Effect of a confocal pinhole in two-photon microscopy

Gauderon, R.; Lukins, P. B.; Sheppard, Colin J. R.

doi:10.1002/(sici)1097-0029(19991101)47:3<210::aid-jemt7>3.0.co;2-h

Cited by 43 publications

(27 citation statements)

References 9 publications

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“…Whereas the nondescanned mode collects the most signal, the addition of a pinhole in the emission path of a point-scanning system can improve resolution when the pinhole is closed (9). In practice this is seldom done for biological specimens because signal-to-noise decays as the pinhole diameter decreases (9)(10)(11).…”

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confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

112

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Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 μm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.multiphoton | superresolution F luorescence microscopy is an invaluable tool for biologists.Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200-300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 μm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.

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confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

112

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“…Besides using a high numerical aperture objective and shorter excitation wavelengths, the optical resolution of the TPE system can be increased by using descanned detection in conjunction with a confocal pinhole in the emission path (see also the online data supplement). 13 However, a confocal pinhole will reject scattered emissions, even though they originated in the focal plane, thereby reducing fluorescence collection efficiency, and thus signal-to-background ratio.TPE events are exceedingly rare at light intensities typically used for epifluorescence microscopy. To generate enough fluorescence practical for TPE laser scanning microscopy, sample illumination is typically provided by pulsed lasers such as the mode-locked Ti:Sapphire laser, which generates pulses of Ϸ100 fs duration at a repetition rate of Ϸ80 MHz.…”

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confidence: 99%

“…It is therefore surprising that the difference in effective resolving power between near-infrared light TPE microscopy and visible light confocal microscopy has been shown to be much less than one would predict from the differences in excitation wavelength. 13 This finding results from the nonlinear nature of the two-photon absorption process, which in turn limits the spread of the excitation volume, thereby preventing a significant decrease in resolution resulting from the use of longer-wavelength excitation light. …”

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confidence: 99%

Two-Photon Microscopy of Cells and Tissue

Rubart

2004

Circulation Research

287

214

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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...

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“…As the scattered light is unpolarized, the intensities of the parallel-polarized and perpendicular-polarized components are equal, and therefore an e±cient gating method is to subtract the intensity of the perpendicular-polarized component from that of the parallel-polarized component, to give the pure polarized component. 110 Combinations of polarization gating with time-gating, 111 and angular gating have been reported. [88][89][90]95,112 2.…”

Section: Polarization Gatingmentioning

confidence: 99%

“…MPM can be combined with a confocal pinhole, [108][109][110][111][112] to improve the overall optical sectioning strength, but then use of a nondescanned detector is not possible.…”

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confidence: 99%

Advanced optical microscopy methods for in vivo imaging of sub-cellular structures in thick biological tissues

Chen

Rehman

Sheppard

2014

J. Innov. Opt. Health Sci.

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Optical microscopy has become an indispensable tool for visualizing sub-cellular structures and biological processes. However, scattering in biological tissues is a major obstacle that prevents high-resolution images from being obtained from deep regions of tissue. We review common techniques, such as multiphoton microscopy (MPM) and optical coherence microscopy (OCM), for di®raction limited imaging beyond an imaging depth of 0.5 mm. Novel implementations have been emerging in recent years giving higher imaging speed, deeper penetration, and better image quality. Focal modulation microscopy (FMM) is a novel method that combines confocal spatial ltering with focal modulation to reject out-of-focus background. FMM has demonstrated an imaging depth comparable to those of MPM and OCM, near-real-time image acquisition, and the capability for multiple contrast mechanisms.

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Effect of a confocal pinhole in two-photon microscopy

Cited by 43 publications

References 9 publications

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Two-Photon Microscopy of Cells and Tissue

Advanced optical microscopy methods for in vivo imaging of sub-cellular structures in thick biological tissues

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