample axis, which is also moved along an orthogonal axis.

In light-sheet fluorescence microscopy (LSFM), the specimen is illuminated by a thin sheet of light that sweeps across it in one direction. At the same time, a detector scans in the perpendicular direction.

This arrangement makes it possible to acquire images at high speed, with very low photo-bleaching and phototoxicity.

LSFM is well suited to live cell imaging and other applications where dynamic processes need to be observed and recorded over time without damaging the sample.

The speed of lightsheet microscopes is what makes them perfect for studying large tissues or even entire organs such as rodent brains that have been cleared with methods like CLARITY. This is best for studying things such as neuron projections from one part of the organ to another.  While light sheet microscopes are achieving better and better resolution, confocal microscopes still dominate in terms of observing cell detail under very high magnification (up to 150x objective).

Similarities

Both types of microscopes can be used to create image stacks and ultimately 3D volume or surface renderings of the objects studied.

Both light-sheet microscopy and confocal microscopes are types of fluorescence microscopes. Both techniques involve laser scanning of a specimen to produce an image.

The main difference is that confocal microscopes use a series of pinholes to exclude light outside the focal plane. In contrast, light-sheet microscopy combines illumination and detection along the same axis using a thin light sheet.

Light-sheet microscopy uses a thin sheet of laser light to illuminate the specimen below or above rather than illuminating it with a point source (as in a conventional microscope).

The thin layer of light reduces photobleaching and phototoxicity, which are significant problems when imaging live specimens over time. The advantage over confocal microscopy is speed; because fluorescence emitted from all points on the specimen are collected at once.

Both light sheet and confocal microscopes can be used to image cleared specimens.

How old is light sheet microscopy?

The Ultramicroscope was the first light-sheet microscope, and was first created in 1903 by Richard Adolf Zsigmondy and Henry Siedentopf. It consisted of a rectangular slit combined with white light. It produced images of microscopic particles and enabled scientists to closely observe the interaction of individual molecules.  However, it was not a fluorescent light sheet microscope.  Those did not appear until the 1990s.

Price of light sheet vs confocal

When it comes to light-sheet microscopy, a typical system costs around 200,000- $400,000, depending on what features are included. The lattice light-sheet approach for in-cell imaging costs about $700,000.

Confocal microscopes are still more popular and more available than light sheet and can be purchased used for a much lower price, but a modern confocal microscope from a well known brand such as Olympus or Nikon, with an average of 4 lasers, will cost around $300,000.

Pricing of either can vary greatly based on the type of imaging you want to do, the type of specimen holders, objective types which can have different characteristics even with the same magnification, and attachments.  You can even buy a light sheet attachment to a confocal microscope, but those can be pricey at $200,000.

Here is a useful video on light sheet microscopy basics:

I hope you found this article useful. Click the following link to learn the maximum magnification of a confocal microscope.

Do Microscopes Invert Images? Why Does it Happen?


Modern microscopes are marvels of optics and technology which allow us to view minute details of cells with great clarity while others permit us to observe living organisms at incredible magnification.

man on microscope

Have you ever wondered the following simple questions:  Do microscopes invert images?  Which kind of microscopes inverts images?  This article discusses answers to these questions and much more. Read on.

Some microscopes invert images because they have multiple lenses and an increased level of magnification, including compound microscopes. They form enlarged, inverted, and real images. However, quite a few microscopes do not invert images including dissecting microscopes. The term inverted microscope does not refer to image inversion.

Why Do Microscopes Invert Images?

An image is inverted because of the reflection of light rays and because it goes through two lens systems: the objective lens and the ocular lens.

Let’s discuss these in detail.

The most essential components of an optical microscope are objectives. These microscope objectives are responsible for the creation of a primary image. Plus, they play a very important part in determining the quality of images that the optical microscope can produce.

The objective is positioned near the specimen being observed and produces the first enlarged image of the object.

Also called an eyepiece, an ocular lens is where you look through to observe the sample. It enlarges the image formed by an objective lens so that you can see it.

The image will first pass through the objective lens and then the ocular lens, and the image will be inverted because of the objective lens’ curvature. The second lens further enlarges the inverted image that is projected from the first lens.

microscope parts

Both objective and ocular lenses are important to have to produce the proper magnification of the image. Most of the magnification happens within the objective lens.

Light Sources and Condensers

The light source, such as a light-emitting diode or an electric lamp, is there under the slide on which the specimen is being enlarged, and it illuminates the sample on the stage.

Condensers are lenses that gather and concentrate light from the illuminator into the sample. They are located below the stage and ensure that sharp images are created with a high magnification.

The light source under the stage helps you view the sample better. This light is then refracted. The light rays converge to produce an upside-down image once the light comes out of the other side.

The focal lengths of a lens determine the magnification of an image. The focal length is a measurement of how powerfully a system diverges or converges light. Magnifying power shares an inverse relationship with the focal length of the lens. The longer the focal length of the lens, the smaller the magnifying power of the microscope.

There are negative and positive focal lengths. Lenses are diverging lenses if they yield a negative focal length. And converging lenses always have positive focal lengths. A positive focal length forms a real image.

Do Dissecting Microscopes Invert Images?

Dissecting microscopes, also called stereo microscopes, do not invert the image of the sample under view because they have a lower total magnification. A dissecting microscope has two separate objectives and eyepieces and a low magnification range (between 10x and 40x).

It gives you an enlarged three-dimensional view of the sample so that you can see more fine details. The two eyepieces see the same object at a different angle, producing the 3D effect.

Compared to other microscopes, the stereo microscope utilizes the reflected light from the sample.

A dissecting microscope is called so because it is often used to conduct the dissection of a sample. This low-power microscope lets you see live samples and carry out dissections under the microscope.

It is often used for the purposes of examining large samples, such as plant parts, insects, and rocks.

Applications of a dissecting microscope

  • Stereo microscopes are used in forensic engineering.

  • Dissecting microscopes are used for examining fractures.

  • These low-power microscopes are used for inspecting circuit boards and watches.

  • Stereo microscopes are used for studying the surfaces of solid objects.

  • Dissecting microscopes are used for microsurgical procedures and dissection.

Advantages of a dissecting microscope

  • Stereo microscopes are used to observe complete samples and not in pieces.

  • Dissecting microscopes are easy to use and carry.

  • It is an important microscopic technique because it can be used in a variety of fields.

Disadvantages of a dissecting microscope

  • Dissecting microscopes are expensive to buy.

  • These microscopes cannot be used to observe tissue structures because they have a low magnification power.

Is an Inverted Microscope One That Inverts Images?

In an inverted microscope, the condenser and the light source are above the stage pointing downwards, while the objectives are hidden under the stage pointing up.

This is a reverse construction of normal conventional microscopes, where the objective lenses are on top of the specimen stage while the source of light and the condenser are under the specimen stage. So, with this microscope, you see the image from down upwards instead of seeing the image from up downwards.

The inverted microscope uses light rays to focus on a sample to create an image that you can see through the objective lenses. The condenser lens focuses the light on the sample or object.

inverted microscope example

The objectives, which are situated below the specimen stage, collect light from the condenser, enlarging the image, which is then sent to the eyepiece lens. The eyepiece lens reflects the light through a mirror. You can easily see the cells through the bottom of the cell culture vessel.

Uses of an inverted microscope

An inverted microscope is mainly used for diagnosis, identification, and sample preparation. Because of its versatility, it can be used for a wide range of tasks. Disintegrated or dried parts of organisms that cannot be seen using the conventional microscope methods can be seen using an inverted microscope.

  • Inverted microscopes are used to view living organisms or cells found at the bottom of Petri plates or cell culture flasks.

  • They are useful in assessing nematology specimens.

  • Inverted microscopes are used in fungal cultural diagnoses.

Advantages of an inverted microscope

  • An inverted microscope lets you view more specimens in a shorter period of time.

  • The risk of crashing an objective into the specimen is less with an inverted microscope.

  • These microscopes save you money and time in the preparation of a specimen.

  • You have larger working distances and can image big specimens as the specimen is above the objective.

  • You can use an inverted microscope to observe cells in large amounts of the medium.

Disadvantages of an inverted microscope

  • The biggest downside is the cost. An inverted microscope is costly to construct and is more complex.

Check out this video to learn about inverted microscopes.

Do Compound Microscopes Invert Images?

Compound microscopes invert images because of their increased level of magnification and two lenses. The image viewed with compound microscopes is two-dimensional.

Also known as a biological microscope, a compound microscope uses a compound lens system to offer magnification in the ranges of 40x-1000x. It consists of two lenses, an objective lens (near the object) and an eye lens (near the eye). While these two lenses produce high magnification, a condenser below the sample stage concentrates the light directly into the specimen.

A compound microscope is also called a high-power microscope because of its high magnification, and it’s often used to observe living cells.

Uses of a compound microscope

  • Compound microscopes are used for medical and forensic research. They help in the identification of diseases in pathology laboratories. Forensic laboratories use these high-power microscopes to identify human fingerprints.

  • Compound microscopes are used in schools for educational purposes.

  • These biological microscopes are used to detect the presence of metals.

  • They are used to observe viruses and bacteria.

Advantages of a compound microscope

  • Compound microscopes are easy to handle.

  • They have their own light source in their base.

  • You can get detailed information about the specimen due to the usage of two or more lenses.

Disadvantages of a compound microscope

  • The specimen can be magnified to a certain extent. It cannot be observed once this limit is achieved.

Learn more about compound microscopes in this video.

Final Thoughts

Microscopes lets you view things you could never see before. We use them to study molecular structures, microorganisms, and cells and their components. In fact, these optical instruments are not just used to observe microbes and bacteria but are also used in several industries.

A lot of industries use these tools for several purposes like manufacturing processes, quality control, and inspection. Additionally, microscopes are used to diagnose diseases and other conditions in hospitals.

There are many different types of microscopes, such as inverted microscopes, dissecting or stereo microscopes, and compound microscopes, to name a few. And some microscopes produce an inverted image like compound microscopes.

Click on the following link to learn how to make microscope slide mounting medium.

What is the Best Way to Normalize Fluorescent Images?


Fluorescence microscopy has become an essential procedure for understanding cellular structures, physiology, and medical diagnoses. It provides clear and quite colorful images of the sample under study. This makes identification and understanding of the biological processes so much easier. However, there are some challenges with fluorescence microscopy when it comes to obtaining good quality and reproducible images.

fluorescent photo

To counter these issues, normalization of the images is done.

Normalization of images in fluorescence microscopy can be done by (a) calibration using a sample of reproducible fluorescence field, and (b) using 10-50% w/v of concentrated Fluorescein or rhodamine between the cover glass and the slide (c) using the same microscope settings which show a good image across all samples.  Anti-quenching agents (eg. AuNP) are also helpful.

Fluorescence microscopy has many applications in the field of biology and medical science. However, it’s quite often difficult to get identical images of the same slide in different microscopes and even in the same microscope if the images are being taken over some time (say, to study a biological process that might complete over a few days).

This is a major drawback of fluorescence microscopy, and it’s often the major reason why it can’t be used under various circumstances (ex. to make certain medical diagnoses), even though it’s an excellent tool to study various structures, proteins, DNA, and biological processes.

The quenching (or photobleaching) of the sample can be prevented by using anti-quenching agents like AuNP (gold nanoparticles). This safeguards the sample’s fluorescence for a longer duration of time and therefore will provide a slightly longer time for the examination of the sample with minimal changes in the fluorescence quality.

Normalization Of Images

Some of the major challenges with the fluorescence microscopy include:

(A) various instrumental factors, like different – microscopes may use different optics and different light detectors, and so the emitted light may have slight variations in different microscopes, 

(B) shading. Because of shading, some areas of the field may appear brighter while some may appear darker, despite having the same amount of fluorophore (the dye), and

(C) Photobleaching. This can be controlled to some extent by reducing the duration of light exposure, and by using anti-quenching agents like AuNP.

The variation between different instruments is managed by calibration using a sample of a reproducible fluorescence field. Thin and uniform fluorescing reference layers are used here for the comparison of the image for image intensity correction and reproducing imaging conditions and quality control. The characteristics of the imaging situation can be summarized in a SIP (sectioned imaging property) chart for future use and comparison.

Shading is managed by applying a concentrated solution of fluorescence dye. Doing this brightens the image uniformly, and also prevents photobleaching of the sample to some extent. This is a simple, pocket-friendly, and reproducible method— making it a convenient method for routine fluorescence microscopy.

Photobleaching can be prevented by using anti-quenching agents (AuNP). Additionally, as mentioned above, the use of concentrated fluorescein dye also provides a certain degree of protection from photobleaching.

The normalization can also be done using certain imaging programs like metamorph, Isee, and Slidebook.

When performing measurements of intensity on a microscope such as a confocal to determine the amount of a protein for which you stained using an antibody, normalization is required because you will not be able to reproduce conditions from one day to another which would result in erroneous intensity values.  In addition, picking an intensity value that is too high would result in washed out images that could not be used for measurements.

In this case you would want to go through your samples and set your laser amount, gain, etc to a value where all the images can be readable for measurements and not blown out.

Some Background on Fluorescence Microscopy

Fluorescence microscopy is the type of microscopy which uses the fluorescence produced by fluorophore stained specific structures/proteins in the specimen to visualize these structures. 

In a regular light microscope, white light is used to illuminate the background. But in fluorescence microscopy, instead of using white light, a specific color of light is used— depending upon what structure is being studied. More on how it works is discussed below.

The sample is stained with a fluorescent dye (flours chrome/fluorophore) and these emit fluorescent light which helps the structure being studied visualized. Alternatively, the gene under study can also be GFP tagged. A GFP tagged DNA will produce a GFP tagged protein, and thus this protein can be visualized easily as well. 

Principle Of Fluorescence Microscopy

Fluorescence microscopy works on the principle that when a certain wavelength of light is directed on the sample, the electrons in the fluorophore (dye) absorb the energy, and then emit light to get stabilized. 

After absorbing light, the electrons in the atoms of the dye jump from the innermost shell to the outer shell due to excitation. But electrons are unstable in the outer shell. To get stable again, they emit the energy out and jump back to the innermost shell again. 

The energy/light/photon thus emitted by the electrons in the dye is the fluorescence light that is observed in fluorescence microscopy.

 

How Fluorescence Microscopy Works

 

  1. The light source in the fluorescence microscopy generates the white light, which then goes through the excitation filter.

  1. The excitation filter allows the light of a certain wavelength to pass only (say, blue light or UV light).

  1. This light is then reflected by the dichroic mirror to the sample.

  1. When the light reaches the sample, the dye absorbs and then emits the energy.

  1. The released energy from the sample goes via the objective lens, passes through the dichroic mirror, then to the emission filter, and lastly to the ocular lens where the observer can view the result.

  1. Fluorescence microscopes usually also have a camera attached to them at the top, which transfers the image to a computer attached.

 

The light given to the sample is of high energy, short wavelength, and the energy emitted by the electrons is low energy and long wavelength. This is the reason that the light given and the light emitted are of different colors (ex. if blue light is given, green light is emitted). 

The following video briefly explains how a fluorescence microscope works:

 

Applications Of Fluorescence Microscopy

 

Some of the advantages of using fluorescence microscopy include:

 

1. The biggest advantage of fluorescence microscopy is that different structures/organelles/proteins within a cell emit different colors, so it’s easier to see these structures and their locations distinctly.

 

2. Since this process does not kill the cells under study, various biological processes can be studied using fluorescence microscopy. Similarly, various pathological phenomena can also be studied.

 

3. Images of the subcellular structures formed by fluorescence microscopy are more magnified and clear when compared to other light microscopes. So, we can obtain much clearer images of these microscopic structures.

 

Disadvantages Of Fluorescence Microscopy

 

1.The major disadvantage of fluorescence microscopy is photobleaching. The fluorophores on repeated exposure to light lose their ability to fluoresce. So, the shelf life of the slides may not be very long (varies from dye to dye).

 

2. Quenching can also occur due to other factors. Quenching refers to the loss of fluorescence due to various factors, like pressure and temperature. This can be dealt with by using anti-quenching agents to some extent.

 

3. Only those structures that fluoresce can be visualized using this microscopy. 

 

Points To Remember About Normalization In Fluorescence Microscopy

 

Due to various microscope-related factors, photobleaching, and shading, obtaining reproducible images in different and even the same microscopes is usually quite difficult. To combat these issues, the following measures can come in handy:

 

1.To deal with the instrument to instrument variation– uniform, and thin fluorescing reference layers can be used. These can be used for comparison and reproducing imaging control.

 

2. The best way to tackle shading is by using a concentrated solution (10-50% w/v) of fluorescein or rhodamine dye. Out of the two, fluorescein dye gives the best result. Using these concentrated dyes also prevents photobleaching to a certain extent.

 

3. Photobleaching can be reduced by limiting the exposure time of the sample with the light, or by focusing the light on an area next to the sample. Anti-quenching agents like AuNP (Gold Nanoparticles) can also help prevent rapid photobleaching.

 

Read the following article to learn the maximum magnification of a confocal microscope.

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