Trends in Microscopy

Goeggel, Daniel; Schué, Anja

September 19, 2012

Trends in Microscopy

How Much "Digital" Do You Really Need?

There are digital cameras, digital TV sets, digital picture frames, digital schools on the internet. Cryptologists design digital signatures, communication researchers speak of digital identity. Digital may be an overused buzzword, but digital technology has undeniably revolutionized our world ever since the invention of the computer and will continue to do so in future. The digital revolution has also had a radical impact on the field of microscopy. The beginnings were made by digital cameras, which provided users in all fields of microscopy with better documentation and analysis facilities. Today, there are digital microscopes on the market which are a far cry from traditional microscopy methods. New technologies always attract plenty of attention, and after all, digital microscopes offer a host of advantages. However, this does not mean they can simply replace all the world’s traditional microscopes. It’s worth taking a closer look to identify the limitations of digital microscopy as well as the benefits.

Quality for surface analysis

Digital microscopy offers clear advantages for a large number of industrial quality inspections, particularly for surface analysis. Fracture analysis and the analysis of inclined or vertical surfaces, or in situ inspection of large components such as turbine rotors are just a few examples of applications where digital microscopes can show their full potential. For some applications, however, a traditional solution with stereo- or light microscopes is more practical and cost-effective. Qualified advice and extensive application know-how are therefore essential for choosing the right solution. But what are the key criteria for the successful use of digital microscopes, and what are the differences between digital and traditional microscopy?

What is a digital microscope?

First of all, a digital microscope has no eyepiece to look through. The sample is directly imaged on the monitor. This enables the user to view and analyze the sample in a single pass using the software, sitting in a comfortable and relaxed position. The individual components of a digital microscope are chosen according to the particular application: zoom optics for low to ultra high magnifications, stands, sliding stages, etc. A digital microscope system should have a modular design so that it can be exactly configured to suit its intended use and flexibly adapted to changes in general conditions. To offer users real value added compared with traditional set-ups, a digital microscope has to meet the five following technological requirements:

Optimized digital imaging
Dynamic viewing of processes or objects
Qualitative and quantitative sample analysis
Display and analysis of samples with high dynamic range
Lean optics for flexible orientation to sample and for mobile use

Fig. 1: Digital microscopes with a flexible tilting stand and a rotary x/y stage allow reliable inspection and analysis of the sides of samples or inclined surfaces.

Optimized digital imaging

Typical digital microscopes are equipped with a 2.11 megapixel CCD camera that is perfectly tuned to the high-resolution optics. The camera delivers the best possible information yield while keeping the amount of data of each picture manageable. However, digital cameras tend to be judged by the number of megapixels they offer. The more the better, many people think. But in microscopy, the camera with the most megapixels is not necessarily the best. The key criteria for deciding which camera will produce the best imaging results are the application and the optical performance of the microscope. Long before the triumph of digital photography, the US-American scientist Harry Nyquist proved that cameras in the double-digit pixel range do not offer more image information, but only fill the hardware with useless data more quickly.

A measure of the resolution in a digitized image is the maximum number of black-and-white line pairs that are imaged in sharp focus. The rule of thumb for calculating the line pairs per millimeter for microscopes is 3000 x NA (numerical aperture). This number is divided by the reproduction ratio of the object on the camera sensor to obtain the number of line pairs per millimeter that are actually captured by the camera sensor. This shows that a camera with, for instance, 12 megapixels, is no advantage at high magnifications. The resolving power of such a camera is then higher than that of the optical system. The image produced would be larger, but not better. Instead of resolving additional detail, the camera just produces more data.

As a digital microscope does not have an eyepiece, it has to be able to display the live image on the monitor at a high refresh rate. An ideal refresh rate is at least 15 frames per second, which ensures that the image can be viewed in comfort even when the stage is moved in xy direction. The faster image processing offers a further advantage during an experiment: The image capture rate is accelerated and therefore the overall sample processing time is shorter.

Illumination is another important issue for a microscope without an eyepiece. The light source should be as powerful and durable as possible and should have a color temperature similar to that of daylight to ensure that a realistic image of the sample is obtained. Metal-vapor lamps are ideal for this job. The long-life, maintenance-free LED-based systems now available on the market offer a convincing alternative.

Fig. 2: a) High Dynamic Range imaging reveals all the details of a solder joint;

b) Long exposure times are better for visualizing dark areas,

c) while bright areas show up better with short exposure times.

Dynamic viewing of processes or objects

Compared with traditional stereomicroscopes, zoom systems have the disadvantage of not being able to provide a three-dimensional image. With a digital microscope, this can be more than compensated with a smart accessory: A 360° rotary head enables the user to view the sample from all sides and even record this panorama view as a movie. This literally opens up new perspectives and viewing opportunities. Also, the 360° rotation clearly shows the three-dimensionality of the sample. The standard software suite should also feature time series recording to document dynamic processes.

Qualitative and quantitative sample analysis

Fig. 3: 3D model of a plastic component, displayed in pseudo colors

One of the key strengths of digital microscopy is the fast creation and analysis of 3D surface models. Using the motorized focus drive, an image is recorded in every focal plane in z direction. Then the focus is determined in every single image and for each pixel. The pixel with the best definition determines the focused texture, from which an optimized 3D model is computed. In addition, a topographical profile can be produced from the information on the distance from which the sharply focused points were recorded.

This method is versatile and can be used at low magnifications (macro objective) as well as high magnifications (high-performance 7000x objective) for precise topographical surface measurement. Besides 3D profiles it is also possible to measure roughness, geometries and volumes.

In the interests of reliability as well as precision, digital microscopes should be equipped with an electronically coded zoom. The digital image is then automatically given the correct calibration during the recording. Incorrect image values are a frequent source of error with conventional systems.

Display and analysis of samples with high dynamic range

Most digital microscope cameras use 16-bit single color capture (equivalent to 65,536 colors) to exploit the entire dynamic range of the image. The problem is that most computer screens and printers can only display 8 bits per channel, which is equivalent to 256 brightness levels. This means that all the natural nuances in brightness that our eye is able to differentiate are not always reproduced.

For capturing images with a high dynamic range, the HDR (High Dynamic Range) method is used. These high-contrast images capture the whole range of natural brightness nuances. The pixel values are in proportion to the actual luminous density. Despite the fact that HDR images have to be adapted to the lower brightness range which is still a feature of almost all screens, they offer advantages nevertheless. In particular, details remain visible even in extremely dark and bright areas of the screen image.

Fig. 4: HDR image of an electronic component

Lean optics for flexible and mobile use

Fig. 5: Measurement of wear on a hard metal indexable insert

For examinations of extremely small structures on inclined or vertical surfaces, conventional microscopes come up against their limits. For example, a sideways view of a solder joint on a large circuit board can only be obtained with an adventurous setup of equipment. Previously inaccessible areas of samples are no problem for digital microscopes.

A flexible tilting stand combined with the rotary x/y stage allows reliable inspection and analysis in virtually any position. Some products, however, can neither be transported, nor can samples be taken from them. For modern, portable digital microscopes, non-destructive inspection even of stationary objects poses no challenge. And of course, mobile digital microscopes are also useful for performing quality control tasks at multiple sites.

Conclusion

Digital microscopes are a particularly good alternative to traditional microscopes for the frequent inspection of difficult-to-document samples or fast 3D surface quantification. However, if optical brilliance and variety of contrasting techniques are more important, stereo or light microscopes are superior. Before investing in a new instrument therefore, it always pays to weigh up the benefits carefully and obtain comprehensive and objective advice on the alternatives.