Visualization 1: Label-free imaging of thick tissue at 1550 nm using a femtosecond optical parametric generator

Z stack of brain tissue. Originally published in Optics Letters on 01 August 2015 (ol-40-15-3484

Widefield two-photon microscope and image capture system.

<p>An upright microscope was modified for two-photon excitation by changing the excitation, emission and dichroic filters. A cooled sCMOS camera was used to detect fluorescence from the specimen. A femtosecond-pulsed Ti:Sapphire laser was used as the excitation source. The weakly divergent output from the Ti:Sapphire laser was coupled into the microscope and focused to provide a beam waist close to the back aperture of the objective lens. This produced a wide and weakly-focused beam in the specimen plane which did not contribute significantly to the optical sectioning power. HR = highly reflecting mirror, ND = neutral density filter, SWP = short-wave pass filter.</p

Resolution measurements and evaluation of the uniformity of the illuminated field.

<p><i>(a)</i> Lateral intensity profile through a 200 nm bead imaged using a 60x/1.35 NA oil immersion lens at an excitation wavelength of 820 nm, showing the lateral resolution to be 0.55 <i>μ</i>m from the full width at half maximum. <i>(b)</i> Axial intensity profile of the same bead, showing that the axial resolution is 1.5 <i>μ</i>m. <i>(c)</i> Widefield two-photon image of a fluorescent Perspex block used to evaluate the uniformity of the illuminated field. Scale bar = 15 <i>μ</i>m. <i>(d)</i> Intensity profile along the diagonal line in <i>(c)</i>, showing the fluorescence intensity varied by less than 10% across the field of view.</p

Reduced photo-bleaching using widefield two-photon excitation.

<p>(<i>a</i>) Single-photon and two-photon excited widefield images of live neuronal cells loaded with Fluo-4 AM, taken at frame rates of 1 Hz, 10 Hz and 100 Hz with continuous irradiation for 590 seconds. The normalised average fluorescence intensities are plotted versus time in (<i>b</i>) for single-photon excitation and in (<i>c</i>) for two-photon excitation. Photo-bleaching was observed when using single-photon excitation with image acquisition rates of 10 Hz and 100 Hz because of the higher light doses required to compensate for the short exposure times, whereas no photo-bleaching was observed when using two-photon excitation with an acquisition rate of 10 Hz, and there was weak photobleaching in fewer than half of the cell specimens imaged at a rate of 100 Hz, with the main source of error being time-averaged fluctuations in fluorescence signal intensity arising from Ca²⁺ signalling events. Scale bar = 15 <i>μ</i>m.</p

Localisation of changes in fluorescence intensity.

<p><i>(a)</i>, <i>(b)</i> and <i>(c)</i> show widefield two-photon excited fluorescence intensities within three adjacent live neuronal cell bodies loaded with Fluo-4 AM, acquired at a frame rate of 100 Hz over a duration of 45 seconds. This confirms that the measured change in fluorescence signal intensity with time was not global across the image, but was instead localised to individual cell bodies at different times. <i>(d)</i> An image of the field of view and accompanying ROIs from which the measurements were taken.</p

Widefield two-photon excitation for recording synaptic activity.

<p>(<i>a</i>) Fluorescence recording at a frame rate of 10 Hz shows spontaneous changes in fluorescence intensity in live neuronal cell bodies loaded with Fluo-4 AM, which were abolished in the presence of the glutamatergic antagonists DL-AP5 and NBQX (<i>b</i>). The elevated Ca²⁺ level seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147115#pone.0147115.g005" target="_blank">Fig 5b</a> fits with the observation of an overall increase in two-photon excited fluorescence signal over time at an image acquisition rate of 100 Hz shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147115#pone.0147115.g004" target="_blank">Fig 4</a>. The expanded region of (<i>a</i>) shows the envelope of events indicating synaptically-driven activity. (<i>c</i>) Whole-cell current clamp recordings from hippocampal neurones revealing spontaneous action potential firing which is abolished in the presence of DL-AP5 and NBQX (<i>d</i>).</p

Photo-bleaching experiments using primuline.

<p>(<i>a</i>) Single-photon and two-photon-excited widefield images of lens tissue fibres stained with 10 <i>μ</i>M primuline in tap water, taken at image acquisition rates of 1 Hz, 10 Hz and 100 Hz with continuous irradiation for 600 seconds. The normalised fluorescence intensities, averaged over 18 ROIs from 6 recordings made using 3 specimens for each image acquisition rate are plotted over time in (<i>b</i>) for single-photon excitation and in (<i>c</i>) for two-photon excitation. Photo-bleaching was consistently reduced with widefield two-photon excitation at all image acquisition rates. Scale bar = 15 <i>μ</i>m.</p

Photo-bleaching experiments using acridine orange.

<p>(<i>a</i>) Single-photon and two-photon excited widefield images of lens tissue fibres stained with 120 <i>μ</i>M acridine orange in PBS, taken at image acquisition rates of 1 Hz, 10 Hz and 100 Hz with continuous irradiation for 600 seconds. The normalised fluorescence intensities, averaged over 30 ROIs from 10 recordings made using 5 specimens for each image acquisition rate are plotted over time in (<i>b</i>) for single-photon excitation and in (<i>c</i>) for two-photon excitation. Photo-bleaching rates were similar for single-photon and two-photon excitation at image acquisition rates of 1 Hz and 10 Hz, but with an image acquisition rate of 100 Hz, photo-bleaching is markedly reduced with widefield two-photon excitation, with the normalised average fluorescence intensity having levelled off at 75% the initial value, while with single-photon excitation the fluorescence was at 45% after 600 seconds, and still decreasing. Scale bar = 15 <i>μ</i>m.</p