Abstract
Abstract Two-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. Two-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been postulated that the two-photon depth limit, beyond which results become inaccurate, is where in-focus and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ∼600 µm in mouse cortex. Neither the two-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth of two-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous two-photon and three-photon excitation. Two-photon and three-photon results from superficial locations were almost identical. two-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for two-photon excitation. However, the two-photon depth limit and balance depth were at 450 µm, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the two-photon depth limit in mouse visual cortex.
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📋 Methods
Basic three-photon microscope
Our three-photon microscope was built around a Coherent Monaco/Opera-F laser source (≤2 nJ, 50 fs pulses at 1 MHz; Coherent Inc.) and a modified MIMMS microscope manufactured by Sutter Instrument. We replaced the scan and tube lenses (respectively, Thorlabs SL50-3P and a Plössl pair of achromatic doublets, Thorlabs AC254-400-C) to improve transmission at 1300 nm. The primary dichroic mirror was FF735-DI02 (Semrock). We used an Olympus 25×/1.05 objective (75% transmission at 1300 nm) or Nikon 16×/0.8 objective (50% transmission at 1300 nm) and image acquisition was controlled by ScanImage (Vidrio Technologies LLC) with acquisition gating for low rep rate lasers. We estimated group delay dispersion (GDD) through the microscope at ∼15,000 fs 2 , approximately half of which was attributable to the Pockels cell (360-40-03-LTA, Conoptics Inc). To compensate, we built a four-pass pulse compressor using a single SF-11 glass prism (Thorlabs PS-853) and a two hollow roof prism mirrors (Thorlabs HRS1015-P01 and HR1015-P01). Compression was tuned by maximizing brightness with a fluorescein sample. Here, 400–500 mW of 1300 nm illumination was available after the objective, corresponding to transmission from laser source to sample of ∼20%. The maximum field of view of three-photon excitation was 360 × 360 µm. Images were acquired with dual linear galvanometers at a frame rate of ∼8 Hz. Illumination intensity Photodamage is often a concern in light microscopy. Photodamage can result from linear processes, principally heating (resulting from the absorption of infrared light by water in brain tissue) and from non-linear processes. Non-linear processes are of particular concern with high-energy pulsed sources such as those used for two-photon and three-photon fluorescence microscopy. Heating-related photodamage often occurs with >250 mW of prolonged illumination at 800–1040 nm ( Podgorski and Ranganathan, 2016 ) and the molar extinction coefficient of water at 1300 nm is ∼2× that at 900 nm ( Curcio and Petty, 1951 ; Hale and Querry, 1973 ; Bertie and Lan, 1996 ), suggesting that heating-related tissue damage may occur at more than ∼100–150 mW of prolonged illumination at 1300 nm. To avoid damage, we used illumination intensities
Show full methods section
Basic three-photon microscope
Our three-photon microscope was built around a Coherent Monaco/Opera-F laser source (≤2 nJ, 50 fs pulses at 1 MHz; Coherent Inc.) and a modified MIMMS microscope manufactured by Sutter Instrument. We replaced the scan and tube lenses (respectively, Thorlabs SL50-3P and a Plössl pair of achromatic doublets, Thorlabs AC254-400-C) to improve transmission at 1300 nm. The primary dichroic mirror was FF735-DI02 (Semrock). We used an Olympus 25×/1.05 objective (75% transmission at 1300 nm) or Nikon 16×/0.8 objective (50% transmission at 1300 nm) and image acquisition was controlled by ScanImage (Vidrio Technologies LLC) with acquisition gating for low rep rate lasers. We estimated group delay dispersion (GDD) through the microscope at ∼15,000 fs 2 , approximately half of which was attributable to the Pockels cell (360-40-03-LTA, Conoptics Inc). To compensate, we built a four-pass pulse compressor using a single SF-11 glass prism (Thorlabs PS-853) and a two hollow roof prism mirrors (Thorlabs HRS1015-P01 and HR1015-P01). Compression was tuned by maximizing brightness with a fluorescein sample. Here, 400–500 mW of 1300 nm illumination was available after the objective, corresponding to transmission from laser source to sample of ∼20%. The maximum field of view of three-photon excitation was 360 × 360 µm. Images were acquired with dual linear galvanometers at a frame rate of ∼8 Hz. Illumination intensity Photodamage is often a concern in light microscopy. Photodamage can result from linear processes, principally heating (resulting from the absorption of infrared light by water in brain tissue) and from non-linear processes. Non-linear processes are of particular concern with high-energy pulsed sources such as those used for two-photon and three-photon fluorescence microscopy. Heating-related photodamage often occurs with >250 mW of prolonged illumination at 800–1040 nm ( Podgorski and Ranganathan, 2016 ) and the molar extinction coefficient of water at 1300 nm is ∼2× that at 900 nm ( Curcio and Petty, 1951 ; Hale and Querry, 1973 ; Bertie and Lan, 1996 ), suggesting that heating-related tissue damage may occur at more than ∼100–150 mW of prolonged illumination at 1300 nm. To avoid damage, we used illumination intensities
📊 Figures
Figure 1.
Contrast declines with depth with two-photon excitation. A , Example three-photon images from 300, 600, 900, 1100, and 1400 u00b5m below the pial surface of visual cortex. Emx1-IRES-Cre;CaMK2a-tTA;Ai9...
Figure 2.
Implementation of near-simultaneous two-photon and three-photon excitation. A , Schematic of the optical layout for near-simultaneous two-photon and three-photon excitation; 1300-nm beam (black) passe...
Figure 3.
Changes in two-photon image quality and apparent u0394F with depth. Au2013D , Plots of image brightness ( A ), contrast ( B ), corrected motion ( C ), and ROI count ( D ) for two-photon (red) and thre...
Figure 4.
In-focus and out-of-focus fluorescence. A , Percentage of total fluorescence that originates from the focal plane, plot as a function of depth of the focal plane below the brain surface. Each point re...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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