Automating Fluorescent Imaging Techniques

For scientist who uses fluorescence microscopy techniques in their research, National Instruments (NI) provides a flexible and low cost image processing alternative to expensive stand-alone systems. Unlike embedded, inflexible alternatives, NI products provide graphical programming and have built-in compatibility with a broad range of image acquisition and motion control hardware.

Fluorescence Imaging Technologies

There are many benefits to using fluorescence instead of regular visible light. To understand these, we first need to know the basic principles of fluorescence excitation and emission. Fluorescent compounds, or fluorophores, absorb light at one wavelength and emit at another wavelength. In a fluorescence microscope, this ability to isolate the exciting light from the fluorescence provides a method of contrast that can be used for imaging structures at a molecular level in both live and fixed cells and tissue. Most fluorophores are inert, but some change their fluorescence properties in response to the chemistry of the local environment. When coupled with a scientific grade digital camera, we can image and measure biochemical processes in subcellular structures in single cells.

First, we label the sample that we want to image with a fluorophore (adding the fluorophore). Often, the molecular structure of a fluorophore is modified to target specific regions or compounds within a cell. Or a fluorophore may be attached to a protein such as an antibody to select the target region within the cell. A recent and now widely practiced method is to transfect or genetically modify cells to insert a fluorescent peptide such as green fluorescent protein, GFP, in a native protein.

Fluorescence compounds or fluorophores are capable of absorbing light at one wavelength and emitting at a longer wavelength. For example, fluoroscein, a commonly used fluorophore absorbs blue light and emits green, and rhodamine, absorbs green and emits red. When a fluorescent compound absorbs a photon, it’s outer shell of electrons move from a ‘ground’ state to an ‘excited’ state. It can release this energy as thermal radiation and return to the ground state. Or it can release part of the energy to its molecular environment, and then emit a photon with less energy and consequently a longer wavelength. The ratio of transitions emitting a photon to those without an emission is known as the quantum efficiency of the fluorophore. The frequency (wavelength) of this released photon is determined by Planck’s constant and will depend on the energy released by the electron returning to its base level. Depending on the temperature and microenvironment, different molecules of the same fluorophore release different amounts of energy before emitting a photon, creating a range of emission wavelengths or spectrum. The absorption spectrum predicts the probability of photon at a given wavelength being absorbed, and the emission spectrum predicts the probability of emission at a given wavelength.


As mentioned, the light emitted as fluorescence is at a higher wavelength (lower energy) than the incoming excitation light. The following graph illustrates this concept:


[+] Enlarge Image

Light emitted as fluorescence is at a higher wavelength (lower energy) than the incoming excitation light


Fluorescence imaging has many uses. For example, cells expressing green fluorescent protein can be imaged and counted. Adding a computer controlled X-Y stage and focus stepper motor allows scanning multiwell plates enabling measurements over varying populations or drug conditions. More complicated methods include adding a computer controlled emission filter to measure multiple fluorophores allowing comparison between controls and experimental probes. Another example for this is in the use of fluorescent microarrays for gene expression analysis. Fluorophores whose excitation or emission spectra change with local ion concentration or pH can have pairs of images ratioed and using appropriate controls, provide a reliable, quantitative measurement of the corresponding ion or pH.

Many cells move or migrate in response to chemical stimuli. Tracking fluorescent cells is easily performed with National Instrument’s analysis tools. The most useful technique is by setting a region of interest and use blob analysis. Then, by updating the coordinates of the moving particle, detect if the particle has moved closer to any of the borders. The ROI should then be updated appropriately. Additionally, Edge detection, and even pattern matching can be used. As we will see below, intensity measurements to find concentrations of tagged molecules can be utilized in a variety of very interesting applications. Many of the vision processing algorithms for counting and taking measurements of particles are similar to regular cell counting (see application note: Counting Particles or Cells Using IMAQ Vision)

Recently, National Instruments, Olympus Corp., and DVC Co., configured a microscope system for automated analysis. For more information, please see the Microscope Start-Up Kit.

The biggest advantage of using a fluorescence method is the ability to detect extremely small amounts. This is because fluorescence, with the appropriate filters, measures a light source on a perfectly black background. With a high gain CCD camera such as DVC’s Intensicam, one can easily detect the light emitted from a single molecule as a near real-time “live” image.

While performing Fluorescence imaging of live cells, researchers are acutely aware of the fact that stronger excitation light (usually in the UV spectrum) will destroy the cells at a much faster rate. Therefore, it is usually not possible to increase the excitation and/or the emission intensity beyond a certain level.

Camera Technologies

From the above, it is clear that Fluorescence Imaging, as all scientific Low-light imaging applications, carry a unique set of challenges. These must be addressed by cameras and imaging systems with a feature set that is specifically designed for these applications. A common problem with live cell imaging is most cells cannot tolerate high intensity excitation. To keep from killing the cells, and inadvertently measuring the effect of phototoxicity, a camera has to be extremely sensitive. While it is intuitive that the addition of gain (or a long exposure) can make any camera more sensitive, it is the noise in the system that precludes the use of generic cameras in such applications. Cameras such as the DVC1412 series are recommended for these applications because of their low noise specifications.

If the field of view presents an image that is low in intensity but static, a low noise camera can use long-exposures to integrate an image directly on the CCD. A lower noise image is obtained when integrating long exposures on the CCD rather than by summing multiple frames to “integrate” an image off-camera (e.g. in a frame buffer). However, as the exposure is increased the effects of thermal noise begin to dominate the photon noise. For this reason, cameras in this class are usually available with thermo-electric cooling. As a rule of thumb, low-noise scientific cameras can produce usable fluorescence images without cooling for exposures less than about 1 second. Exposures greater than 1 second will certainly benefit from cooling – but cooling is usually deemed as mandatory for exposures longer than a few seconds. Cameras in the DVC1412 series are designed to accommodate TE cooling (to -20°C) as an upgrade that may be provided as a retrofit to a previously non-cooled camera. This allows the capabilities of the camera to grow with the needs of the application.

If the field of view presents an image that is low in intensity AND transient, the previously described technique of increasing the exposure is no longer usable. In these cases, it is necessary to use some form of image intensification. In an intensifier, every incident photon generates several electrons (this multiplicative effect is referred to as Intensifier Gain), which produce light when they bombard the phosphor. In DVC’s Intensicam, a gated GenIII intensifier is fiber-optically coupled to a high Quantum Efficiency CCD. Care is taken to provide a match between the emission wavelength of the phosphor (of the intensifier) and the spectral response of the CCD – resulting in an optimal coupling, which maximizes the sensitivity of the system.


[+] Enlarge Image
DVC camera using Image Intensifying Technologies

Due to the importance of discriminating between different emission wavelengths, some of which may be separated by only 10-20nm, another technique that is frequently employed is to introduce an emission filter in the viewing path. While simple in concept, the switching of these filters under software control adds complexity to the overall system design. Liquid Crystal Tunable Filters (LCTF) provide one way to solve the problem – however their downside is that the transmission of these filters (in the pass band) is only about 30%. Since most fluorescence experiments involve low-light conditions this transmission loss is not acceptable to most users.

Optical filters can be specified to have as much as 95% pass-band transmission characteristics. Therefore, filter wheel technology is seen as a better solution for these applications. A system solution may take the form of a low-noise camera and an external filter wheel. The DVC1412-MS multi-spectral camera provides an integrated solution in the form of a built-in 4-position filter wheel module. The design allows the rapid removal & replacement of the entire filter wheel module by the user, as well as the ability to quickly install a standard 1” filter to any one of the 4 positions that are available. For applications such as FRET (see below), the filter-wheel may also be combined with the Intensicam model – allowing software control of image intensification as well as the emission filter within the same camera.


[+] Enlarge Image

Filter Wheel Camera from DVC

Specialized camera parameters such as Intensifier Gain, Intensifier Gate “ON” time, Intensifier Gate Delay, multi-spectral filter position as well as generic parameters such as camera gain, offset and exposure are available within the LabVIEW interface driver for these DVC cameras. This allows the developer or end-user to utilize these enhanced camera features through the familiar graphical LabVIEW user interface.

New Advances in Microscope Lens Technologies

The basics of fluorescence work require a means of hitting the object with the proper wavelength and collecting the emissions in a usable format suitable for the intended purpose. Research grant money is often limited. A fluorescent microscope from one of the major microscope houses might be too expensive, so Navitar has created a family of components that permits OEMs or system integrators to perform specialized tasks.



[+] Enlarge Image

NAVITAR ZFL SCOPE

One can use incoming and exiting illuminating filter wheel systems, but the optimum in performance is achieved with a “filter cube” containing a matched excitation filter, beam splitter, and emission filter. There are hundreds of off-the-shelf cubes on the market and Navitar chose to utilize the Olympus BX-2 format available from a number of suppliers.

Most fluorescent packages are intended to be used in the high magnification microscope mode. While this mode is accommodated, Navitar also offers the ability to operate in a “macro” (zooming or fixed focal length) mode with fields of view up to 15mm and 165mm working distances.

Included in the family of components are a “quick change” cube holder, fine focus capability, plug in UV or T-H light sources, 6.5X zoom system, macro lenses and optics required to display the image at the camera sensor plane.

These components are rugged and portable enough to permit “in the field” operation, with results linked back to a central location.

The customer provides the objective (if desired), the appropriate filter cube for his application, and a suitable camera.

Measurement Techniques

Below is a description of two common techniques used widely in microscopy. Using National Instruments IMAQ Vision tools, these measurements can be performed easily and with a great deal of flexibility to make changes to the algorithm used:

FRAP:
To measure the ability of a molecule to move around over time, a technique called FRAP (Fluorescence Recovery After Photobleaching) is used. As mentioned above, all fluorescent dyes emit light of one wavelength (e.g. green) after they have absorbed light of another higher energy wavelength (e.g. blue). If a very high intensity blue light is delivered to the dye, the dye can "photobleach", which renders the dye unable to fluoresce (This also is the case if the dye is exposed to normal intensity light over time). Photobleaching occurs when a fluorophore is photochemically converted to a non-fluorescent compound. When a fluorophore is excited, it undergoes a transition from a ‘ground’ state to an ‘excited’ state. If the excited state chemically reacts with other compounds, a non-fluorescent compound may be generated, resulting with a lower fluorophore concentration and a loss of fluorescence. To perform FRAP analysis, a fluorophore must be attached to the molecule (i.e. protein, lipid, carbohydrate).

The following steps perform the analysis:

• First, using a fluorescence microscope, visualize the fluorescently tagged molecule using a low light intensity.
• Then, flash a very intense light onto these same molecules - close to 100% of the fluorescent molecules will be photobleached
• When the molecules are monitored again, the area will appear black due to the loss of fluorescence. Now there should be a black area filled with photobleached molecules surrounded by fluorescently tagged molecules that have not been photobleached. If these molecules are able to diffuse (move), they will.
• As these molecules move around, the photobleached molecules and the fluorescent molecules will begin to mix. This will cause the fluorescent area to become a little less bright, but that is difficult to measure. However, the blackened area will gradually increase in brightness as fluorescent molecules migrate into this area. Hence, the moving of the molecule is detected!

With National Instruments Vision products, intensity measurements can be used for certain areas of interest to perform this analysis.

FRET:

Another important technique for investigating a variety of biological phenomena that produce changes in molecules that are very close to each other (less than 1 to 2 nanometers apart) is called FRET (Fluorescence Resonance Energy Transfer). With visible light, the resolution limit of a microscope is a function of the wavelength and the numerical aperture of the objective. This translates in a typical microscope to a limit of 200 nanometers. Using FRET, we can measure interactions between macromolecules.

A good analogy here is to think of two magnets suspending on springs. If one magnet starts oscillating, and is moved close to the second magnet, the second magnet will start to oscillate. The oscillation energy of the first magnet is transferred to the second. Fluorescent molecules are oscillators, and if placed close together with their dipoles oriented in the same direction, can transfer energy in the same manner.

To use FRET for molecular analysis, the emission spectra of the donor fluorophore must overlap the excitation spectra of the acceptor fluorophore. The detection mechanism utilizes that the farther apart the two molecules are, the weaker the transfer efficiency between the two. Also here, National Instruments tools can be utilized to detect intensities from donor and acceptor, and hence determine the distance between the two.

3D Imaging with Microscopes
Obtaining 3D imaging with a microscope can be done in a couple of different ways. First, using a stereo microscope (and your two eyes), you can obtain a 3D representation of the object that you are looking at. The downside of this method is that you cannot project that 3D information onto a camera. Another method is to put a high resolution stepper motor on the focus wheel. By applying software that detects which parts of the image that is in focus (such algorithms are available for autofocus on ni.com using National Instrument’s Vision Software modules), you can turn the focus wheel and apply this algorithm each time you reach a new depth. The frequency of applying this procedure depends on the axial (z-axis) resolution that you require.

Confocal Microscopes and Deconvolution:
Images taken with a microscope usually include information above and below the focal plane, which is very narrow for a microscopic imaging system. To avoid blurring of the image and to extract information from parts of the object that are closer to or further away from the focal plane than half of the depth of field, a laser scanning confocal microscope can be used. A confocal microscope rejects out-of-focus information by using a laser to control the illumination plane and a pinhole in front of the detector. More specifically, a confocal microscope rejects out of focus information by illuminating a single point and placing a pinhole in front of the detector. A laser is often used to since it is easier to focus at a single point, but not required. The trick is, from a point source, out of focus information spreads laterally in the image plane. By placing a pinhole in the image plane, only in-focus information is detected.

Today, the most expensive confocal microscopes are so advanced that they offer real time imaging. Confocal microscopes are very expensive, and an alternative solution to the problem has in recent years been developed using deconvolution algorithms. The same information can be obtained and utilized by digital deconvolution of the optical-sectioned images.

Using National Instruments software and hardware products with cameras from DVC and the ZFL lens systems from Navitar, customers can develop a powerful and cost efficient fluorescent imaging system. As mentioned above, recently National Instruments, Olympus Corp., and DVC Co., configured a microscope system for automated analysis. For more information, please see the Microscope Start-Up Kit.

19 ratings | 4.26 out of 5

Print

Reader Comments | Submit a comment »

Link to the Microscope Start-Up Kit
Link to the Microscope Start-Up Kit http://zone.ni.com/devzone/cda/epd/p/id/4 714
- May 7, 2009