Fabian F. Voigt

Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens

Functional two-photon Ca2+-imaging is a versatile tool to study the dynamics of neuronal populations in brain slices and living animals. However, population imaging is typically restricted to a single two-dimensional image plane. By introducing an electrically tunable lens into the excitation path of a two-photon microscope we were able to realize fast axial focus shifts within 15 ms. The maximum axial scan range was 0.7 mm employing a 40x NA0.8 water immersion objective, plenty for typically required ranges of 0.2–0.3 mm. By combining the axial scanning method with 2D acousto-optic frame scanning and random-access scanning, we measured neuronal population activity of about 40 neurons across two imaging planes separated by 40 μm and achieved scan rates up to 20–30 Hz. The method presented is easily applicable and allows upgrading of existing two-photon microscopes for fast 3D scanning.

Rapid 3D light-sheet microscopy with a tunable lens

The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques.

Miniaturized selective plane illumination microscopy for high-contrast in vivo fluorescence imaging

Light-sheet-based fluorescence imaging techniques rely on simultaneous excitation of a single optical plane and thus permit high-contrast optically sectioned imaging of extended tissue samples. Here, we introduce a miniaturized fiber-optic implementation of a selective plane-illumination microscope (miniSPIM). The excitation light was delivered through a single-mode optical fiber, and a light-sheet was created with a cylindrical gradient-index lens and a right-angle microprism. Fluorescence emission was collected orthogonally to the light-sheet through a gradient-index lens assembly and a coherent fiber bundle. The end face of the fiber bundle was imaged onto a charge-coupled device camera. The spatial resolutions of the miniSPIM were 3.2 microm laterally and 5.1 microm axially. Images of fluorescent beads and neurons in mouse neocortex exhibited superior axial resolution and contrast in the miniSPIM-mode compared to images recorded in epi-illumination mode. The miniSPIM may thus enable novel in vivo imaging approaches.

Long-range population dynamics of anatomically defined neocortical networks

The coordination of activity across neocortical areas is essential for mammalian brain function. Understanding this process requires simultaneous functional measurements across the cortex. In order to dissociate direct cortico-cortical interactions from other sources of neuronal correlations, it is furthermore desirable to target cross-areal recordings to neuronal subpopulations that anatomically project between areas. Here, we combined anatomical tracers with a novel multi-area two-photon microscope to perform simultaneous calcium imaging across mouse primary (S1) and secondary (S2) somatosensory whisker cortex during texture discrimination behavior, specifically identifying feedforward and feedback neurons. We find that coordination of S1-S2 activity increases during motor behaviors such as goal-directed whisking and licking. This effect was not specific to identified feedforward and feedback neurons. However, these mutually projecting neurons especially participated in inter-areal coordination when motor behavior was paired with whisker-texture touches, suggesting that direct S1-S2 interactions are sensory-dependent. Our results demonstrate specific functional coordination of anatomically-identified projection neurons across sensory cortices. DOI: http://dx.doi.org/10.7554/eLife.14679.001

Online correction of licking‐induced brain motion during two‐photon imaging with a tunable lens

•  In order to understand the underlying behaviour of neuronal circuit dynamics, it is necessary to monitor brain activity in the awake, behaving animal. •  Licking to obtain water reward is an approach that is often used to measure an animals decision during reward‐based behaviour tasks. •  In head‐fixed mice, licking produces stereotyped brain motion that interferes with two‐photon calcium imaging of neuronal activity. •  We describe a method to provide online optical correction of licking‐induced brain motion during two‐photon imaging using refocusing with an electrically tunable lens. •  Online correction of licking‐induced brain motion improves the measurement of neuronal activity during reward‐based behaviour.

An R-CaMP1.07 reporter mouse for cell-type-specific expression of a sensitive red fluorescent calcium indicator

Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6.

Multiphoton in vivo imaging with a femtosecond semiconductor disk laser

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

A modular two-photon microscope for simultaneous imaging of distant cortical areas in vivo

We have designed and built a two-photon microscope which allows calcium imaging in awake, behaving animals across field-of-views (FOV) of up to 1.7 × 1.7 mm. A special scan system enables independent x,y, and z-positioning of two smaller sub-areas within this FOV for simultaneous functional recordings. This microscope enables us to optically record neuronal activity with cellular resolution across much larger spatial scales than previously possible and should help in deciphering the behavior-dependent flow of information within the neocortex. The microscope hard- and software are modular and can be extended to other imaging and photostimulation modalities.

Multiphoton microscopy in every lab: the promise of ultrafast semiconductor disk lasers

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.