Optical microscopy – Not just for making pictures

The study of natural materials at varying scales – from molecular to millimetric- is an increasingly necessary field of research. By observation of the interaction of light with natural materials at different scales through an optical microscope, we can further understand their organization and properties.  This understanding leads to the development of new technologies for practical applications from medicine to the processing of natural resources.

Although optical microscopes are often thought of only as instruments for making pictures they are actually multi-functional instruments which can also use light to probe structures across many length scales.

Imaging requires contrast: some way of distinguishing where something is located in space and where it is not. Modern developments in microscopy have provided us with new tools for gaining contrast of natural materials as small as 0.1 to 1 nanometer based on chemical or other atomic-scale structural properties.  This is still too small for an optical microscope to let us actually “see” anything but it does enable us to infer something about their structures based on other measurements done on the same instrument.

Moreover, careful measurement in an optical microscope of the motion of small particles in biological and other “soft” materials gives us important understanding of their integration at the nanometric level.  Therefore, modern optical microscopy-based instruments are not just about taking pictures of small things; they are an ideal experimental platform to make measurements across many time and space scales up to a few millimeters size and help us better understand the organization of natural materials.

A view of the oil sands after processing, through an optical microscope. The blue and magenta areas in the image show quartz sand grains. Adhering to these grains, the fluorescent (green) areas are a residual oily material. Additionally some nanometer size carbon particles are found on the surfaces in some places and show up as bright yellow colored dots – some are indicated by the white arrows. Credit: National Research Council of Canada.

A view of the oil sands after processing, through an optical microscope. The blue and magenta areas in the image show quartz sand grains. Adhering to these grains, the fluorescent (green) areas are a residual oily material. Additionally some nanometer size carbon particles are found on the surfaces in some places and show up as bright yellow colored dots – some are indicated by the white arrows. Credit: National Research Council of Canada.

A bit of history

We understand well that most things in nature – animal, vegetable and mineral – are composed of molecules formed through tight, chemical bonding of atoms. Deriving macroscopic properties of materials is more difficult.  A process which is most successful in cases where there is little interaction between particles, or highly ordered lattice or crystal structures. Very few natural materials, however, conform nicely to these states of matter. In most materials there is aggregation and granularity occurring at many different spatial and time scales. But by using optical microscopy we can make difficult multi-scale measurements and have been doing so over the course of history.

Around 1830 the Scottish botanist Robert Brown observed the ceaseless agitated motion of small particles of about 1 micron in preparations of pollen grain specimens suspended in water using a simple optical microscope. This type of movement of microscopic particles in a liquid or gas is now called Brownian motion. Early in the 20th century, in work derived from his doctoral thesis, Albert Einstein showed that it was possible to estimate the size of the molecules of water, which we cannot observe with an optical microscope, from the careful measurements of Brownian motion of particles about 10,000 times bigger.

At that time the idea that there was a discontinuous/granular nature in the structure of matter was controversial.  It wasn’t until a few years after Einstein’s work —not much more than 100 years ago—   that the careful measurements of Brownian motion by the French physicist Jean Perrin, again using an optical microscope, provided experimental confirmation that lead to the wide acceptance of the atomic theory of the organization of matter.

This story from the history of science reveals the important role of optical microscopy in the experimental confirmation of theories of the organization of matter; something that is continuing today.

Enabling technology:  how different types of light have let us see new things

Many important insights can be made into the organization of matter, especially at the atomic and molecular scale by carefully managing the light we use to illuminate a sample.

One method is to use nonlinear optical signals.  Normally, light interaction with matter will generate a response which is linearly proportional to the intensity of the light, where an increase in one will lead to an increase of one in the other.  However, many revealing responses in materials are made through nonlinear illumination. To give a better picture, mathematically y = x, is a linear relationship while y=x2 or y=x3 etc. are nonlinear.

Continuous illumination levels required for observable nonlinear optical responses would rapidly destroy samples.  These light intensity levels can be obtained, however, by using a laser that produces a stream of very short light pulses. In this case, while the transient intensity is sufficient for nonlinear optics, the average power can remain within a tolerable level. The squeezing of light in time and space confines the non-linear optical response to a very small volume by tightly focusing it through the microscope. This volume is about one billion times smaller than a 1 millimeter grain of sand but still contains around a billion atoms.

Using a variety of these nonlinear optical techniques, we are able to discern specific properties about the organization of matter. The optical signals provide the contrast needed for imaging and multi-scale measurements.

The most basic organization of atoms into matter is the chemical bonding of two atoms together to form molecules.  This primary level of organization creates a new property that we can probe with light. As a mechanical analogy, atoms can be thought of as little weights and the bond holding them together as a stiff spring. This simple mechanical system will vibrate at a well-defined frequency as a characteristic of the two atoms and how they are bonded together.  The frequency will be shifted slightly when more atoms join together or when the molecule is close to others. Very generally measuring the frequency responses of something is called spectroscopy.

Saving lives and resources one microscope at a time

At the National Research Council of Canada (NRC) some of the techniques we use to make these kinds of measurements are called coherent-anti Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS).  CARS and SRS provide contrast in imaging based on the density of vibrating chemical bonds in the sample.  These occur at the same time and can often be collected simultaneously.

Using the CARS/ SRS techniques NRC is studying microorganisms which can accumulate high quality oil.  With these methods we are able to take many microscopic pictures, each one depicting a different part of the vibrational frequency spectrum (see below) to help us better understand how the micro-organisms generate the oil drops

 Some microorganisms can accumulate a lot of high quality oil.  Pictured above is molecular scale information (spectrum on right) from 1 micron scale oil drops (circled in picture) that are accumulated in a single-cell organism, using CARS/SRS microscopy. Credit: National Research Council of Canada.

Some microorganisms can accumulate a lot of high quality oil. Pictured above is molecular scale information (spectrum on right) from 1 micron scale oil drops (circled in picture) that are accumulated in a single-cell organism, using CARS/SRS microscopy. Credit: National Research Council of Canada.

Another nonlinear optical signal we can readily see in the microscope is second harmonic generation (SHG). This is a signal which occurs in materials where there is some periodic ordering (crystal-like) but where there is a particular asymmetric arrangement.  SHG arises from the atomic level structure of materials.  Nature seems to have contrived to provide us with SHG as a means of microscopic study as it is very common in natural materials. For example, collagen—which is the major structural component of most tissues of animals as well as being the most abundant protein in their bodies—provides a very strong SHG. By understanding the origin of the SHG signal from collagen from microscopy, we may be able to relate it to the diagnosis of disease in collagen containing tissues.

SHG is also very strong from the mineral quartz which, again, is one of the most abundant terrestrial minerals and so is a major component of many soils and rocks.

Another example of a project taking place at NRC’s CARS laboratory is the observation of oil sands using optical microscopy to let us understand how the different components of the raw material interact during processing. Here we use SHG as well as the natural fluorescence of the oil component. We also use strong signals originating from nanometer sized particles which are much smaller than can be resolved by the optical microscope but still interact strongly with the laser light.

Sand ore consists of grains that are usually less than 200 microns or so in size as well as a lot of much smaller mineral material. Process developers are working to reduce the amount of wet waste produced by oil extraction from the sands. They would like to know how the different components migrate during oil extraction. This information is an important step towards developing new natural resource processing technologies. The Figure at the beginning of the post depicts a view of an oil sand after processing.

Optical microscopy at the heart of innovation

We now can see how structure at the atomic scale can be inferred by examining the interaction of a material with light. Let’s return to Robert Brown and what he saw when he examined plant material with his microscope.  The agitated random motion of small particles when pollen grains were put into water was only one of many forms of motion he saw. The motion inside the cells of living plants was quite different.  Particles embedded in living cells and tissues demonstrate movement which deviates from that which is characterized by Einstein’s theory.

Careful characterization of this type of motion is one of a number of new hardware and analytical tools,  again based on optical microscopy, that are leading  to a significant deepening of our understanding of the mechanical integration of cells and tissues. These insights should, in turn, provide advances in diagnosis and treatment of disease.

New optical microscopy techniques based on the intimate understanding of the interaction of light with matter are finding other important applications across science and technology. For many measurement types there are overlapping methods that may be superior to optical microscopy. However, I believe that some of our best hope for breakthrough understanding in science that leads to life-enhancing technology will involve the careful multi-scale measurement of intact living things and other complex materials. Optical microscopy will continue to be at the heart of discovery and innovation.


D214-16-0351-6014Andrew Ridsdale is a technical officer at the National Research Council of Canada.  Since 2007 he has been part of the team responsible for the setting up the CARS laboratory, NRC’s multi-user, nonlinear optical microscopy facility.  He started research in the area that is now known as epigenetics but has always been interested in the physics of living matter and especially material-level approaches to measuring and understanding the dynamics living cells.  He likes the engineering and physics of optical microscopy and worked with some of the first nonlinear optical microscopes.

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