Multiphoton microscopy: A growing — and diversifying — area of critical science

The Women in Multiphoton Microscopy conference debuted this year at SPIE Photonics West, with keynote presentations from an international roster
22 March 2023
Karen Thomas
SPIE Fellow Melissa Skala, professor of biomedical engineering at Morgridge Institute for Research
SPIE Fellow Melissa Skala, professor of biomedical engineering at Morgridge Institute for Research, served as session chair for the Women in Multiphoton Microscopy conference. Credit: Morgridge Institute for Research

The quantum mechanical formulation of two-photon (multiphoton) molecular excitation was first conceived by Maria Goeppert Mayer in her doctoral thesis in the 1930s. Professor Watt Webb’s group at Cornell University later put the theory into practice by coupling an infrared laser with laser scanning confocal microscopy. This work was published in 1990 in the journal Science.

Today, multiphoton microscopy (MPM) is considered an imaging workhorse in life-science laboratories. There has been tremendous growth in the commercialization and building of MPM for various applications in labs around the world. The technique is known for its high-quality imaging of living cells, particularly within intact tissues (> 1 mm) such as brain slices, embryos, and whole organs. Goeppert Mayer, who was awarded the Nobel Prize in Physics for her work in 1963, is the second of only four women so far to receive the honor. But there are many in the optics and photonics community working to increase that number.

“The theory behind the multiphoton was developed by Maria Goeppert Mayer over 90 years ago,” says SPIE Fellow Ammasi Periasamy, professor of biology and biomedical engineering at University of Virginia. “The unit for the two-photon absorption cross section was named “GM” for Goeppert Mayer.” (1 GM is 10−50 cm4 s photon−1.)

Periasamy, an internationally recognized expert in advanced microscopy techniques and founder and director of the W.M. Keck Center for Cellular Imaging, started the multiphoton conference at Photonics West in 2001 with the help of Professor Peter So of MIT and later Karsten Koenig of Saarland University joined as a cochair. Since then, they have organized many events to promote MPM, including initiating the JenLab Young Investigator Award with JenLab GmBH in 2012. The annual JenLab award is granted to scientists under the age of 32 who are doing research in MPM.

SPIE Fellow Ammasi Periasamy, professor of biology and biomedical engineering at University of Virginia

SPIE Fellow Ammasi Periasamy, professor of biology and biomedical engineering at University of Virginia, started the multiphoton conference at Photonics West in 2001. Courtesy of Ammasi Periasamy.

To celebrate the achievements of Goeppert Mayer and other women working in MPM, Periasamy organized the first Women in Multiphoton Microscopy conference at this year’s SPIE Photonics West.

The three-part conference took place January 29, and featured keynote presentations and invited papers from renowned women researchers and scientists in the field of MPM.

What multiphoton microscopy can do

When asked about the applications of MPM, session chair Dr. Adeela Syed, project scientist and manager of the Optical Biology Core at University of California, Irvine, offers a still-growing list of applications in biology and medicine:

Live imaging of cells and tissues: MPM allows researchers to visualize the dynamics of cells and tissues in real time, providing important insights into the functions of various cellular structures and processes.

Metabolic Imaging: MPM, with the added modality of fluorescence lifetime imaging (FLIM), provides a technique for metabolic imaging that is non-invasive and does not require the use of exogenous labels or probes, making it a useful tool for studying metabolism in living systems.

Studies of neural activity: MPM can be used to study the activity of neurons in the brain, providing a detailed look at the complex networks that underlie brain function.

Cancer research: MPM can be used to study the behavior of cancer cells and the microenvironments in which they grow, providing important insights into the development and progression of cancer.

Drug development: Researchers can use MPM to study the effects of drugs on cells and tissues, helping to identify potential new treatments and to optimize existing therapies.

Skin imaging: MPM can be used to study the structure and function of the skin in great detail, providing important insights into skin diseases and the aging process without the need for biopsies.

Dr. Adeela Syed, project scientist and manager of the Optical Biology Core at University of California, Irvine

Adeela Syed, project scientist and manager of the Optical Biology Core at University of California, Irvine. Credit: University of California, Irvine.

 “This was my first time chairing a session at SPIE,” adds Syed. “I was very excited and honored to chair a session with so many women from across the world who have contributed to the field of MPM!”

Syed is in good company with SPIE Fellow and session chair Melissa Skala, professor of biomedical engineering at Morgridge Institute for Research. Skala’s lab at Morgridge develops biomedical optical-imaging technologies for cancer research, cell therapy, and immunology. “MPM has exciting applications, such as imaging dynamic behaviors of cells in their native environment, monitoring cell-cell interactions in 3D, and ultimately improving treatments for human disease,” says Skala.

Gail McConnell, professor of physics and director of the Centre for Biophotonics at the University of Strathclyde, gave a keynote presentation on her group’s progress with Mesolens, a giant custom-built objective lens with an unusual combination of low magnification and high numerical aperture (4x/0.47), for widefield multiphoton excitation microscopy. McConnell’s group works with the design, development, and application of linear and nonlinear optical instrumentation and new methods for biomedical imaging.

“As with the light microscope, which has become an icon synonymous with all sciences, MPM has found application in many different technical studies,” says McConnell. “There are other advantages of MPM, such as deeper imaging, which is needed for imaging thick, highly scattering tissue such as lung, and the greater difference between excitation and emission wavelength makes for better spectral separation, which can improve the quality of multiplex imaging. I’m particularly excited by the possibilities offered by mesoscale multiphoton imaging of clinical tissue, and the role of multiphoton imaging in single-cell sequencing and spatial transcriptomics.”

Women in MPM

As noted earlier, Periasamy began the Women Multiphoton Microscopy to celebrate the work of Maria Goeppert Mayer and other women working in this field. Considering the notable lack of women in many areas of science, this begs the question: Are more women working in this area?

“I have trained many male and female students and scientists in the area of MPM,” says Periasamy. “I have noticed a tremendous increase in women’s participation, year-byyear, in MPM and they have presented fantastic work at our conference covering technology development and applications in biomedical sciences.”

Skala has also noticed an increase in woman participating in MPM. “I train a lot of women in MPM and have a lot more women colleagues in this area now than when I started,” says Skala. “I think we see a general trend of more women in science, including MPM.”

McConnell is optimistic about the increasing numbers of women in science — with a strong caveat: In recent publications in MPM, most of the women publishing in the field are in the early stages of their careers. As an example, she points to a December 2022 article in Nature ( that noted a male:female authorship ratio of approximately 10:1 in recent work. “It’s improving, but far too slowly,” says McConnell. “We are still too far from gender-balanced representation. As a community — from student recruitment, through to fellowship appointment committees and funding panels — we must do all we can to ensure that we don’t lose this expertise and talent.”

Gail McConnell, professor of physics and director of the Centre for Biophotonics at the University of Strathclyde

Gail McConnell, professor of physics and director of the Centre for Biophotonics at the University of Strathclyde. Credit: University of Strathclyde.

McConnell points to the work of a fellow scientist Jayne Squirrell as one of the most elegant studies of in vivo microscopy. In a paper published in 1999, Squirrell and her colleagues at University of Wisconsin-Madison first demonstrated the potential of two-photon microscopy for more gentle three-dimensional imaging of living embryos — compared with its single-photon confocal imaging counterpart — and applications of in vivo imaging have continued apace since.

“It is generally the case that women are underrepresented in many STEM fields,” says Syed. “However, it is by efforts such as this conference that highlight and promote the participation of women in these fields, that this number grows. It is likely that the number of women working in MPM, as well as other areas of science and technology, will continue to increase in the future. It is important to promote diversity and inclusion in all fields, as diverse perspectives and experiences can lead to new ideas and approaches, and ultimately lead to greater scientific advances.”

Session Chair Margarida Barroso, professor of molecular and cellular physiology at Albany Medical College is researching the application of fluorescence lifetime microscopy to the measurement of drug-target engagement in pre-clinical tumor models. She notes that there are many women producing great work in the area of advanced microscopy, including multiphoton, FRET, FLIM, and many others. “I believe the reason why women are getting into MPM is because of the numerous role models that have opened up the field for the younger women coming up,” says Barroso.

Advice to students

For those interested in investigating or starting a career in MPM, McConnell suggests jumping right in, if possible, and visiting an imaging facility to see MPM in action. “You’ll hear from researchers about the challenges they face, and you may be able to consider new solutions that will improve MPM for many people,” says McConnell. She also suggests reaching out to other students, researchers, group leaders, and companies working in MPM technologies and applications.

“My advice is for young women to collaborate and work at the interface of biological applications and advanced imaging techniques,” says Barroso. “It’s always exciting to have great collaborators helping you to develop and apply your research to interesting and impactful biomedical applications.” Syed suggests starting with relevant coursework, such physics, biology, chemistry, and math, to build a strong foundation in the scientific principles that underlie MPM. She also advises considering doing research in a laboratory that uses MPM, as this can provide valuable hands-on experience and help develop the skills needed to work with this technology. For guidance and advice, she says, find a mentor who is experienced in MPM or in a related field. It’s also important to keep abreast of developments in the field by reading relevant scientific literature and attending conferences and workshops. This helps students stay current and competitive in the field.

“Don’t be discouraged,” adds Syed. “It is normal to encounter challenges and setbacks as you pursue a career in science. Research is just that, you have to search, and then “re” search again and again! Don’t let negative results discourage you and remember that perseverance and hard work can ultimately lead to success.”

Related SPIE content:

Join the JBO Hot Topics Webinar: Women Pioneering Multiphoton Microscopy in April.   

Enjoy this article?
Get similar news in your inbox
Get more stories from SPIE
Recent News
Sign in to read the full article
Create a free SPIE account to get access to
premium articles and original research