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Advanced Imaging Microscopy

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Microscopes are a quintessential scientific tool, and their applications span well outside of science. People use them in hospitals, labs, and high tech manufacturing. Despite the pervasiveness, the conventional microscope, called a wide-field optical microscope, has major weaknesses.

Three of these weaknesses are: 1) Samples that are not cut extremely thin scatter light so that layers outside of the focus blur the image. Biological samples such as human tissue biopsies and structures embedded in a material are therefore difficult to measure with a wide-field microscope. 2) High contrast in a conventional microscope image requires high color variance over the image. This is a huge problem when cancer has the same color as normal tissue, or a semiconductor defect looks the same as normal semiconductor. It may seem like this is just a fact of life, but there exist other imaging metrics that can provide high contrast without high color variance. 3) A wide-field image does not provide any direct information about the function of the imaged object.

Confocal laser scanning microscopy (CLSM) overcomes the first challenge, depicted below, by filtering out most light not emitted from the focal point of the excitation light. Nonlinear techniques, including 2-photon absorption and four-wave mixing further improve the resolution in the z-direction.

Comparison of widefield microscopy (left) and confocal laser scanning microscopy (right). Figure adapted from Ishikawa-Ankerhold, et al. Molecules 17, 4047-132 (2012).

To overcome the other two weaknesses of wide-field microscopy, we need a new metric that is more descriptive than color of reflected or transmitted light. Advanced optical methods include Raman imaging, photoluminescence (or fluorescence) imaging, fluorescence lifetime imaging, two-photon imaging, four-wave mixing imaging, and ultrafast lifetime imaging microscopies. Researchers have demonstrated each of these methods for a subset of materials and applications. What method is best for your application?

In the table below we list five advanced imaging microscopy techniques with known applications. The functional info for each technique is the material or sample parameter that the technique can measure. For instance Raman imaging microscopy is useful for identifying composition, and four-wave mixing imaging microscopy is sensitive to the presence of defects and strain. We estimate the imaging speed by taking the specifications of known industry or laboratory systems.

Imaging microscopy MethodApplicationsFunctional InfoSpeed (1MP image)
Fluorescence, PhotoluminescenceBiology, semiconductor inspectionIdentifies trapped states>1 s
RamanGeology, chemistry, material science researchSubstance ID, vibrational modes>10 min
Fluorescence lifetime (FLIM)Biology researchSlow (nanosecond) energy transfer>10 min
Four-wave mixing (FWM)Semiconductor inspection, paint inspectionDefect and strain presence, material degradation>1 s
Ultrafast lifetime, coherence timeSemiconductor inspection, material science, biology, chemistry researchDefect identification, fast (femtosecond) energy transfer>10 s

Detection techniques can each be classified as either incoherent or coherent. Incoherent techniques including fluorescence/photoluminescence and conventional Raman are isolated with spectral filters and require measurement of very low intensity signals. Coherent techniques including stimulated Raman, four-wave mixing, and ultrafast techniques all involve nonlinear processes that enhance the signal emission. For low density samples, such as gases, incoherent detection schemes will typically provide a better signal-to-noise ratio (SNR). For dense samples a coherent method can improve the SNR by many orders of magnitude.

Here at MONSTR Sense we are particularly interested in the coherent imaging techniques because of the signal enhancement and the universal nature of MDCS to measure ultrafast lifetimes, coherence times, and vibrational modes.

Four-wave-mixing and Ultrafast Imaging Microscopy

At right is an example of four-wave-mixing (FWM) imaging microscopy from the research group of Prof. Jacek Kasprzak. Here the FWM energy reveals defects and how the defects affect the local potential. The researchers further measure the ultrafast decay time at specific points to determine how the defects affect the sample excitation. For more details about FWM imaging microscopy check out this article measuring encapsulated WSe2 and MoSe2 and this article to measure bare MoSe2 from the Kasprzak group. The technique was originally used to measure coupling between quantum dots.

The next figure is a FWM image of a strained III-V semiconductor (GaAs quantum well). It is evident in the image that inhomogeneous strain has enhanced and shifted the signal.

Common semiconductor defects (localized states, strain, and nanostructures) have enhanced optical dipole moments compared to 2D or 3D excitations in those materials. Because four-wave mixing scales cubically with the dipole, the four-wave mixing signal is very sensitive to these defects

In contrast to this four-wave-mixing detection, photoluminescence detection schemes scale linearly with the dipole moment and only identify defects that lower the local potential.

Defects and strain characterized in a monolayer semiconductor with four-wave mixing imaging. Four-wave-mixing image in (b) is much more revealing than an optical image (a) or photoluminescence map. Figure adapted from C. Boule et al. Phys. Rev. Materials 4, 034001 (2020).
Strain in III-V semiconductor (GaAs quantum well) revealed by four-wave-mixing imaging. Figure adapted from Dr. Brad Smith (U-Michigan).

The greatest challenge for laser-scanning microscopes coupled with spectroscopy is acquiring images quickly. For many spectrometers, 1 ms is a very short acquisition time. Unfortunately, if an imaging microscope measures 1 pixel every millisecond, the acquisition of a 1 MP image will require over 15 minutes. Though this timescale is acceptable for some research applications, it does not scale acceptably for most industry applications of imaging. To overcome these challenges researchers have made substantial efforts for each technique to reduce pixel acquisition time and increase the rate of the laser-scanning hardware. Despite these efforts, the most fundamental parameter to consider is the achievable SNR for each technique. Since SNR is technique and application dependent, it is not possible to report universal acquisition times for any advanced microscopy.

Disclaimer: MONSTR Sense has received an NSF SBIR Phase I award to develop a rapid-scanning imaging microscope based on four-wave-mixing and ultrafast metrics. The abstract can be found here.

Applications and Links

Advanced optical imaging techniques have transformed research in biology, physics, and material science. The techniques are also beginning to find industrial applications as experts verify their reliability. Here we focus on nonlinear technique where the emitted signal is stimulated. Stimulated techniques typically generate more intense signals, and therefore the image acquisition times are faster. Here we list applications with some links to the original work. Most of these researchers are physicists and chemists that have invested tremendous time and effort into building the setups they use to achieve their results.

Ultrafast imaging microscopy measures decay rates on a femtosecond timescale over an image. Four-wave mixing signals are typically excited by ultrafast pulses, this section also includes imaging of those nonlinear signals. Because coherent detection of nonlinear signals is a relatively recent technology advance, only a few of the measurements are single color. The traditional way of isolating the signal from the excitation beams in a microscope was to spectrally distinguish the excitation beams (two color). Recently single color has become possible by frequency tagging each excitation beam as discussed in the Collinear Advantage resource page. Though two-color experiments have been sufficient to demonstrate ultrafast microscopy, these methods excite phonons that artificially reduce the measured decay times and degrade the signal.

These cutting-edge techniques have proved useful in this small subset of applications. We are just starting to learn what is possible, and MONSTR Sense wants to help you start exploring faster. What application do you have in mind? Contact us to discuss how we can help.

Stimulated Raman is a coherent technique that, like four-wave mixing, measure the third-order-nonlinear response of a material. Because Raman spectra provide high specificity for substances and does not require fluorescent tags, Raman imaging is often synonymous with “label free”. For dense sample, stimulated Raman will provide a higher SNR than the traditional Raman imaging. Below is an incomplete list of applications:

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We offer compact and collinear all-in-one spectroscopy systems for transient absorption (pump-probe), stimulated Raman, and multidimensional spectroscopies. We invite you to learn more about the techniques’ capabilities and check out our products.

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