Light Sources

When designing a microscope for imaging, the choice of light source is critical. In general, an ideal imaging system will provide fast spectral switching, high light throughput, provide stable power emission, have a long lifetime, be cost-effective, and be low complexity.

Several light have been developed for use in microscopy, including arc lamps, light emitting diodes (LEDs), solid state lasers (including fiber lasers, optically pumped semiconductor lasers, diode pumped solid state lasers), supercontinuum lasers, and ultrafast lasers (including ti-sapphire lasers). A few key parameters to consider are discussed below.

Linewidth

One key aspect of any light source is the linewidth of its emission. This is the spectral width of a light source in nanometers of the spectral density of the emitted electric field. It can also be reported in wavenumbers (cm -1) or frequency (Hz). Narrow linewidths enable more aggressive detection bands, reduced bleedthrough, and a reduced background. LEDs typically have a linewidth of 20-50 nm, which is much broader than the 0.1-1 nm linewidth of a laser. As such, we prefer lasers for our imaging systems.

M 2 Value

The M 2 value of a beam is the degree at which it can be focused to a given beam divergence angle (e.g., numerical aperture). If you have a higher M 2 value of ~1.6, it will focused to a laser spot that is 1.6x larger than the diffraction limit. As such, for high-resolution imaging, it is important to have a low M 2 value. We prefer lasers with an M 2 value of <1.2 for our imaging systems. Fiber lasers, such as those offered by MPB Communications, provide very low M 2 values. However, these systems require external mechanisms for controlling the power output, which is a major disadvantage. For lasers with high M 2 values, additional optics may be necessary to clean up the beam profile, such as a spatial filter.

Modulation

The ability to modulate the power of a light source is important for many imaging applications. It is not uncommon to use an acousto-optic modulator (AOM), acousto-optic tunable filter (AOTF), or electro-optic modulator (EOM) to modulate the power of a laser. AOMs induce sound waves within a crystal to change the refractive index of the crystal, which alters the angle of the light at a single wavelength. Likewise, AOTFs use a variable frequency sound wave to operate at multiple wavelengths. EOMs, such as a Pockels cell, induce a directionally dependent change in refractive index in a crystal, which can change the polarization of the light passing through it. These are especially common in ultrafast laser systems. Both AOMs and EOMs can be used to modulate the power of the laser, with rise times of ~5-50 ns and Acousto-optic devices offer rise times 0.25 - 1 ns, respectively. However, especially for continuous wave (CW) lasers, these devices serve as a source of additional complexity, cost, and potential failure points. For example, we have noted that AOMs can be sensitive to temperature changes, which can lead to drift in the power output. As such, we prefer lasers with built-in modulation capabilities.

Vibrations

Many lasers require active cooling, and are often placed directly on the optic table, which can lead to vibrations. These vibrations can be detrimental to imaging quality, especially when imaging in soft contexts such as expanded tissues. Nonetheless, if insufficient cooling is provided, fluctuations in laser emission or instability can occur.

Power

The power of the laser that is needed depends critically on the light throughout of the illumination train, as well as the size of the field of view. While some light sources such as supercontinuum lasers can provide high power (e.g., >3W), their emission is spread across a large spectral range, reducing their spectral power density at any given wavelength to a few mW/nm. We typically recommend lasers with a power of 100 mW or greater for imaging.