Media 1: Light-induced Ca²⁺ transients observed in widefield epi-fluorescence microscopy of excitable cells

Originally published in Biomedical Optics Express on 01 June 2012 (boe-3-6-1266

Immunological parameters before Ig-replacement treatment.

<p>Serum IgG (A), IgA (B) and IgM (C) levels in the year before Ig-replacement are shown for the primary (n = 58) and secondary groups (n = 27). Each symbol represents the mean value over the year for one subject and the bars represent the group median. The frequency of switched memory B cells (CD19⁺CD27⁺IgD⁻IgM⁻) as a proportion of peripheral blood B cells is shown for the primary (n = 50) and secondary (n = 10) groups (D). Dotted lines indicate the normal reference ranges for each. Data in panels A–C were analysed by a two-tailed unequal variance t-test and data in panel D were analysed by a two-tailed Mann-Whitney test; * p<0.05, ** p<0.01; n.s. non-significant.</p

Diagnostic delay and the presence of bronchiectasis.

<p>Diagnostic delay (time between symptom onset and antibody deficiency diagnosis) was determined for the primary (n = 58) and secondary (n = 25) groups (A). The percentage of subjects with or without bronchiectasis (determined by high-resolution CT scan) is shown for each group (B). Diagnostic delay by bronchiectasis presence or absence is shown for the primary (n = 45) and secondary (n = 21) groups (C). The bars in panels A and C represent median values. Data in panel A were analysed by a two-tailed unequal variance t-test and data in panel C were analysed by a two-tailed Mann-Whitney test; n.s. non-significant (p values <0.05 were considered significant).</p

Ig-replacement therapy in primary and secondary antibody deficiency patients.

<p>Data were analysed with a two-tailed Mann-Whitney test; n.s., non-significant. IV indicates intravenous; and SC, sub-cutaneous.</p

Number of immunosuppressive therapies used by each group before symptom onset.

<p>Number indicates the number of therapies used (a single subject may have had more than one therapy).</p

Number of serious and non-serious infections before and after Ig-replacement treatment.

<p>The number of serious infections requiring hospitalisation or IV antibiotics and the number of patient-reported non-serious infections in the year preceding Ig-replacement treatment (A–B) and in the year 2012/2013 (C–D) is shown. The bars represent the group medians. Serious (E) and non-serious infections (F) are shown for each patient before (filled symbols) and after (open symbols) treatment. Data in panels A–D were analysed by a two-tailed unequal variance t-test and data in panels E–F were analysed by a two-tailed paired t-test; * p<0.05, ** p<0.01, *** p<0.001; n.s. non-significant.</p

Immunodeficiency cohort on Ig-replacement treatment.

<p>CVID indicates common variable immune deficiency; ALPS, autoimmune lymphoproliferative syndrome; and WHIM, warts hypogammaglobulinaemia infections and myelokathexis syndrome.</p

Likely cause of secondary antibody deficiency in each subject.

<p>CLL indicates chronic lymphocytic leukaemia; MM, multiple myeloma; MGUS, monoclonal gammopathy of unknown significance; RTX, Rituximab; RA, rheumatoid arthritis; and SLE, systemic lupus erythematosus.</p

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