- Maximize Image Contrast from Multiphoton Microscopes
- Designed for Ti:Sapphire Multiphoton Imaging Lasers
- Adjustable Compensation from -12,500 fs2 to 0 fs2 at 800 nm
As shown by the sample images below, pulse width broadening can lead to significantly decreased image contrast in two-photon microscopy. Multiphoton microscopes like Thorlabs' Bergamo® II rely upon femtosecond lasers that emit near-infrared pulses with durations of roughly 100 fs. In order to be short in duration, the pulses consist of a broad range of wavelengths.
Ideally, the pulse width at the sample would be as short as the pulse emitted by the laser. However, when propagating through the optical elements in the microscope (such as fluorescence filters, dichroic mirrors, scan lenses, and objectives*), the pulse width broadens: each wavelength travels at a different velocity because the glasses' indices of refraction vary as a function of wavelength. In contrast, when propagating through air, the pulse width is conserved: all wavelengths within the pulse effectively travel together because air's index of refraction is relatively constant as a function of wavelength.
This broadening phenomenon is known as group delay dispersion (GDD). The femtosecond pulse compressor compensates for GDD imparted by the microscope on the laser pulse, ensuring that the pulse arriving at the sample is as short as possible, thereby maintaining high contrast.
Shorter Laser Pulses Provide Increased Image Contrast
Above top: Without Dispersion Compensation (Long Pulse at Sample)
Above bottom: With Dispersion Compensation (Short Pulse at Sample)
Adjustable Dispersion Compensation
The pulse compressor supports operating wavelengths from 700 to 1050 nm, making it compatible with many common Ti:Sapphire laser systems The plot below shows the range of possible dispersion compensation values as a function of wavelength.
The shaded region in this plot denotes the precompensation values that the Femtosecond Pulse Compressor can provide.
To provide optimal GDD compensation for a wide range of microscope configurations, the dispersion can be adjusted by rotating a knob on the outside of the housing, shown in the photo below. The knob controls the insertion of a prism into the beam. This feature helps ensure that performance is not compromised as you change filters, objectives, or the laser wavelength.
As the knob is rotated, a display shows how far the prism has been inserted. Higher values correspond to more glass in the beam path and hence more positive dispersion. The side of the unit contains a female 3.5 mm audio jack, which outputs a voltage that is proportional to the prism displacement for use as a diagnostic. A plot that converts between the prism insertion and dispersion is available in the Group Delay tab.
|Wavelength Range||700 - 1050 nm|
|Dispersion Range at 800 nma||-12,500 fs2 to 0 fs2|
|Transmission at 800 nmb||>85%|
|Input Pulse Width (Recommended)||>50 fs|
|Input Beam Diameter (1/e2)||2 mm (Max)|
|Pointing Stabilityc||<100 µrad|
How the Femtosecond Pulse Compressor Improves Image Contrast
When laser pulses are sent into a multiphoton microscope without dispersion compensation, the pulse experiences positive dispersion as it propagates through the optical elements in the microscope. In a positively dispersed pulse, the longer wavelengths contained within the pulse travel ahead of the shorter wavelengths (that is, red travels ahead of blue). As a result, the laser pulse at the sample is lengthened in duration, leading to reduced image contrast.
The Femtosecond Pulse Compressor (FSPC) compensates for this effect by applying negative dispersion to the pulse before it is sent into the multiphoton microscope. In a negatively dispersed pulse, the shorter wavelengths contained within the pulse travel ahead of the longer wavelengths (that is, blue travels ahead of red). The microscope then applies positive dispersion to this pulse, and as a result, the pulse is short again at the sample, improving contrast.
The FSPC provides negative group delay dispersion (GDD) for 700 - 1050 nm laser pulses, adjustable by rotating the knob on the side of the unit. Because the dispersion is adjustable, the unit is easily re-optimized when an objective, fluorescence filter, or other optical element is changed in the setup. The contour plot below shows the achievable GDD values as a function of wavelength. It provides a conversion between the prism displacement (shown on the display) and the dispersion applied to the pulse. (The prism displacement magnitude is defined by a calibration procedure, which is detailed in the manual.)