Two-Photon Microscopy of Cells and Tissue

Abstract

Abstract-Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering specimens by way of restricting fluorophore excitation (and thus emission) to the focal plane of the microscope. Spatial confinement of two-photon excitation gives rise to several advantages over single-photon confocal microscopy. First, penetration depth of the excitation beam is increased. Second, because out-of-focus fluorescence is never generated, no pinhole is necessary in the detection path of the microscope, resulting in increased fluorescence collection efficiency. Third, two-photon excitation markedly reduces overall photobleaching and photodamage, resulting in extended viability of biological specimens during long-term imaging. Finally, localized excitation can be used for photolysis of caged compounds in femtoliter volumes and for diffusion measurements by two-photon fluorescence photobleaching recovery. This review aims to provide an overview of the use of two-photon excitation microscopy. Selected applications of this technique will illustrate its excellent suitability to assess cellular and subcellular events in intact, strongly scattering tissue. In particular, its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep within intact, buffer-perfused hearts is demonstrated. Potential applications of two-photon laser scanning microscopy as applied to integrative cardiac physiology are pointed out. Key Words: two-photon excitation Ⅲ laser scanning microscopy Ⅲ calcium imaging T wo-photon excitation (TPE) microscopy 1 has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling longterm imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. For example, TPE of the calcium-sensitive fluorophore rhod-2 has been used to resolve differences in the kinetics of intracellular calcium ([Ca 2ϩ ] i ) transients in donor myocytes and juxtaposed host cardiomyocytes deep in Langendorff-perfused mouse hearts following intracardiac transplantation of fetal cardiomyocytes and skeletal myoblasts. 2,3 For neuroscientists, TPE microscopy has become an invaluable tool for studying calcium dynamics in thick brain slices and live animals 4,5 and for long-term imaging of neuronal development. 6 The spatial confinement of TPE has also been used for three-dimensional photolysis of caged compounds in femtoliter volumes [7][8][9] or diffusion measurements by two-photon fluorescence photobleaching recov- ery. 10,11 This article describes the basic physical principles of TPE and reviews the advantages and...

2‐photon excitation fluorescence microscopy enables deeper high‐resolution imaging of voltage and Ca2+ in intact mice, rat, and rabbit hearts

“…A high sensitivity of Di-4-ANEPPS to changes in V m was confirmed in the present study, whereas Fura-2 has a dissociation constant to free Ca 2+ of 224nM in the presence of 1mM Mg 2+ [16], which is close to diastolic intracellular [Ca 2+ ] in cardiomyocytes (150-300nM) [1]. This makes Di-4-ANEPPS and Fura-2/AM amenable to prolonged experiments in cardiac preparations, which coupled with the minimally invasive nature of 2P excitation [10][11][12] precedes other available techniques.…”

“…As such, laser power may be increased as focal depth is increased in order to maintain signal, as out-of-focus signal does not interfere [10,11]. Thus, deep imaging of multi-cellular preparations with high spatial resolution optical sectioning including along the Z-axis is achieved without the need for a F o r P e e r R e v i e w 4 detector pinhole (which is required in 1P excitation confocal microscopy to eliminate out-of-focus fluorescence emission), which further increases the fluorescence collection efficiency [11,12]. This, coupled with the availability of voltage-and Ca 2+ -sensitive dyes that fluoresce after 2P excitation [13,14] allowed us to develop protocols for high-resolution deep imaging of AP and intracellular Ca 2+ characteristics of in situ cardiomyocytes in intact, perfused whole hearts.…”

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

“…When the fluorescence techniques are applied on the level of tissues or whole organisms, strong limitations appear due to low penetration of light in these media because of high absorbance and light-scattering. Presently in this case the 'labeling' technology has definite advantages, since it allows free selection of fluorescence reporters including the dyes excited in the near-IR range 800-1,000 nm (the wavelength range of maximal transparency of living tissues) (Ntziachristos et al 2004;Smith et al 2011b), the fluorescent nanoparticles exhibiting upconversion phenomenon (Nagarajan and Zhang 2011) or applying two-photon excitation (Rubart 2004). In the case of deeply located cells and dense tissues the probing with radioactive or MRI contrast agents (Blankenberg and Norfray 2011) or smart nanoparticles that combine MRI and fluorescence responses (van Tilborg et al 2006) was suggested.…”

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…The first 2-photon excitation of electronic states was found for blue fluorescence emission with CaF 2 :Eu 2+ crystals based on the availability of lasers in 1961 (Kaiser & Garrett, 1961). To date, tunable mode-locked Ti:sapphire lasers operating at high repetition rate (typically 80-90 MHz) and a ultrashort pulse duration (less than 200 fs) are typically used as near infrared (NIR) sources in multiphoton excitation imaging (König & Riemann, 2003;Zipfel et al, 2003b;Rubart, 2004;Wang et al, 2005a). The required transient high irradiance can be obtained by tightly focusing ultrashort laser pulses within a sub-femtolitre focal volume provided by a special objective with high numerical aperture.…”