The field of optical microscopy experienced significant gains in resolution and speed following the introduction of lasers. Unfortunately, these gains came at the expense of sample degradation caused by the continuous flux of intense light. Taking advantage of the two-photon absorption process, Webb and Denk implemented a microscope based on the use of near-IR light pulses capable of causing simultaneous multiple fluorophore excitation. Two-photon microscopy is now widely applied in the biomedical imaging field due to the absence of out-of-focus photobleaching and reduced photodamage and fluorescence scattering. These advantages are brought about collectively by the inherent instantaneous peak intensity and narrow focal plane of excitation. Given that peak intensity increases with decreasing laser pulse duration, one would expect extensive use of available ultrashort (sub-10 fs) pulse laser systems in the field of biomedical imaging. However, most two-photon microscopes still use the same pulse duration that Webb and Denk used in 1990 (≈150 fs).

Figure 1. The electric field of an ultrashort pulse (top) shown after group velocity dispersion (middle) causing significant broadening, or after third order dispersion (bottom) causing sub-pulses and signal loss. The experimental setup used for pulse characterization and dispersion compression at the focal plane of the microscope uses an adaptive pulse shaper between the laser and the microscope.

In principle, replacing a laser producing 150 fs pulses with one that produces 10 fs pulses should result in a factor of 15 improvement in signal. However, experimentally one would observe a net decrease of 30% in signal, with the loss of signal being caused by chromatic dispersion introduced by the high numerical aperture microscope objective lens (Figure 1). It is a common practice to distinguish between group velocity dispersion (GVD) and a third order dispersion (TOD). GVD causes different frequency components of the pulse to arrive at the sample at different times, effectively increasing the pulse duration, while TOD breaks the pulse into sub-pulses. A simple prism pair can compensate GVD. Such correction would cause a 2.2× increase in signal when using 10 fs instead of 150 fs pulses; unfortunately, the prism pair introduces a significant amount of additional TOD. Only by correcting both GVD and TOD, ensuring transform limited (TL) pulses (i.e. pulses with no dispersion), would the expected 15× improvement in signal for two-photon microscopy and an impressive 225× improvement for third-harmonic generation microscopy be observed.

Figure 2. Greater signal with MIIPS compensated pulses (TL) compared to GVD-only compensated pulses in images from mouse intestine tissue. Cross section of fixed intestine, stained with Alexa Fluor 350 wheat germ agglutinin (goblet cells), Alexa Fluor 568 phalloidin (actin) and SYTOX Green nucleic acid stain (nuclei). The image obtained using TL pulses had ≈5 times greater intensity than that taken with GVD-only compensated pulses. Image size 75μm top, 100 μm bottom.

For a given laser system with a fixed set of optics, it is possible to design a static optical device to eliminate GVD and TOD. However, practical correction for GVD and TOD for microscopy must take into account that the laser parameters and objective lenses are usually changed during imaging. Therefore, the ideal device must be able to measure and correct the femtosecond pulses at the focus of the microscope. Here we present the use of such an adaptive system capable of measuring and correcting all orders of dispersion, thus delivering TL pulses at the focal plane.

Figure 3. Higher depth capabilities with MIIPS compensated pulses (TL) over GVD compensation in mouse heart tissue. Section of mouse heart tissue (over 100μm thick) stained with Phaloidin 568 (actin). The images show actin fibers in heart muscle cells. The images obtained using TL pulses show increased penetration depth compared to the images taken with GVD-only compensated pulses. Image size 100μm.

The use of an adaptive optical device, as shown in Figure 1, allows one to measure and correct the chromatic dispersion at the focal plane without the use of interferometry. Such a device was first demonstrated by the Dantus group in 20041. The principle of operation is similar to that used in noise canceling earphones - the average noise is sampled and then the negative of the average noise is actively introduced, so that the average noise at the ear is cancelled. In this optical example, the dispersion is sampled and once measured, its negative is introduced to cancel all orders of chromatic dispersion at the focal plane. This entire process takes approximately one minute. The reader is referred to recent articles that describe this method, known as MIIPS, in greater detail [2, 3]. This award wining technology4, developed by the Dantus group at Michigan State University, has been patented and is now being commercialized by BioPhotonic Solutions Inc. and Coherent in their Silhouette pulse shaper.

The use of MIIPS enables optimization of the use of sub-15 fs pulses in bio-medical imaging. The signal gain is significant when images acquired using MIIPS are compared to images acquired using GVD correction at the same location as shown in Figure 2.

To study the implications of spectral phase correction for two-photon depth resolved imaging, we imaged a thick section of mouse heart tissue (Figure 3). The results shown here demonstrate increased penetration depth when higher-order dispersion is compensated by applying MIIPS, compared to GVD-only compensation. Additionally, our group has found that the increased efficiency gained by using ultrashort, TL pulses allows one to reduce the laser intensity incident on the sample leading to a significant reduction of light-induced damage and photobleaching 5.

In conclusion, automated phase measurement and compensation using MIIPS allows the use of ultrashort laser pulses for nonlinear optical imaging. Here we have demonstrated significant improvement (>5×) when compared against GVD corrected pulses. More significant enhancements are expected when the laser is delivered through an optical fiber, because of the additional dispersion, or when the laser is used for higher order imaging modalities such as third-harmonic generation imaging. We believe that the work presented here can help set the conditions needed for the implementation of ultrashort pulses, which will improve image resolution and penetration depth while minimizing photodamage and photobleaching in biomedical imaging.

This article was written by Marcos Dantus, University Distinguished Professor and Yair Andegeko, Research Associate, Michigan State University (East Lansing, MI). For more information, contact Mr. Dantus at This email address is being protected from spambots. You need JavaScript enabled to view it., Mr. Andegeko at This email address is being protected from spambots. You need JavaScript enabled to view it. or visit .


  1. J. M. Dela Cruz, I. Pastirk, V. V. Lozovoy, K. A. Walowicz, and M. Dantus, “Multiphoton Intrapulse Interference 3: probing microscopic chemical environments.” J. Phys. Chem. 108, 53 — 58 (2003).
  2. M. Dantus, V.V. Lozovoy, and I. Pastirk, “MIIPS characterizes and corrects femtosecond pulses,” Laser Focus World 43, 101-104 Feature Article, (2007)
  3. Vadim V. Lozovoy, Bingwei Xu, Yves Coello, and Marcos Dantus, “Direct measurement of spectral phase for ultrashort laser pulses,” Optics Express 16, 592-597 (2008).
  4. PhAST/Laser Focus World Innovation Award (2007), Honorable Mention, presented to BioPhotonic Solutions Inc. for “For developing a pulse shaper that can automatically measure and compensate phase distortions that broaden femtosecond laser pulses.”
  5. P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281, 1841—1849 (2008).