Multiphoton Microscopy Applications

Label-Free Imaging

Two-photon microscopy and second- and third-harmonic generation methods allow for visualization of cells that have not been labeled or altered in any way. This approach of label-free imaging has greatly improved our understanding and ability to diagnose cancers.

Advancing Cancer Research with Multiphoton Microscopy

Cancer is a major cause of death worldwide and, given the aging global population, the number of deaths due to cancer is expected to continue to increase. The relationships between cancer, immune cells, and the collagen matrix are complex, and analyzing them requires the implementation of new technologies and ideas. Understanding the mechanisms behind these relationships will enable the development of more precise therapeutics and faster diagnostic tools.

The application of MEM with label-free fluorescence lifetime imaging microscopy and second-harmonic generation microscopy helps to move cancer research forward by providing highly precise data about these relationships.

Two T cells (green) infected with a human immunodeficiency virus (HIV) amidst collagen fibers (blue, SHG) and autofluorescent cells (white/orange hues) in a lymph node. Data courtesy of Dr. Thorsten Mempel, MGH Harvard.

Explore Bruker MEM Technology

Over the past two decades, multiphoton excitation microscopy (MEM) has enabled detailed intravital imaging of metastasis in animal models of cancer with supreme optical sectioning and minimal phototoxicity. In addition, MEM offers the possibility of fast imaging of unstained tissue based on its morpho-chemistry. As a result, MEM imaging has already greatly improved our ability to diagnose and treat cancer and will continue to play a significant role in preventing cancer-related deaths going forward.

Cancer researchers have successfully used Bruker multiphoton imaging systems to study the structure and physiological state of cells and tissues in normal and pathological conditions.

Cancer Research Applications of Multiphoton Microscopy

Imaging at single-cell resolution

Cancer researchers consider metabolic changes to be one of the key hallmarks of cancer. Consequently, imaging and assessing cellular metabolic changes has become critical to advancing cancer research. The assessment of such cellular metabolic responses can be achieved by two-photon imaging two endogenous, auto-fluorescent redox cofactors, reduced NAD(P)H, and oxidized FAD involved in multiple metabolic processes. In doing so, fluorescence lifetime imaging microscopy (FLIM) estimates the time the fluorophore is in the excited state before returning to the ground state and differentiates between free and bound NAD(P)H and FAD. Imaging the fluorescence intensity of NAD(P)H and FAD and measuring their fluorescence lifetime provides quantitative information about the cellular metabolism (oxidative phosphorylation and glucose catabolism) of cancer tissue. Combining FLIM with MEM allows for single-cell resolution. This is essential to collecting accurate and reliable results because various populations of cells may respond differently to for example a drug treatment.

Live cell metabolic imaging of cancer cells

Researchers' interest in monitoring cellular metabolic changes extends to cells participating in the immunological response to cancer. Using two-photon FLIM, researchers have found that immune cells — such as microphages and T-cells — and microglia cells in the brain respond to their microenvironment by taking adaptive or injurious phenotypes. For example, after activation, T-cells have increased metabolic demands. This metabolic state of increased aerobic glycolysis is required for T-cells to maintain their function. FLIM allows researchers to distinguish between quiescent and activated populations of T-cells, providing information they can then use to characterize immune cells in-vivo and in clinical applications focused on bio-manufactured cell lines for immune therapies.

Assessing alterations in cancer cell collagen organization

Changes in collagen organization are also associated with cancer. Oddly, the extracellular collagen matrix has been found to promote the progression of many types of cancer. The visualization of the spatial organization of collagen is based on a two-photon non-linear optical effect known as second-harmonic generation (SHG). SHG is a scattering process where two photons of the same wavelength interact simultaneously with certain biological structures, leading to the emission of a single photon with twice the energy and half the wavelength. Using this approach, researchers have found that it is possible to predict the prognosis of breast, ovarian, and pancreatic cancers depending on the alignment of collagen fibers with respect to each other or with respect to associated tumor boundaries.
Metabolic autofluorescence imaging in macrophage in 2D culture. Representative optical redox ratio, and NAD(P)H and FAD mean lifetime images. Data courtesy of Dr. Melissa Skala’s lab, Univ. of Wisconsin, Madison. Find more details by clicking on the image.


Mean NAD(P)H lifetime from T cells. First two images on the left present non-activated vs. activated cells in donor C, next two images show non-activated vs. activated cells in Donor D. Data courtesy of Dr. Melissa Skala’s lab, Univ. of Wisconsin, Madison. Find more details by clicking on the image.
Collagen fibers observed with SHG imaging match the geometry of implanted neural devices independent of probe size/type in silicon NeuroNexus probes. Fibers consistently conformed to the implant shape. Data courtesy of Dr. Kevin Eliceiri’s lab, Univ. of Wisconsin, Madison. Find more details by clicking on the image.