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Integrated Photoacoustic, Confocal, and Two-Photon Microscope Integrated photoacoustic, confocal, and two-photon microscope Bin Rao Florentina Soto Daniel Kerschensteiner Lihong V. Wang Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 06/17/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Journal of Biomedical Optics 19(3), 036002 (March 2014) Integrated photoacoustic, confocal, and two-photon microscope Bin Rao,a Florentina Soto,b Daniel Kerschensteiner,b and Lihong V. Wanga,* aWashington University in St. Louis, Optical Imaging Laboratory, Department of Biomedical Engineering, One Brookings Drive, St. Louis, Missouri 63130 bWashington University School of Medicine, Department of Ophthalmology and Visual Sciences, Box 8096, St. Louis, Missouri 63110 Abstract. The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacous- tic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality micro- scope provides a platform for future biological and medical discoveries. © 2014 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.19.3.036002] Keywords: confocal microscopy; two-photon microscopy; photoacoustic microscopy; optical-resolution photoacoustic microscopy. Paper 130894R received Dec. 19, 2013; revised manuscript received Jan. 27, 2014; accepted for publication Jan. 28, 2014; published online Mar. 3, 2014. 1 Introduction provided in a modern fluorescence microscope, and (2) they Fluorescent proteins (FP) are structural homologs of Aequorea do not provide two-photon microscopy, the most important fluo- green fluorescent protein (GFP), and they are able to form an rescence microscopy technology. No modern microscope that can image both fluorescent proteins and nonfluorescent chromo- internal visible wavelength fluorophore from their own polypep- phores with confocal, two-photon, and PA technologies exists tide sequence. Since Chalfie et al. first successfully demonstrated for biologists and physicians. Here, we report the development the significant potential of GFP as a molecular fluorescent probe,1 of an automatically coregistered trimodality microscope that confocal fluorescence microscopy has been widely used to mon- fills this gap. In a familiar microscopic fluorescence imaging itor gene expression and protein localization in living organisms. setting, this microscope provides the research community with The development of genetic mutation technologies resulted in a novel platform for future biological and medical discoveries. enhanced GFP and variously colored mutants, such as blue (BFP), cyan (CFP), yellow (YFP), and their enhanced versions (EBFP, ECFP, and EYFP).2 The enriched contrast mechanisms 2 Material and Methods provided by these fluorescent proteins further promoted the usage of confocal fluorescence microscopy. The evolution 2.1 Microscope System from confocal technology to two-photon microscopy provided We built this microscope on a commercial Olympus inverted higher contrast, lower toxicity, and deeper imaging depth due setup (IX81). It integrates confocal, two-photon, and OR- to the unique two-photon excitation approach. Even more PAM imaging in a single platform, as shown in Fig. 1(a). recently developed optical-resolution photoacoustic microscopy A femtosecond laser (Mai Tai®, Spectra-Physics, Santa Clara, (OR-PAM) extends microscopy technology further to image California) is used for two-photon excitation. Several continu- nonfluorescent chromophores, such as hemoglobin, melanin, ous-wave (CW) laser beams of 405, 488, 543, and 635 nm 3–5 cytochromes, and lipids. Functional OR-PAM reveals the wavelengths are provided by a CW laser unit (not shown in physiological status of an organism by probing the optical absorp- Fig. 1) and coupled to a microscope visible port via a single- 2 tion of nonfluorescent proteins at different wavelengths. There mode optical fiber (not shown in Fig. 1). An acousto-optic should be other, unknown, nonfluorescent light absorbing chro- tunable filter in the CW laser unit can simultaneously output mophores that can serve as reporters for various vital biological any combination of these laser lines with different attenuations. processes. Our previously reported integrated confocal and The collimated laser beams for confocal imaging share the same – – OR-PAM imaging system6 8 and other similar systems4,9 11 are visible port with a tunable dye laser (Credo, Sirah Lasertechik not suitable for biologic discovery because (1) they do not GmbH, Grevenbroich, Germany) operating at a pulse repetition have flexibility in the selection of objectives, fluorescence dyes, rate of 10 kHz for PA imaging. Currently, with a pump laser of filter sets, and some of the complementary imaging modes, such 532 nm, a 541- to 900-nm wavelength range is covered with as bright-field imaging, wide-field epi-fluorescence imaging, different dye settings. Alternatively, a dye laser range between and differential interference contrast (DIC) imaging, which are 372 and 722 nm could be acquired if a pump laser wavelength of *Address all correspondence to: Lihong V. Wang, E-mail: [email protected] 0091-3286/2014/$25.00 © 2014 SPIE Journal of Biomedical Optics 036002-1 March 2014 • Vol. 19(3) Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 06/17/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Rao et al.: Integrated photoacoustic, confocal, and two-photon microscope platform (IX81, Olympus America Inc, Center Valley, Pennsylvania). The inset in Fig. 1(a) is a schematic of the IX81 platform. The forward-propagating PA wave excited by the dye laser beam is detected by acoustic transducer 2 whose focus overlaps the region of interest in the sample. The backward-propagating fluorescent light excited by either the confocal or two-photon laser beam is detected by one of the two groups of photomultiplier tube (PMT) detectors. One group of optical filters and photomultipliers detects fluorescence excited by the confocal lasers, and the other group detects fluo- rescence excited by the two-photon laser. A pin-hole at the focus of a confocal lens is optically conjugated with the focus of the objective in the sample. The pin-hole improves fluorescence image contrast by rejecting background fluorescence signals. The pin-hole is not necessary for two-photon fluorescence detection, which excites fluorescence signals only from the tight focus of the objective. Dichroic mirrors (DM 7 and DM 8) can be selected in the microscope’s software. For two-photon microscopy, dichroic mirror RDM690 is selected at the DM 8 location, and a second dichroic mirror is selected at the DM 7 location. For confocal fluorescence microscopy, no dichroic mirror is selected at the DM 8 location, and different dichroic mirrors are selected at the DM 7 location according to the fluo- rescence dye settings. When a 20∶80 (reflection:transmission) beam splitter is chosen at the DM 7 location and no filters are selected in front of the confocal PMT detectors, the confocal fluorescence microscope senses optical scattering instead of fluorescence. For PAM, no dichroic mirror is selected at the DM 8 location, and a highly reflective mirror is selected at the DM 7 location. Similarly, different dichroic mirrors will be selected in the software settings for DM 1 to 4 fluorescence detection locations. For two-photon microscopy, three different dichroic mirror sets can be selected and manually installed at Fig. 1 (a) Schematic of the integrated confocal, two-photon, and optical-resolution photoacoustic microscope. BS, beam splitter; DM, the DM 5 and DM 6 locations. dichroic mirror; DMW, dichroic mirror wheel; HWP, half-wave plate; In conventional modes, this microscope also supports bright- PBS, polarization beam splitter; PMT, photomultiplier tube. (b) Logic field imaging, wide-field epi-fluorescence imaging, and DIC imag- function of the integrated confocal, two-photon, and optical-resolution ing, which are not shown in the schematic. A beam profiling device photoacoustic microscope system. is used to align the OR-PAM imaging focus with the confocal laser beam focus and two-photon laser focus. The aligned trifoci 355 nm was used. This wide laser wavelength tuning capability guarantee the coregistration of three modality images. is the key to exploring both endogenous and exogenous optical The OR-PAM imaging mode is implemented passively as absorption contrast mechanisms in biological applications. shown in Fig. 1(b): an independent PA computer (Dell worksta- In this new microscope, a polarization beam splitter (PBS 1) tion T1600) listens to the synchronization signals sent from the allows the insertion of a dye laser beam for PAM into the optical analog box of the Olympus microscope system and acquires PA path of the confocal laser beams. A two-photon laser beam is signals from the acoustic transducers. Pixel, line, and
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