The holy grail of neuroscience, and medicine in general, is the detailed accurate imaging of cells and their contents in real time. A previous post on mapping the brain described many problems with current imaging technology in seeing details of the neuronal network. Even with accurate tiny slices of brain tissue observed through light and electron microscopes, only tedious human observation can untangle the jungle of axon connections. Another post described the many problems using fMRI of brain regions to study behavior. Brain events stimulate activity in large circuits connected by individual neurons across the entire brain. This whole brain activity occurs in milliseconds. Unfortunately, the smallest measurement in the fMRI, the pixel of light in the image, comes from blood flow in a region of 100,000 neurons summed over a second. This blood flow is controlled by astrocytes, not neurons. In fact, all correlations of fMRI and behavior are speculative.

But, now comes a totally new type of technology that promises incredibly accurate imaging of cells, including intracellular organelles and even individual molecules inside and outside of cells. This technology uses an analysis of the vibrations from specific molecules when they are stimulated by light and sound—a new way to image cells with vibrational spectroscopy.

Molecules in cells vibrate and can be picked up by their electromagnetic spectra when stimulated by a light beam. The vibrations and their spectra have been studied for molecules in a test tube. Until recently, it hasn’t been possible to correlate the spectra found for the vibrations for the complex multi layered molecules in living cells. The vibrations can interfere with each other and “scatter,” making it hard to go deep into tissues. Vibrations from the large amount of water can create noise. Also, like other imaging devices, computer data from the readings are summed over a second or less to create the pixel on the screen. But, recent spectacular advances are opening the door to totally new ways to image the details of cellular life, even inside of dense tissue. These advances involve multiple different techniques being added together for new complex systems.

Spectroscopy

The spectrometer measures a spectrum of wavelengths or frequencies. One of the most widely used measures is with infrared light (IR), a light that has a wavelength that is longer than what can be seen. A longer wavelength means a lower frequency. There are many ways that it is used; one common type is measuring absorption patterns for chemicals, which can be gas, liquid or solid. The spectrum is a graph of how the IR is absorbed and transmitted. The graph is plotted with the absorption vertically and the wavelength or frequency horizontally and the number can be called wave numbers.

Molecules absorb the light based on the chemical structures including specific bonds between atoms in the molecule. How the absorption occurs (resonant frequencies) is related to the energy in the chemical bonds of the molecules. Either a group of connected atoms or the bond itself vibrates from the transitional energy. The bonds in different structures can move in many ways to create the vibrations. There are symmetric and un symmetric moves of rocking, wagging, twisting, stretching and scissoring. Bonds in water don’t rock, wag or twist because of their simple bonds using just hydrogen and oxygen. Instead the whole molecule vibrates. Water has its own very unique wave pattern and interferes with the ability to decipher molecules floating in a lot of water.

There are many different chemical factors that can determine this unique vibration, including the mass of the atoms, the way the bonds are formed and the possible energy. There are specific mathematical formulas that approximate harmonic characteristics of the vibration. It becomes quite complex, involving energy surfaces, quantum states, and electronic ground potential. In simple terms, frequencies that resonate are related to the strength of the bonds and the types of molecules on either side of the bonds.

Vibrational Imaging Advances

Advances have been made in the speed that the data can be gathered, the details it can pick up, the spatial separation of different objects and how deep it can go. With these advances, several of these microscope devices—single-frequency coherent anti-Stokes Raman scattering or CARS and stimulated Raman scattering or SRS—can produce videos of molecular movement with the surprising speed of 3.5 milliseconds per pixel for CARS and 32 and 32 ?s for each SRS pixel (for comparison, the average human eye blink takes 350,000 microseconds, or just over 1/3 of one second).

CARS and SRS can even image small molecules. This technology can, also, be integrated with atomic force microscopy to see very tiny Nano scale objects. Merging yet another technology—studying acoustic overtone transitions and detection of diffuse protons allows imaging of single cells in real time, along with observing myelin and lipid droplets. These techniques have observed medications arriving at a target inside of a cell. They are increasingly being used instead of injected dyes to observe cancer and heart cells. Vibrational imaging with acoustic measurements has been able to observe fatty plaques in blood vessels.

There are still some definite limits on this developing technology. But, it is possible that with further research advances, small amounts of specific molecules in cells and tissues could be detected in the nano and micro realms. Another possibility in the future is being able to probe more deeply into tissues up to 20 centimeters.

New Vibrational Imaging Versus Stains, Labels and Tags

A hundred years ago, Golgi stains allowed neurons to be seen under a microscope. Recently, proteins that have the property of green fluorescence allow visualization of proteins inside of cells. More recently, super microscopes have been able to see smaller and smaller objects inside the cell with the fluorescence.

But, there are inherent problems with stains and other labels. They are very specific to certain kinds of molecules. It can’t just see what is there and scan for new molecules. The labeling can alter the cell by its own properties. It is very difficult to apply the specific label to particular places in real life without distorting it. The stains and labels are toxic to humans and therefore limit studies to other animals.

The new vibrational images, finds particular exact patterns of unique vibrations and can, in theory, image almost any protein. This can be found for infrared light or the Raman patterns. If this can happen fast enough inside of a cell, then the molecule’s movements and locations can be observed close to real time.

How Does It Work

Unfortunately, these vibrational techniques are not as simple as attaching a microscope to a spectrometer. Important questions include whether the process can be fast enough and not damage the cell. This has been accomplished for slices of tissue (see post on limits of brain mapping), in real cells. One issue is the amount of water in and around cells and the effects this has on light. In particular water absorption of the infrared light is a problem. The shorter wavelengths that Raman techniques use are much better for imaging small molecules in and around water. There is already a Raman microscope with speed of milliseconds for each pixel. But Raman in larger areas is limited to tens of minutes.

CRS has improved. It uses two different sources of excitation and these are compared by computer for each pixel of the image. In fact, four processes become involved (anti and pro scattering) and it becomes faster and more accurate. It is a four wave mixing process that utilizes quantum calculations. A limitation of both SRS and CARS is that you have to know what you are going to look for and set the device for that molecule. The researcher can’t just look around and see what is there. Another complication comes from data from the non resonant background. There are now devices using both CARS and SRS at the same time instantly recording each pixel. Some of these devices can image particular types of single layers of lipids (membranes).

Using Sound Waves As Well

Another technique uses sound wave patterns. Molecules absorb photons of light and some of this energy becomes heat. Expansion from the heat creates pressure and a sound vibration. A special device measures this photo acoustic signal and from it the nature and location of the absorbing molecule can be determined. One use of this has been observing hemoglobin. Because CRS images such as narrow slice of tissue only, further acoustic techniques are helpful. One uses the absorption of sound overtones along with SRS. SRS is mostly related to photons that have little penetrance into tissue.

But overtone acoustic imaging can see much deeper into tissue. Overtone imaging starts with pulses of infrared light. The absorption produces produces heat, then rebound pressure waves, which are essentially sound waves. Then the overtones are created from this sound wave. This technique uses only the near IR so that there is little water absorption to ruin the signal and it, therefore, can see further into the tissue without the negative effects of too much water signal. There is now a special version for blood vessels called intravascular vibrational photoacoustic (IVPA), a technique that uses a catheter to find atherosclerotic plaques, margins of breast cancers and particular nerves. There are many other combined techniques.

Advances Come From Many Directions

Increased Speed

More speed is dependent on the amount of extraneous signals that are picked up that have to be accounted for (noise). Photons create noise. In the Raman type the photons don’t make much noise, but the process after that does. So, the recording the photon effects take a millisecond, but the analysis to create the image takes minutes. In the CARS technique, the signal is amplified by fields and becomes very focused. This laser effect creates the noise. When the material is very concentrated such as the structures of lipid in fatty tissue, the noise is very low and therefore imaging of lipids in the skin of mice has been done. When only a single vibration frequency is used, it can only be to detect a known entity. Another technique looks at many different frequencies at the same time. This technique takes minutes to take the pictures and then build the images. This can’t be used for live action.

Using both CARS and SRS at the same time, pixels can be instantly imaged. This combined technique uses several different wavelengths at once, such as broadband and narrowband beams. It is able to use the background that doesn’t resonate as an amplifier of the signal. With a third beam, Raman is discerned from the CARS signals. This can produce pixels in milliseconds. Others use multicolored beams and multi channel detectors such as three spectral channels.

Further efforts have been made to understand the exact chemical components of a spectral signature and therefore further analyze the information rapidly. This technique uses pattern mappings of major chemicals involved and the different possible densities. Other statistical techniques are applied if nothing is known of the material being studied. To take a snapshot of the full spectrum using CARS takes milliseconds per pixel. Hundreds of waves are recorded in microseconds with SRS. Single frequency recording for a known molecule is in nanoseconds.

Increased Sensitivity

Using a variety of techniques, single beam CARS can detect 1 million carbon-hydrogen bonds in a lipid membrane. When less strong beams are used, the background often buries the signal. The issue with sensitivity is to avoid the background interference. There are many techniques being researched, but they are very complex to use and therefore have not been useful, yet. One complex multi layered technique images a single layer of lipid molecules. With single frequency beams, much of the background noise can be eliminated but not as rapidly.

One technique uses tags. It takes carbon-deuterium bonds (C-D) the cyano bond (C=N) or the alkyne bond (C with a triple bond to another C) and their spectrum and compares it to Raman bands. This comparison allows more sensitive imaging. Using the alkyne tag, DNA synthesis has been observed and small cellular molecules. The problem with tags, as mentioned earlier, is they can cause disruption in the activity of what is being observed. When labeled with deuterium (heavy hydrogen with extra proton), imaging was rapid for fatty acids, amino acids and medications in live cells.

Spatial Resolution

Some studies benefit greatly from better separation of small objects, such as organelles in a cell, functional activity and arrays of chemicals in an organelle like the Golgi. One technique for more separation has two laser fields at the same time. This superresolution CARS allows better diffraction quality of the beams of light. One version of this used both infrared and an atomic force microscope at the same time to gain great resolution. For this a probe, a tip of silicon with a layer of gold is used instead of a laser. Near the tip, there is better IR absorption and therefore better separation and less limitation on the diffraction patterns. This is still a work in progress for cells and living tissues.

Depth of the Picture into Tissue

When multiple single photons are shot at tissue, they cannot go very deep without dissipating in the complexity of the tissue. Another way that light is used is with diffuse photons and molecules.

Infrared light waves, also, produce overtone vibrational signals. One technique uses these IR optical overtones. Photons interact with the overtones in molecules. Characteristics of diffusion of photons in tissues are used in this calculation of the image. If you would like to find out more about photonic technology, you can read about the helium neon laser and other optical technology online.

Another technique picks up the scattered photons further into the tissue comparing it to Raman signals. Diffuse photons can go deep into the tissue but take different time periods. These characteristics are compared for information. Different amounts of side scattered and deeply scattered signals are picked up. Some of these techniques can gather information that is millimeters deep.

A third technique uses ultrasound vibrations and their overtones. Near IR lasers excite a molecule causing heat immediately and then expansion and contraction causing sound waves. Sound detectors pick these up and the overtones are studied. This has been effective with carbon hydrogen bonds. The stretch of the C-H bond’s first and second overtone are particularly helpful since they are best to avoid water interference as the signal goes deeper into the tissue. The first overtone sees the millimeter and the second with several millimeters deep.

Seeing Cells Working

Small molecules inside cells can not be tagged with a fluorescent label without altering their functions. CRS can image organelles inside of cells without disruption, tracking amino acids, nucleic acids, lipids and carbohydrates. Already, it has helped understand properties of myelin, lipids, cell division and medication delivery.

The most study has been in lipids and myelin because of the common C-H bonds that stretch and send good signals. CRS has tracked dietary fat absorption in the intestine. By correlating with RNA interference, genetic regulation of fat storage was found. Another studied identified the differences between fatty lysosomes, fat droplets and oxidized lipids. Another the difference between organelles with protein contents versus fat. A study found that prostate cancer has a large amount of cholesteryl ester.

Study of the myelin sheath with CARS shows when deterioration occurs in degenerative diseases like multiple sclerosis. Myelin has a great amount of lipid and produces a strong signal to see the detailed structure such as the Node of Ranvier. CARS can, also, study injury to white matter (bundles of axons), such as in spinal injury.

Another type of imaging with SRS shows the metabolic state of cells. One study showed cancer cells changing retinol to retinoic acids and another the conversion of palmitic acid by oleic acid. A study showed pancreatic cells making lipids and another cell making proteins. Raman tags can follow DNA and RNA synthesis.

A striking result of CRS microscopy is observing cell division and apoptosis in real time. Watching the cell cycle used SRS monitoring DNA with a specific vibration of carbon and hydrogen. Recent techniques have allowed three-dimensional sections to tissue to be seen. With multiple signals, multiple chemicals are followed. Another technique follows both lipids with CH3 vibrations and proteins with CH3.

In Medical Treatment

A major advance expected in the future is to be able to eliminate the need for potentially toxic contrast material used with imaging. CARS and SRS can be used accurately for skin and tissue that is exposed. This can allow an alternative to skin biopsies in the future. With one study, squamous cell carcinoma was found. This occurred with two beams mapping both CH2 and CH3 vibrations, which produced maps of fats and proteins for specific organelles and extracellular markings. This same technique identified the margins of a brain tumor in mice, otherwise not identifiable. The blood vessel visualization by catheter was already mentioned. By noting the amount of water and lipid, breast cancer was identified.

A New Way To Image Cells with Vibrational Spectroscopy

Current methods of imaging individual cells and their contents are limited. This includes observing the activity of neurons and glial cells. Dyes and tags can alter the tissue being observed and can be dangerous for humans. The holy grail of neuroscience—in fact, all biology—is the real time observation of individual cells and their communication with other cells, including brain circuits.

These new techniques that measure unique vibration patterns from specific molecules, organelles, cells and tissues could bring us much closer to the goal. There are many complex different techniques that are added together and each presents its own problems. With the intense research that is going on, it looks like real progress with be made soon.