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Optical Microscopy vs. Digital Microscopy: For cell cultures studies

The microscope is a ubiquitous instrument in every research laboratory. Be it a pocket model or a full-sized unit, microscopes are used across diverse settings, including research, healthcare, education, manufacturing, and engineering. The basic premise of all microscopes is to see a cell in far greater detail when compared to the human eye1.

Ever since compound microscopes were invented in the late sixteenth century by the Dutch father-son duo of Hans and Zacharias Jansen, scientists have been improving these devices. The traditional form of microscopy is optical microscopy, also termed as light microscopy2. Herein, the technique used is to use visible light to view a sample through the magnification of a lens system3.

A variation of the optical microscope is the digital microscope. These microscopes use a digital camera instead of eyepieces. Images are seen in real-time on a computer screen. The invention of the USB port in the 1990s has made available a wide variety of USB digital microscopes to suit every need4.

Optical microscopes

Optical microscopes use visible light and a system of lenses that are located in-between the sample and the eye of the viewer. They help magnify objects.

Modern optical microscopes may appear very different in their design and specifications from the traditional simple microscope that used sunlight as a source of light. However, the basic principle is the same.

Principle of optical microscopy

A beam of light (from light-emitting diodes or an electric bulb) is focused on the sample to be studied. The light transmits through it and passes through a set of objective lens and ocular lens (eyepieces) into the viewer’s eyes.

Depending on the type of objective lens used, varying magnifications are obtained. In present-day microscopes, the lenses are fixed both above the slide and below, within the substage condenser. The objective lenses located above the sample are used when additional magnification is required5.

Magnification is dependent on three variables

Three variables play a role in the magnification to be obtained. These are: The angle of incidence, which is the angle at which light enters the lens; the curvature of the lens; and, the refractive index (RI)5.

Digital microscopes

In simple terms, a digital microscope uses a digital camera in place of an eyepiece. The microscope is connected to a computer monitor and results are obtained in real-time.

Principle of digital microscopy

In digital microscopy, optics and a digital camera are used to capture images onto a computer monitor.

Digital microscopes can range in complexity from simple handheld versions to highly advanced systems. The latter uses sophisticated software to perform advanced tasks.

Some of the advanced features available are: Image editing; analysis of 3D samples; taking measurements; report generation6.

Differences between conventional optical microscopes and digital microscopes

Digital microscopes perform the same tasks as optical microscopes. But, there are additional features. Table 1 highlights the key differences between conventional optical microscopy (COM) and digital microscopy (DM)7. The crucial difference between a digital microscope and a conventional optical microscope is that in the former there is no provision to view the sample directly through the eyepiece.

Table 1: Differences between Conventional Optical Microscopy and Digital Microscopy

Feature

Conventional Optical Microscopy

Digital Microscopy

Depth-of-field

With higher magnifications, the depth-of-field becomes shallower.

The depth-of-field in DM is 20-times more when compared to COM.

Variable-angle observation capability

COM lacks this capability. All specimens must be observed from directly above.

DM achieves observation through 360 degrees. The sample need not be tilted.

Three-dimensional imaging

Images using COM are two-dimensional. Surface characteristics are difficult to determine.

DM gives three-dimensional and fully-focused images.

Quantitative data

COM does not provide measurements and other quantitative data.

In DM, built-in measurement tools provide real-time measurements.

Data sharing

Not possible with COM.

Images are easily recorded and stored on-site.

Why should you choose a digital microscope?

Ergonomics

A key reason to choose a digital microscope is the ergonomics of the instrument. If you are working in a research laboratory with a high sample throughput, or, if you use microscopes for hours together, a digital microscope is your best choice.

Since images are instantly displayed on a computer screen, you are will be able to analyze them comfortably8.

Large depth-of-field

Digital microscopes offer a large depth-of-field that will allow you to precisely observe your cell cultures even on an uneven surface. Combine this with the depth composition function that allows for stacking focal positions of numerous images and you will get high-clarity pictures9,10.

Analysis in 360 degrees

Digital microscopes have a variable-angle capability that allows observation through 360 degrees. You do not have to tilt your sample. Additionally, the inspection time is greatly reduced10.

Multi-lighting feature

Some digital microscopes come with a multi-lighting feature. At times, some parts of the image may not be clear due to glare. By changing the direction of the ring light, the glare from the image is removed. All this is possible at just the touch of a button9.

Broad magnification range

You will be able to analyze your cell cultures in depth because of the range of magnifications available in digital microscopes. An assortment of magnifications is available from 0.1X to 6000X9.

Remote working

Digital microscopy can be linked to a cloud-based system to facilitate remote working. What this means is that you can operate the device and observe the samples using a computer.

With remote monitoring, you are able to assess your cell cultures located within the incubator itself. The key advantages here are: You can assess your cells from a remote location regularly; you time your visits to the lab with efficiency; you store all relevant data and can analyze images from your desk.

Integrated filing system

After you have observed and chosen your images, you can store them in different file formats. In certain microscopes, digital images analysis can be integrated into the software. These can help towards automation in e.g. colony detection, wound healing assays, or confluency detection (growth curve plotting). Image formatting is often compatible/optimized (differs per manufacturer) to image analysis software. Moreover, you can prepare on-site reports instantly9.

Live-cell imaging

Your digital microscope plays a crucial role when you want to image cell cultures live, rather than using fixed cells. In live-cell imaging, time-lapse microscopy is used to dynamically observe, track, and quantify processes in living systems. Such information is more likely to be physiologically relevant when compared to the information obtained from fixed cells.

Indeed, it is known that fixation influences the apparent structure of the cellular cytoskeleton11, or even change cellular morphology12.

Digital microscopy in the COVID-19 era

Long-established modes of operation in research and healthcare have been severely disrupted over the last several months due to the COVID-19 pandemic13.

If you are a researcher working in a biosafety level 3 or 4 (BSL 3 or 4) laboratory, you know how essential it is to protect yourself and your colleagues in such an environment.

You are now in an unprecedented position, in which the social distancing rules are directly interfering with routine microscope sessions and in-person interactions.

With remote monitoring of cell cultures in incubators, you continue your vital contributions to science, and, also abide by social distancing guidelines13.

In remote monitoring, the cells are assessed and monitored in a live online environment, without having to physically remove them from the incubator.

A small microscope with a digital attachment is placed inside the CO2 incubator. This allows you to monitor the cells contained in a dish or flask in real-time and over extended periods from the comfort of your desk. We recommend the CytoSMART Lux2 that can facilitate remote monitoring.

Conclusions

Since the advent of microscopy in the late sixteenth century, this technology has come a long way. Traditionally, optical microscopes have been the workhorse in diverse settings, including research. Digital microscopy has got several unique features that make it advantageous in the research setting.

In the current scenario of COVID-19, researchers need to adapt to new rules and regulations. This is made a lot easier with the use of digital microscopy.

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