Modes of Microscopy

Differential Interference Contrast (DIC) mode of the Keck 3DFM

In DIC, two waves (generated by a Tungsten source) propagate through a phase object with a sub-pixel displacement created by a beam-splitting prism. The displacement created is defined as the "shear." The phase object must be transparent, with low amounts of scattering and absorbance (typical of a live cell). The waves are delayed by different amounts if the optical path length through the specimen, at the focal region, varies in the shear direction. The two waves are later combined to create a differential interference. Thus, the source of contrast in a DIC microscope is the phase gradient of the object, transverse to the optical beam, measured by the interference of the two beams.

In addition to DIC, we also have epi-fluorescent imaging capabilities using a mercury arc-source lamp as a standard modality.

Reflectance Confocal Microscopy (CRM) mode of the Keck 3DFM

The advent of the laser scanning confocal microscope created a means to record digitally the image created in a confocal microscope. The RCM detects light that is backscattered into the objective lens. Like other confocal microscopes, only the parts of the cell that are in "focus" are detected. In RCM, a point source, we use an argon-ion laser, is collimated by a lens. The light passes through a beamsplitter, to another lens that focuses the light onto a specimen. The backscattered light then retraces its path, is re-collimated, and then reflects off the beamsplitter. The light is then focused onto a pinhole that is in the same focal plan as the image. Backscattered light from the in-focus plane passes through the pinhole, while the out of focus light is rejected. This allows for a light image of only in-focus back scattered light.

Laser Scanning Confocal Microscopy (LSCM) mode of the Keck 3DFM

Initially, LSCM was used for imaging in materials science applications. The confocal fluorescent microscope allowed confocal microscopy to expand into the field of biology. Much like RCM, LSCM has the ability to create thin optical sections. However, the source of contrast in LSCM is different. LSCM uses specific laser illumination (argon-ion at our facility) to excite an electron of a fluorophore from its ground state to a metastable state. When the electron of the fluorophore relaxes from the metastable state to the ground state, a new photon with equal energy to the difference in energy level between the metastable state and the ground state is given off. This is collected in the same manner as in LSCM.

In addition, LSCM may not need fluorescent labels. Some specimens may already have endogenous auto-fluorescent properties. This could be useful in certain instances such as detecting elastin fibers that auto-fluoresce in the presence of NADH in skin.

Two-photon Microscopy (TPLSM) mode of the Keck 3DFM

As with LSCM, TPLSM uses the principle that an electron excited to the metastable state will release an excited photon when relaxing to the ground state. The difference between TPLSM and LSCM is that the fluorophore simultaneously absorbs two photons to reach the excited state. To illustrate, suppose that a fluorophore needs one blue photon, with an energy equivalent to "1," to be excited to its metastable state (and then release a green photon). Two-photon microscopy uses the principle that two red photons, with energy equivalents to "0.5" each, can simultaneously excite the fluorophore to its metastable state (and then release the same green photon when relaxing). To observe this effect, large amounts of intense near-infrared light is necessary, and this is achieved with a high-power titanium-sapphire laser.

Two-photon microscopy, by definition, uses longer wavelengths. Longer wavelengths inherently penetrate substrates deeper, which allows for deep tissue imaging (up to several hundred microns). The longer wavelengths, of lower power, are also less damaging to the cells by reducing production of oxidative particles and by eliminating exposure to UV light, which damages DNA.

Quadrature Microscopy (QTM) mode of the Keck 3DFM

Quadrature microscopy is a detection technique for measuring phase and amplitude changes to a sinusoidal signal. A signal from a HeNe laser is split into two components, reference and unknown. The unknown signal passes through the sample. The known reference signal is split, with one component being phase shifted by 90 degrees. The unknown signal is then mixed separately with both components of the known reference signal. The merged signal consisting of unknown and non-phase shifted reference is referred to as the I channel, or the in-phase signal, while the unknown signal mixed with the 90 degree phase-shifted reference signal is referred to as the Q channel, or the quadrature signal. By interpreting the I and Q signals as real and imaginary values of a complex number, it is possible to find the amplitude and phase of the unknown signal.