Superresolved multiphoton microscopy with spatial frequency-modulated imaging Jeffrey J. Fielda,b,c,d,1,2, Keith A. Wernsinga,1, Scott R. Dominguea, Alyssa M. Allende Motze,f, Keith F. DeLucab,c,d, Dean H. Levif, Jennifer G. DeLucab,c,d, Michael D. Younge, Jeff A. Squiere, and Randy A. Bartelsa,c,d,g aDepartment of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523; bDepartment of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523; cMicroscope Imaging Network Foundational Core Facility, Colorado State University, Fort Collins, CO 80523; dInstitute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523; eDepartment of Physics, Colorado School of Mines, Golden, CO 80401; fNational Renewable Energy Laboratory, Golden, CO 80401; and gSchool of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523 Edited by Rebecca R. Richards-Kortum, Rice University, Houston, TX, and approved April 19, 2016 (received for review February 23, 2016) Superresolved far-field microscopy has emerged as a powerful electronic states (19), and saturation of scattering due to surface tool for investigating the structure of objects with resolution well plasmon resonance (20, 21). below the diffraction limit of light. Nearly all superresolution Conversely, harmonic generation (HG) results in no net energy imaging techniques reported to date rely on real energy states of transfer to the contrast generating molecules. In this case, the fluorescent molecules to circumvent the diffraction limit, prevent- photokinetics are often described via virtual energy states that ing superresolved imaging with contrast mechanisms that occur drive instantaneous coherent nonlinear scattering. The application via virtual energy states, including harmonic generation (HG). We of most superresolution imaging methods to virtual energy states report a superresolution technique based on spatial frequency- is not possible, including structured illumination microscopy modulated imaging (SPIFI) that permits superresolved nonlinear (SIM) (22). Despite this challenge, examples of HG microscopy microscopy with any contrast mechanism and with single-pixel with subdiffraction-limited resolution have been reported (23, 24). detection. We show multimodal superresolved images with two- In both cases, the polarization state of an illuminating laser pulse photon excited fluorescence (TPEF) and second-harmonic genera- was manipulated to reduce the size of the effective PSF. Although SCIENCES tion (SHG) from biological and inorganic media. Multiphoton SPIFI both methods succeeded in collecting HG images with sub- η APPLIED PHYSICAL (MP-SPIFI) provides spatial resolution up to 2 below the diffrac- diffraction limited resolution, these methods are unlikely to be tion limit, where η is the highest power of the nonlinear intensity robust to imaging at depth in tissue because of effects such as response. MP-SPIFI can be used to provide enhanced resolution in birefringence, circular dichroism, and optical scattering, which optically thin media and may provide a solution for superresolved imaging deep in scattering media. disrupt the input polarization state of the excitation laser pulse. Until now, a technique that provides superresolved imaging of both incoherent and coherent contrast mechanisms in complex superresolution | harmonic generation | multiphoton microscopy media has not yet emerged. Here, we report the use of multi- photon spatial frequency-modulated imaging (MP-SPIFI) (25– f the numerous far-field image formation techniques, mul- 28) for superresolved imaging of second-harmonic generation tiphoton microscopy (MPM) has proved particularly valu- O (SHG) and two-photon excited fluorescence (TPEF). We show able in many biological studies, providing multimodal images from deep within tissues (1), as well as label-free images of complex biological structures (2–4) and inorganic harmonic Significance probes (5, 6). Deep imaging is achieved twofold in laser-scanning microscopy with nonlinear excitation. First, inherent optical Superresolution microscopy is indispensable in biological sci- sectioning provided by the nonlinear intensity dependence (7) ences. The vast majority of superresolution imaging techniques permits removal of the confocal pinhole that is required in lin- exploit real energetic states of fluorescent molecules to break early excited microscopy, and highly scattered signal light can be the diffraction limit. To date, superresolved imaging of second- collected with a single-pixel detector. Second, excitation wave- and third-harmonic generation has been limited to specific lengths in the near infrared (NIR) (8, 9) enhance penetration sample preparations where the polarization state of the exci- depth of the ballistic photons responsible for nonlinear excita- tation laser can be manipulated to overcome the diffraction tion (10). Because of the diffraction limit of light, these longer limit. Here, we describe a method for multiphoton super- excitation wavelengths yield lower spatial resolution than resolved imaging that does not place such restrictions on the wavelengths in the visible region of the spectrum, such as those sample and allows for simultaneous superresolved imaging of required for confocal fluorescence microscopy. An imaging both coherent and incoherent signal light. Combined with method that could meet or surpass the spatial resolution single-element detection, this technique may allow for signif- achievable with confocal fluorescence microscopy while main- icant advances in multimodal multiphoton imaging of highly taining the penetration depth of multiphoton microscopy could scattering biological tissues. dramatically impact biological imaging. The most successful superresolved microscopy techniques Author contributions: J.J.F. and R.A.B. designed research; J.J.F., K.A.W., and A.M.A.M. performed research; S.R.D., K.F.D., D.H.L., J.G.D., M.D.Y., and J.A.S. contributed new reported to date have exploited the real energy states of probe reagents/analytic tools; K.A.W. constructed the laser source; S.R.D. designed and con- molecules to generate image contrast. These real energy states structed the laser source; A.M.A.M. and D.H.L. provided CdTe solar cells for imaging; provide a means to manipulate fluorescent light emitted from K.F.D. and J.G.D. provided HeLa cells for imaging; J.J.F., K.A.W., and R.A.B. analyzed probe molecules, offering a pathway to reducing the effective data; and J.J.F., K.A.W., and R.A.B. wrote the paper. size of the point-spread function (PSF), which sets the spatial The authors declare no conflict of interest. resolution of the microscope. Some examples include saturated This article is a PNAS Direct Submission. structured-illumination microscopy (SSIM) (11), photoactivated 1J.J.F. and K.A.W. contributed equally to this work. localization microscopy (PALM) (12, 13), stimulated emission 2To whom correspondence should be addressed. Email: [email protected]. – depletion (STED) microscopy (14 16), ground state depletion This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (GSD) microscopy (17, 18), saturation of transient absorption in 1073/pnas.1602811113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1602811113 PNAS | June 14, 2016 | vol. 113 | no. 24 | 6605–6610 Downloaded by guest on September 27, 2021 that MP-SPIFI can be used to collect images of both SHG and represents the probability of contrast light generation for a second- TPEF simultaneously and with spatial resolution beyond that of a order nonlinear process, is shown in Fig. 1B.Toforma1D conventional laser-scanning multiphoton microscope. By combin- image of the object, the spatial frequency of the fringes in the ing the relative scattering immunity offered by illumination wave- illumination intensity, which is inversely proportional to the lengths in the NIR with single-pixel detection of signal light, MP- separation of adjacent fringes (Fig. 1C), is varied in time SPIFI has the potential to collect images from unlabeled biological through the range of spatial frequencies supported by the specimens that exhibit harmonic generation. objective lens, i.e., fxðtÞ ∈ ½−NA=λ, + NA=λ (Movie S1). Signal The physical principles of image formation in SPIFI micros- light generated by the interaction of the illuminating light sheet and copy have been rigorously studied in previous reports (25, 27). the specimen is then collected with a single-pixel detector. However, no reports to date have described imaging beyond the The measured signal from the detector, SðtÞ, is represented by diffraction limit with SPIFI. Here, we describe the image for- integratingR the signal light intensity, βðx, tÞ,overallspace: ð Þ = ∞ βð Þ = hβð Þi mation process in SPIFI for both linear and nonlinear excitation S t −∞ dx x, t x, t x. In linearly excited SPIFI, the of signal light generation. signal light is proportional to the product of the illumination The microscope configuration used for this work forms 1D intensity, Iðx, tÞ, and the spatial distribution of contrast-gen- images, similar to the systems used in refs. 25 and 27. Conse- erating molecules, CðxÞ.NotethatwehaveassumedCðxÞ is quently, we constrain the theoretical analysis that follows to a 1D stationary and does not photobleach during the measurement imaging system that collects image data in the x dimension. of a single 1D image. Absorbing proportionality constants However, there